Morphological responses of three contrasting Soybean ( Glycine max (L.) Merrill) genotypes under different levels of salinity stress in the coastal region of Bangladesh

Soil salinity, a global environmental issue, inhibits plant development and production. Soybean is an economically important legume crop whose yield and quality are highly affected by excessive levels of salt in the root zone. A factorial experiment was conducted in a net house from October 2019 to January 2020 to evaluate the performance of three distinct soybean genotypes under varying levels of salinity stress. The experiment followed a completely randomized design (CRD) with three replications. Three soybean cultivars, namely BINA Soybean 1, BINA Soybean 2, and BINA Soybean 4. were used in this experiment. The soil salinity treatments were 0 mM NaCl, 50 mM NaCl, 100 mM NaCl, 150 mM NaCl, and 200 mM NaCl. The electrical conductivity (EC) of the soil sample was 0.91dS/m. Six seeds were sown 3 cm deep in each pot. A total of 45 pots were used in this experiment. The performance of each variety was evaluated based on its germination percentage, time of germination, no. of branches/plant, no. of leaves/plant, no. of flowers/plant, plant height (cm), no. of pods/plant, pod length (cm), seeds/pod, and root length (cm). Based on the results obtained from this research trial, it can be inferred that the BINA Soybean 2 variety along with 0 mM NaCl, 50 mM NaCl, and 100 mM NaCl treatments exhibited superior performance in all parameters compared to the other varieties. This study provides clear evidence that the soybean, particularly the BINA Soybean 2 variety, holds significant promise as a crop suitable for coastal regions. Furthermore, it suggests that the cultivation of soybeans in such areas could potentially enhance agricultural productivity, particularly in the presence of mild saline conditions. Nevertheless, it exhibits limited growth potential in environments with elevated salinity levels.


INTRODUCTION
The issue of salinity stresses poses a continuous and enduring threat to field crops and is widely acknowledged as a significant limitation on global agricultural productivity.It has contrary effects on the plant life cycle, including seed germination, seedling establishment and development, vegetative and reproductive growth, and crop survival (Zhu, 2016;Shu et al., 2017;Islam et al., 2019).Salt stress substantially condenses crop production throughout the arid and semi-arid regions of the world (Carpýcý et al., 2009).Approximately 6% of the total land area and 20% of irrigated land are affected by salinity (Ghonaim et al., 2021) comprising about 1125 million hectares in the world (Hossain, 2019).In Bangladesh, about 2.85 million hectares are of seaside land of which about one million hectares are salt-stressed (SRDI, 2010;Howlader et al., 2018).Saline soils are vastly contained in Na⁺ and Cl⁻ particles which makes water unavailable and negatively impacts crop growth.High salt concentrations adversely affect plant development and growth through various pathways, including water stress, nutritional disorders, ion toxicity, oxidative stress, alterations to metabolic processes, cell membrane disorganization, and reduced cell expansion and division (Hasegawa et al., 2000;Munns, 2002;Zhu, 2002;Shu et al., 2017).
Plants, in general, have established natural morpho-physiological protective mechanisms to conquer stress conditions (Ahmad et al., 2016).Halophytes can cope with the effects of salt stress by developing salt exclusion, salt elimination, and salt succulence (Larcher, 2003).However non-halophyte plants do not grow well in saline soil (Islam et al., 2019) due to the accumulation of reactive oxygen species (ROS) resulting in oxidative damage which disrupts cellular homeostasis and decreases photosynthetic efficiency (Khan et al., 2014).Therefore, salinity tolerance research is crucial to increase the ability of plants to raise in saline soil.Germination is the first criterion for consideration in salt-tolerant research (Fernández-Torquemada & Sánchez-Lizaso, 2013).In this study, we investigated the performance of salt stress on germination, growth, and development of three cultivars of soybeans.Soybean (Glycine max (L.) Merrill) as a key commercial crop, is an important and foremost edible oil crop, widely adopted and cultivated throughout the tropics and subtropics of the world.It has good nutritional quality for humans including proteins, lipids, and carbohydrates.Additionally, Soybean has a lot of saturated, monounsaturated, and polyunsaturated fatty acids and includes beneficial secondary metabolites such as isoflavones, phenolic components, and saponins (Sakthivelu et al., 2008;USDA, 2018).Over time, the global demand for soybeans is continuously rising.Soybean is a moderately saltsensitive crop with a salinity threshold of 5.0dS/m (Pavli et al., 2021).These plants are generally very sensitive to salinity stress during the seedling stage and less so for reproduction.That's why high salinity stress has expressively declined soybean yield by inhibiting seed germination and post-germinative growth.
Several key reasons are responsible for decreasing soybean production.High and uniform germination and emergence in the field are the key determinants of soybean yield, especially under salt stress conditions (Shu et al., 2017).Hence, the main objective of the current investigation was to assess the morphological responses of three distinct soybean genotypes under different salinity levels.The current observations have the potential to provide a conceptual framework for the identification and selection of soybean cultivars that exhibit tolerance to salinity.

