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Full Research Article

Exploring Flood Resilience: Analysing Root Images for Physiological Responses to Flood Stress

T
Tejaswini Warik1
G
Godawari Pawar1,*
S
Shivaji Mehtre2
H
Hirakant Kalpande1
S
Sunil Umate3
A
Ambika More1
A
Ashwini Pawar1
1College of Agriculture, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani-431 402, Maharashtra, India.
2Officer Incharge Soybean Research Station, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani-431 402, Maharashtra, India.
3Wheat and Maize Breeder, Wheat and Maize Research Unit, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani-431 402, Maharashtra, India.

ABSTRACT

Background: The experiment followed a randomized block design with two replications, including control and waterlogging treatments. After 40 days of normal growth, waterlogging was applied for two weeks with a 12 cm water level above the soil. Observations were recorded eight days after drainage, identifying four tolerant and four sensitive genotypes based on physiological and anatomical traits.

Methods: The experiment was conducted during the kharif season of 2022-23 for one year at the field of All India Coordinated Research Project (AICRP) on Soybean, VNMKV, Parbhani. Among the 27 soybean genotypes evaluated under waterlogging, MAUS 710, JS-20-76, KDS-726 and NRC 257 exhibited high tolerance, showing improved growth and root traits like aerenchyma formation and root diameter. While MAUS 725, MAUS 731, EC250591 and JS 97-52 were susceptible.

Result: Among 27 soybean genotypes tested under waterlogged conditions, MAUS 710, JS-20-76, KDS-726 and NRC 257 observed high tolerance, excelling in traits like aerenchyma formation, root surface area and average root diameter. JS-20-116, KDS-726, JS-20-76 and NRC-257 exhibited moderate tolerance, while MAUS 731 and MAUS 725 were more susceptible. These findings highlight the importance of root traits for flood tolerance and suggest that the tolerant genotypes should be prioritized in breeding programs. Waterlogging resulted in average yield loss, with reduction in root architecture.

INTRODUCTION

Soybean [Glycine max (L.) Merrill] is a self-pollinated, diploid leguminous crop (2n = 40), originated in China. It is often referred to as the “cow of the field” or “gold from the soil” due to its high oil and protein content (Sivabharathi et al., 2025). Soybean belongs to the Leguminosae family and exhibit a symbiotic relationship with many microorganisms, including rhizobium (Budiastuti et al., 2025). Soybean contributes about 25% of the world’s edible oil and is a key source of protein for animal feed, especially in poultry and aquaculture. It also provides important raw materials for the food, pharmaceutical and other industries (Alnuaim et al., 2025). Reduced tillage improved soybean yield, nutrient uptake, protein, oil content and economics. Applying 100% of the recommended fertilizer dose gave the highest productivity and profitability under the mustard-soybean system (Singh et al., 2025).
       
Brazil leads in soybean production, followed by the United States, Argentina and China, with India ranking fifth-Madhya Pradesh alone contributing 78% of the country’s output (SOPA, 2022). Despite its adaptability, soybean faces significant challenges from abiotic stresses, particularly flooding and drought, which adversely affect growth and yield (Khatoon et al., 2012). Flooding is a critical stressor, impacting about 10% of the Earth’s land area. (Leng et al., 2019; Gibbs and Greenway, 2003). As climate change projected to increase flood risks, the development of resilient soybean varieties is crucial. Nevertheless, certain soybean varieties exhibit flood tolerance through adaptations such as aerenchyma formation and enhanced root elongation, mediated by phytohormones like ethylene and abscisic acid (Bailey-Serres and Voesenek, 2008).
       
This study aimed to understand how soybean plants adapt to flooding stress, focused on analyzing root responses to flood stress using image analysis. The goal of study was to identify flood-tolerant genotypes for sustainable farming and food security.

MATERIALS AND METHODS

The experiment was conducted during the kharif season of 2022-23 for one year at the field of All India Coordinated Research Project (AICRP) on Soybean, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani. The site is located at 19°16'N latitude, 76°47'E longitude and an altitude of 409 meters above mean sea level. The experiment followed a randomized block design with two replications, comprising of 27 genotypes (Table 1) under control and waterlogging treatments. After 40 days of normal growth, a waterlogging treatment was imposed for two weeks, maintaining a water level of 12 cm above the soil surface. Observations were recorded eight days’ post-drainage, leading to the identification of four tolerant and four sensitive genotypes based on physiological and anatomical traits.

Table 1: List of genotypes included in the study.


 
Pot experiment
 
Parallel to the field study, a pot experiment was conducted using plastic bags filled with a 50:1 (v/v) mixture of vertisols and farmyard manure. Each genotype was represented by ten bags, with two subjected to waterlogging and two as controls. Plants were thinned to three per bag at 21 days post-sowing and treated similarly to the field experiment. Soil moisture was maintained at 60-90% field capacity.
 
Assessments
 
Root length
 
Root lengths of the main roots were measured with the help of measuring scale after uprooting the plants (Chintey et al., 2025).
 
Average diameter of the root
 
Root images were analysed using ImageJ software by calibrating the scale with a reference marker, converting the images to binary and using the measurement tools to determine root area and length, from which average diameter was automatically obtained (Schneider et al., 2012).
 
Root surface area
 
After calculating root length and root diameter, root surface area was calculated by following formula:
 
Root surface area = Root length x Root average diameter x π
 
Number of seminal roots
 
The number of seminal roots per plant was visually counted after carefully uprooting the seedlings and gently washing the roots to remove soil without causing damage.
 
Assessment of root anatomy: TS of root
 
Medium-sized, freshly harvested Glycine max roots were manually sectioned into extremely thin cross-sections, stained with Acetocarmine stain (45% w/v) for 30 to 90 seconds and images were taken under a compound light microscope (Rajan et al., 2025).
 
Statistical analysis
 
The data collected from the experiment were statistically analyzed using a completely randomized design, following the procedure described by Panse and Sukhatme, 1954; (Ramanadane et al., 2025).

RESULTS AND DISCUSSION

Impact of waterlogging on soybean growth
 
Waterlogging reduces soil oxygen, negatively impacting soybean growth and yield. Tolerant genotypes adapt through increased root formation and aerenchyma (Shimamura et al., 2010), while early reproductive stages are particularly sensitive, affecting pod and seed development (Ploschuk et al., 2022).
 
Aerenchyma formation in soybean roots
 
Flood stress induced a notable enhancement of aerenchyma formation in soybean roots, indicating an adaptive response (Fig 1). Cross-sectional analysis showed significant aerenchyma in tolerant genotypes (MAUS 710, JS 20-76, KDS-726, NRC-257) and minimal to absent in susceptible ones (MAUS 725, MAUS 731, EC 250591, JS 97-52). Extending from cortex to medulla, aerenchyma supports oxygen transport under flooding (Shimamura et al., 2010). Ethylene accumulation during waterlogging restricts gas diffusion, increases ROS and triggers cell death, forming aerenchyma lacunae (Hayashi et al., 2013). This adaptation enhances flood tolerance in soybeans, supported by earlier studies (Hossain and Uddin, 2011; Hingane et al., 2015; Ryser et al., 2011; Visser et al., 1997; Sakazono et al., 2000; Hayashi et al., 2013; Solaiman et al., 2007).

Fig 1: TS of root-Aerenchyma formation in roots.


 
Root length responses to flood stress
 
Significant reduction in root length was observed under flood stress compared to control across genotypes (Table 2). It highlights the impact of flood stress on soybean root length. Under control conditions, root lengths ranged from 37 cm (MAUS 725) to 49.1 cm (NRC 256), while waterlogging reduced them significantly-down to 10 cm in EC 457464 and EC 250591 and 22.6 cm in NRC 257. These findings align with earlier studies showing that flooding during the reproductive stage limits root growth due to anaerobic conditions (Jhorar et al., 1995; Sorte et al., 1996; Sallam and Scott, 1987, Jitsuyama, 2015; Figueroa-Bustos  et al., 2018). Similar reductions were reported by Solaiman et al., (2007) and Sakazono et al., (2014), with losses ranging from 2.7% to 83.1%.

Table 2: Root morphological traits and yield performance of soybean genotypes under control and flood conditions.


 
Root average diameter responses to flood stress
 
Root average diameter varied among genotypes, with higher values recorded under flood stress relative to control (Table 2). It shows that root diameter varied significantly among soybean genotypes under control and flood conditions. Under control, diameters ranged from 0.22 mm (EC 457464) to 0.36 mm (NRC 256, NRC 257), while flooding increased them to 0.43-0.57 mm in NRC 186, NRC 257. These findings support Keeley (1979), who noted that flood-tolerant genotypes develop larger, more branched roots and align with reports that aerenchymatous roots have greater diameters (Visser et al., 2000a, b; Matsui and Tsuchiya, 2006; Grimoldi et al., 2005).
 