Location and Period of the Experiment
The experiment was conducted during the period from October 2019 to January 2020 in a net house of the Department of Agriculture at Noakhali Science and Technology University, Bangladesh.

Soil Sampling
The soil sample used in the experiment was examined by the Soil Resource Development Institute (SRDI) located in Noakhali, Bangladesh (Table 1).The soil exhibited a sandy loam in texture and had a pH value of 6.7 which was measured by a portable pH meter (Hach sensION+ PH1 Basic Portable pH Meter).The electrical conductivity (EC) of the soil sample was 0.91dS/m (deci Siemens per meter) using an EC meter (Hach sensION+ EC7) in the laboratory of the Department of Agriculture.So, the soil was determined to possess a low salinity level.

Climate Condition
The climatic conditions in the locality were suggestive of the winter season, showing both low temperatures and limited precipitation.The average air temperature at the experimental site (Figure 1) was measured throughout the duration of the experiment using a digital thermometer (Digital Thermometer JR-1).

Planting Materials
Genetically diverse three genotypes of soybean viz.BINA Soybean 1, BINA Soybean 2, and BINA Soybean 4 were used in this experiment.These respective soybean cultivars were developed by the Bangladesh Institute of Nuclear Agriculture (BINA).

Experiment Design and Treatments
The factorial experiment involving two factors was conducted using a Completely Randomized Design (CRD) with three replications.The experiment consisted of three varieties viz.
V 1 = BINA Soybean 1, V 2 = BINA Soybean 2, and V 3 = BINA Soybean 4, along with five different salt concentrations (Table 2).Treatment combinations 15, the total number of pots 45, and the total number of seeds 270 were sown.

Pot Preparation and Fertilizers Application
In the pot experiment, soil that was not saline in nature was collected from the field and subsequently spread out on polythene sheets to go through the process of sun drying.The drying process lasted for a duration of one week.After that, the soil sample undergoes a process of crushing and screening, wherein a sieve is used to eliminate large particles and unnecessary substances.The soil was enriched with well-decomposed cow dung.The experiment utilized a total of 45 pots, each measuring 20 cm in width and 25 cm in height.Each pot contained 8 kg of soil that had been previously treated.A total of six seeds were planted at a depth of 3 cm in each individual pot.The soil was consistently kept moist to facilitate optimum germination, with water being applied as necessary.Afterward, the pots were placed inside the net house.Before starting the process of seeding, the soil was amended with fertilizers including Triple Super Phosphate (TSP), Muriate of Potash (MOP), and Gypsum.However, urea fertilizer was applied 25 days after seed germination (Table 3).All other agronomic practices were done when necessary.