Root surface area responses to flood stress
 
Flood stress caused a notable decline in root surface area, reflecting restricted root development (Table 2). It shows that root surface area varied significantly between control and waterlogged conditions. Under control, it ranged from 27.63 cm2 (EC 457464) to 57.54 cm² (NRC 256), while flooding reduced it to 13.5 cm² (NRC 257) to 39.5 cm² (NRC 186).
 
Number of seminal roots responses to flood stress
 
Significant increase in the number of seminal roots was observed under flood stress compared to control across genotypes (Table 2). It shows that flood-tolerant soybean genotypes increased seminal root numbers under waterlogging (Fig 2). In control conditions, root counts ranged from 6 (JS 20-76, MAUS 71) to 12 (JS 20-116, KDS 726, NRC 256), while flooding increased them up to 19 in MAUS 710 and 18 in JS 20-76. This adaptive response aligns with studies reporting that waterlogging promotes adventitious root and aerenchyma formation for survival (Hossain and Uddin, 2011; Jhorar et al., 1995; Keeley, 1979). Ethylene plays a key role in this response by enhancing adventitious and lateral root development under low oxygen (Malik et al., 2001; Sauter, 2013).

Fig 2: Comparison of number of seminal roots in control and flood condition.


 
Average yield (g/row)
 
Significant reduction in average yield was observed under flood stress compared to control across genotypes (Table 2). It highlights yield differences among soybean genotypes under control and flood conditions. Under control, NRC 256 had the highest yield (248 g/row) and MAUS 725 the lowest (61 g/row). Under flooding, MAUS 710 (112.5 g/row), KDS-726 (108.5 g/row) and JS 20-76 (106 g/row) performed best, while MAUS 731 (32.5 g/row) and MAUS 725 (38.5 g/row) showed the lowest yields. These findings confirm genotype-dependent yield variation under flooding, with tolerant genotypes experiencing less yield loss (Kumar et al., 2013; Ren et al., 2014; Zhou et al., 2021).

CONCLUSION

Waterlogging significantly affects soybean root traits and yield performance. Tolerant genotypes showed well-developed aerenchyma, supporting internal oxygen transport. These genotypes also maintained better root length under stress compared to susceptible ones. Flooding led to a notable increase in root diameter, especially in tolerant lines. Root surface area decreased under stress, but tolerant genotypes retained relatively higher values. Seminal root number increased in tolerant genotypes, indicating adaptive root proliferation. Ethylene-induced responses played a role in enhancing root development under waterlogging. Tolerant genotypes also produced higher yields under flood conditions. Yield losses were more pronounced in susceptible genotypes with poor root traits. Root morphological traits proved critical in determining flood tolerance. These findings provide a valuable basis for future breeding programs aimed at developing flood-resilient soybean cultivars.

ACKNOWLEDGEMENT

The present study was supported by Department of Plant Physiology, VNMKV, Parbhani.
       
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

REFERENCES


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    Full Research Article

    Exploring Flood Resilience: Analysing Root Images for Physiological Responses to Flood Stress

    T
    Tejaswini Warik1
    G
    Godawari Pawar1,*
    S
    Shivaji Mehtre2
    H
    Hirakant Kalpande1
    S
    Sunil Umate3
    A
    Ambika More1
    A
    Ashwini Pawar1
    1College of Agriculture, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani-431 402, Maharashtra, India.
    2Officer Incharge Soybean Research Station, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani-431 402, Maharashtra, India.
    3Wheat and Maize Breeder, Wheat and Maize Research Unit, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani-431 402, Maharashtra, India.

    ABSTRACT

    Background: The experiment followed a randomized block design with two replications, including control and waterlogging treatments. After 40 days of normal growth, waterlogging was applied for two weeks with a 12 cm water level above the soil. Observations were recorded eight days after drainage, identifying four tolerant and four sensitive genotypes based on physiological and anatomical traits.

    Methods: The experiment was conducted during the kharif season of 2022-23 for one year at the field of All India Coordinated Research Project (AICRP) on Soybean, VNMKV, Parbhani. Among the 27 soybean genotypes evaluated under waterlogging, MAUS 710, JS-20-76, KDS-726 and NRC 257 exhibited high tolerance, showing improved growth and root traits like aerenchyma formation and root diameter. While MAUS 725, MAUS 731, EC250591 and JS 97-52 were susceptible.