Data Collection
The assessment of variety performance encompassed several parameters, including germination percentage, time of germination, no. of branches/plant, no. of leaves/plant, no. of flowers/plant, plant height (cm), no. of pods/plant, pod length (cm), seeds/pod, and root length (cm).The number of germinated seeds was recorded 10 days after sowing.The results were converted into a percentage.Germination percentage was determined by the following formula: The duration of germination was measured from the time of sowing until the last seed germinates.The total number of branches per plant, number of leaves per plant, and number of flowers per plant were recorded 70 days after sowing.Plant height (cm), number of pods per plant, pod length (cm), seeds per pod, and root length (cm) were recorded during harvesting.The soil temperature was recorded for each pot by using a digital thermometer.

Statistical Analysis
The data were statistically analyzed following the analysis of variance (ANOVA) and mean differences were adjudged by Tukey's HSD test at p<0.05 using MSTAT-C statistical software.

Soil Temperature
The soil temperature was recorded for each pot by using a digital thermometer during the experimental period.Then, it was converted into a month-wise average temperature (Figure 2).The optimum soil temperature for soybean germination is 25-28 °C (Tyagi & Tripathi, 1983;Bohner, 2003).In this experiment, in the month of December, the average soil temperature was found to be 25.2-26.9°C which was suitable for soybean seed sprouting.That's why the maximum germination was observed in the month of December.

Germination Percentage
In terms of germination percentage, substantial variation was found among the varieties.At 10 DAS, the V 2 (BINA Soybean 2) variety exhibited the highest germination percentage (71.11%)compared to the V 1 (BINA Soybean 1) and V 3 (BINA Soybean 4) varieties (Figure 3a).In addition, Table 4 reveals that both T 0 and T 1 treatments showed greater than 75% germination at 10 DAS.Maximum germination (90.74%) was observed in the T 0 (0 mM NaCl) treatment, which was statistically similar to the T 1 (50 mM NaCl) treatment, while the T 4 (200 mM NaCl) treatment provided the lowest germination rate (27.78%).Substantial variation was obtained by combined effects of variety and different levels of salt concentration on germination percentage at 10 DAS (Table 5).All the combined treatments were statistically different.V 2 (BINA Soybean 2) along with  T 0 (0 mM NaCl) provided the highest germination percentage (94.44%)which was statistically similar to the V 1 T 0 and V 3 T 0 treatment and minimum germination (16.67%) was recorded in V 1 T 4 treatment.In addition, up to 70% germination was observed in the V 2 T 2 (BINA Soybean 2×100 mM NaCl) treatment group.In contrast, less than 70% germination was observed for treatments V 1 T 2 (BINA Soybean 1×100 mM NaCl) and V 3 T 2 (BINA Soybean 4×100 mM NaCl).Among the three genotypes, BINA Soybean 2 performed better in the case of germination.Farhoudi and Tafti (2011) revealed that the germination rate and germination percentage of soybeans decreased as salinity levels increased during the growth   (2021) claimed that more than 100 mM NaCl concentration affected the seed sprouting of okra and gradually decreased the emergence of seeds with the increase of salt concentration.

Time of Germination
Based on the data presented in Figure 3b, it has been observed that BINA Soybean 4 exhibited a rapid germination rate (10.87 DAS), which was found to be statistically equivalent to the other two varieties.Late germination (14.56 DAS) was observed in the presence of a 200 mM NaCl concentration, indicating salinity stress.Conversely, the absence of NaCl (0 mM) resulted in fast sprouting (8.11 DAS) (Table 4).Significant variation was observed at the time of germination when considering the combined effects of variety and different levels of salt concentration, as indicated in Table 5.The treatment combination V 1 T 0 showed significantly fast germination (7.67 DAS) which was similar to the V 1 T 1 , V 2 T 0 , V 2 T 1 , V 3 T 0 , and V 3 T 1 treatment combinations.Inversely, the treatment combinations V 1 T 4 , V 2 T 4 , and V 3 T 4 showed a slower rate of germination.According to the study conducted by Farhoudi and Tafti (2011), it concluded that applying salinity stress resulted in a significant prolongation of the average germination period.

Number of Branches per Plant
The quantification of the number of branches per plant was done and the findings revealed inequalities among the different varieties (Figure 3c).The varieties BINA Soybean 2 and BINA Soybean 4 showed the highest number of branches per plant, with recorded values of 2.58 and 2.62, respectively.These two varieties were found to be statistically similar.However, the number of branches per plant gradually declined with the increase in the concentration of salt.In 0 mM NaCl showed maximum branching in the plant instead of other treatments (Table 4).The combination treatment V 2 T 0 showed a maximum number of branches per plant (4.87) whereas the minimum number of branches per plant was found in V 1 T 4 , V 1 T 3 , V 2 T 4 , and V 3 T 4 treatments (Table 5).El-Sabagh et al. (2015) observed that the control condition resulted in the highest average number of branches per plant.Furthermore, the researchers found that an application of salinity stress caused a significant decrease in the number of branches per plant compared to the control condition.Islam et al. (2012) reported a similar finding, observing a decrease in the number of branches in lentil plants exposed to salinity.The inhibition of new branch formation may be assigned to the presence of salinity.

Number of Leaves per Plant
Considering the number of leaves per plant, significant variations were noticed among all treatments and genotypes.Variety V 2 and V 3 showed maximum leaves per plant whereas variety V 1 showed minimum (Figure 3d).High salt concentrations decline the leaves number in plants.The highest number of leaves per plant (15.44) was found in 0 mM NaCl treatment (Table 4).In combination treatments, V 2 T 0 (BINA Soybean 2×0 mM NaCl) showed maximum leaves per plant (19.11), and the minimum leaves were found in V 1 T 3 , V 1 T 4 , V 2 T 3 , V 2 T 4 , and V 3 T 4 treatment respectively (Table 5).According to Mishra et al. (1995), the presence of a low concentration of NaCl did not have a significant impact on the leaf count.However, a higher concentration of soluble salt in water was found to have a notable influence on the number of leaves.

Number of Flowers per Plant
The superiority of the V 2 (BINA Soybean 2) variety in the number of flowers per plant (17.84) was more evident compared with other varieties (Figure 3e).The treatment with 0 mM NaCl resulted in the highest number of flowers per plant, while the number of flowers exhibited a decreasing trend as salt stress levels increased (Table 4).Combining the effects of variety and various levels of salt concentration on the number of flowers per plant produced substantial variation.V 1 (BINA Soybean 1) along with T 0 (0 mM NaCl) provided the highest number of flowers (32.33) which was statistically similar to the V 2 T 0 (32.11)treatment and the minimum flower number was recorded in V 1 T 4 (1.33) treatment (Table 5).Previous studies have demonstrated that the presence of high salinity levels has a substantial negative impact on the abundance of inflorescences and flowering (Ventura et al., 2014;Tayyab et al., 2016).

Plant Height
The data presented in Figure 4a clearly indicates that the various cultivars had significant effects on plant height.The highest plant height (20.64 cm) was obtained from V 1 (BINA Soybean 1).The results also showed that salt stress reduced the plant height.The maximum plant height (32.63 cm) was found in 0 mM NaCl salt concentration and the minimum plant height (2.52 cm) was found in 200 mM NaCl salt concentration (Table 6).The combined effects of variety and different levels of salt concentration in terms of plant height exposed significant variation (Table 7).The highest plant height was found in V 1 T 0 (BINA Soybean 1×0 mM NaCl) treatment which was statistically similar to the V 1 T 1, V 2 T 0, and V 3 T 0 treatments, and the lowest plant height was found in the V 3 T 4 treatments which was statistically similar to the V 3 T 3 , V 2 T 3 , V 2 T 4 and V 1 T 4 treatment combinations.Begum et al. (2022) reported that salt stress reduced the plant height, biomass, and relative water content.Amirjani (2010) observed that increasing salinity levels of 50, 100, and 200 mM NaCl resulted in a reduction of plant height of 30, 47, and 76%.

Number of Pods per Plant
Statistically significant differences existed among the varieties for the average number of pods per plant (Figure 4b).From the results, the maximum number of pods per plant (13.38) was found in V 2 (BINA Soybean 2) variety.Different salt stress also affects the pod quantity in the plant.Maximum pods per plant (23.26) were found in T 1 (0 mM NaCl) treatments while the minimum (1.3) was found in T 4 (200 mM NaCl) treatment (Table 6).The combined effects of variety and salinity also showed significant variation (Table 7).The extreme number of pods per plant (25.89) was collected from the combination of BINA Soybean 2×0 mM NaCl (V 2 T 0 ) which was statistically similar to the V 1 T 0 treatment combination and the lowest number of pods per plant (0.79) was collected from the V 3 T 4 which was statistically similar to the V 3 T 3 and V 1 T 4 treatment combinations.Ghassemi-Golezani et al. (2009) revealed that salinity stress limited the production of pods and grains per plant in soybeans.Mannan et al. (2013) observed that the reduction in the number of pods per plant at 50 mM NaCl ranged from 29% to 61%, and at 100 mM NaCl ranged from 48% to 80%.

Pod Length
The pod length was measured, and the results showed a significant difference among the varieties.The highest pod length (4.02 cm) was recorded in V 2 (BINA Soybean 2), whereas the lowest pod length was recorded as 2.99 cm and 3.04 cm in V 1 and V 3 varieties (Figure 4c).However, the pod length was statistically different and showed a significant reduction under high salinity stress.The highest pod length (5.59 cm) was noted in T 0 (0 mM NaCl) treatment and the lowest pod length (1.09 cm) was noted in T 4 (200 mM NaCl) treatment (Table 6).Considerable variation was obtained by combined effects of variety and different levels of salt concentration on pod length (Table 7).The highest pod length (6.41 cm) was observed in V 2 T 0 (BINA Soybean 2×0 mM NaCl) treatment and the lowest pod length (0.78 cm) was observed in the V 3 T 4 treatment combination.In another leguminous crop, the most extended pods were found in the control condition compared to the other treatments (Düzdemir et al., 2009).

Seeds per Pod
The findings revealed statistically significant differences in the number of seeds per pod across the three varieties (Figure 4d).The maximum number of seeds (2.39) was produced in V 2 (BINA Soybean 2), which was significantly higher than the other two  varieties.Seeds per pod were meaningfully influenced by the different concentrations of NaCl.Seeds per pod reduction were more in T 4 (200 mM NaCl) treatment and less in T 0 (0 mM NaCl) treatment (Table 6).The combined effects of variety and salinity also exhibited significant variation (Table 7).V 2 (BINA Soybean 2) along with T 0 (0 mM NaCl) provided the maximum number of seeds per pod (4.77) and the minimum number of seeds per pod (0.29) was recorded in V 1 T 4 treatment which was statistically alike to the V 2 T 4 , V 3 T 3 , V 3 T 4 , and V 1 T 3 treatment combinations.Consequently, BINA Soybean 2 outperformed the other two varieties.Ghassemi-Golezani et al. (2009) discovered that salinity stress limited the production of pods and grains per plant in soybeans.Similar results were found by Islam et al. (2012) in lentil genotypes and Mannan et al. (2013) in BARI Soybean 5 under salt stress.

Root Length
The progressive decrease in soybean root length was caused by the deleterious effect of high salinity levels (Figure 4e).The variety V 2 (BINA Soybean 2) exhibited the highest root length (11.51 cm), which was significantly higher compared to the other two varieties.However, the T 0 treatment exhibited the greatest root length, measuring 18.19 cm, while the remaining treatments demonstrated a notable decrease in root length when given high salinity stress (Table 6).In the case of a combination effect, the V 2 T 0 (BINA Soybean 2×0 mM NaCl) treatment generated the longest roots (19.67 cm) in comparison to the other combination treatments (Table 7).Chowdhury et al. (2018) observed a significant reduction in root and shoot length of plants in response to increasing NaCl concentration, which aligns with the findings of our experimental study.Islam et al. (2012) reported comparable findings in their study on the impact of salt stress on lentil genotypes.

CONCLUSION
The impact of salinity stress on various soybean cultivars was assessed during both the vegetative and reproductive stages.
In the presence of stressful conditions, the BINA Soybean 2 variety along with 0 mM NaCl, 50 mM NaCl, and 100 mM NaCl treatments demonstrated superior performance across all measured parameters in comparison to the other varieties.The variety of BINA Soybean 2 with a concentration of 100 mM NaCl resulted in seed germination exceeding 70%.Nevertheless, seed germination was reduced under salt treatment as the salt concentration increased.Additionally, the current study revealed that BINA Soybean 1 and BINA Soybean 4 exhibited greater susceptibility and lower tolerance to salinity stress, particularly at a concentration of 100 mM NaCl, compared to BINA Soybean 2. The findings of this study contribute to the advancement of our understanding regarding plant responses to salt-induced stress.These findings are particularly useful for the cultivation of soybean cultivars in coastal areas of Bangladesh.

Figure 3 :
Figure 3: Genotypic performance effect on (a) germination percentage; (b) time of germination (DAS); (c) no. of branches/plant; (d) no. of leaves/plant; (e) no. of flowers/plant; V1: BINA Soybean 1; V2: BINA Soybean 2; V3: BINA Soybean 4. The values are the averages of three replicates ± SE (standard error).One-way ANOVA (analysis of variance) was used to find the mean, standard error mean, and p-value.Tukey's HSD of post-hoc test was used to find the significant differences between treatments.Different letters (a, b, c) show significant differences according to the homogenous subsets of Tukey's HSD test at 0.05 level of significance

Figure 4 :
Figure 4: Genotypic performance effect on (a) plant height (cm); (b) no of pods/plant; (c) pod length (cm); (d) seeds/pod; (e) root length (cm); V1: BINA Soybean 1; V2: BINA Soybean 2; V3: BINA Soybean 4. The values are the averages of three replicates ± SE (standard error).Oneway ANOVA (analysis of variance) was used to find the mean, standard error mean, and p-value.Tukey's HSD of post-hoc test was used to find the significant differences between treatments.Different letters (a, b, c) show significant differences according to the homogenous subsets of Tukey's HSD test at 0.05 level of significance

Table 1 : Physio-chemical properties of the soil sample Measured soil parameters Analytical values
Figure 1:The mean air temperature at the experimental site.The maximum temperature was recorded in the month of October at 31.7 °C, while the minimum temperature was recorded in January at 24.3 °C

Table 3 : Recommended fertilizer doses Name of the fertilizers Dose per pot Total amount
Figure 2: Average soil temperature during the pot experiment

Table 5 : Combined effects of variety and different levels of salt concentration on germination percentage, time of germination (DAS), number of branches per plant, number of leaves per plant, and number of flowers per plant Treatment combination Germination percentage Time of germination (DAS) Number of branches per plant Number of leaves per plant Number of flowers per plant
=Significant at 5% level of probability, CV=Co-efficient of variations, SE(±)=Standard Error.V 1 =BINA Soybean 1, V 2 =BINA Soybean 2, V 3 =BINA Soybean 4, T 0 =0 mM NaCl, T 1 =50 mM NaCl, T 2 =100 mM NaCl, T 3 =150 mM NaCl, T 4 =200 mM NaCl, Here, values in the column having a similar letter (s) are statistically identical (Tukey's HSD test at p<0.05) *