    Result: Among 27 soybean genotypes tested under waterlogged conditions, MAUS 710, JS-20-76, KDS-726 and NRC 257 observed high tolerance, excelling in traits like aerenchyma formation, root surface area and average root diameter. JS-20-116, KDS-726, JS-20-76 and NRC-257 exhibited moderate tolerance, while MAUS 731 and MAUS 725 were more susceptible. These findings highlight the importance of root traits for flood tolerance and suggest that the tolerant genotypes should be prioritized in breeding programs. Waterlogging resulted in average yield loss, with reduction in root architecture.

    INTRODUCTION

    Soybean [Glycine max (L.) Merrill] is a self-pollinated, diploid leguminous crop (2n = 40), originated in China. It is often referred to as the “cow of the field” or “gold from the soil” due to its high oil and protein content (Sivabharathi et al., 2025). Soybean belongs to the Leguminosae family and exhibit a symbiotic relationship with many microorganisms, including rhizobium (Budiastuti et al., 2025). Soybean contributes about 25% of the world’s edible oil and is a key source of protein for animal feed, especially in poultry and aquaculture. It also provides important raw materials for the food, pharmaceutical and other industries (Alnuaim et al., 2025). Reduced tillage improved soybean yield, nutrient uptake, protein, oil content and economics. Applying 100% of the recommended fertilizer dose gave the highest productivity and profitability under the mustard-soybean system (Singh et al., 2025).
           
    Brazil leads in soybean production, followed by the United States, Argentina and China, with India ranking fifth-Madhya Pradesh alone contributing 78% of the country’s output (SOPA, 2022). Despite its adaptability, soybean faces significant challenges from abiotic stresses, particularly flooding and drought, which adversely affect growth and yield (Khatoon et al., 2012). Flooding is a critical stressor, impacting about 10% of the Earth’s land area. (Leng et al., 2019; Gibbs and Greenway, 2003). As climate change projected to increase flood risks, the development of resilient soybean varieties is crucial. Nevertheless, certain soybean varieties exhibit flood tolerance through adaptations such as aerenchyma formation and enhanced root elongation, mediated by phytohormones like ethylene and abscisic acid (Bailey-Serres and Voesenek, 2008).
           
    This study aimed to understand how soybean plants adapt to flooding stress, focused on analyzing root responses to flood stress using image analysis. The goal of study was to identify flood-tolerant genotypes for sustainable farming and food security.

    MATERIALS AND METHODS

    The experiment was conducted during the kharif season of 2022-23 for one year at the field of All India Coordinated Research Project (AICRP) on Soybean, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani. The site is located at 19°16'N latitude, 76°47'E longitude and an altitude of 409 meters above mean sea level. The experiment followed a randomized block design with two replications, comprising of 27 genotypes (Table 1) under control and waterlogging treatments. After 40 days of normal growth, a waterlogging treatment was imposed for two weeks, maintaining a water level of 12 cm above the soil surface. Observations were recorded eight days’ post-drainage, leading to the identification of four tolerant and four sensitive genotypes based on physiological and anatomical traits.

    Table 1: List of genotypes included in the study.


     
    Pot experiment
     
    Parallel to the field study, a pot experiment was conducted using plastic bags filled with a 50:1 (v/v) mixture of vertisols and farmyard manure. Each genotype was represented by ten bags, with two subjected to waterlogging and two as controls. Plants were thinned to three per bag at 21 days post-sowing and treated similarly to the field experiment. Soil moisture was maintained at 60-90% field capacity.
     
    Assessments
     
    Root length
     
    Root lengths of the main roots were measured with the help of measuring scale after uprooting the plants (Chintey et al., 2025).
     
    Average diameter of the root
     
    Root images were analysed using ImageJ software by calibrating the scale with a reference marker, converting the images to binary and using the measurement tools to determine root area and length, from which average diameter was automatically obtained (Schneider et al., 2012).
     
    Root surface area
     
    After calculating root length and root diameter, root surface area was calculated by following formula:
     
    Root surface area = Root length x Root average diameter x π
     
    Number of seminal roots
     
    The number of seminal roots per plant was visually counted after carefully uprooting the seedlings and gently washing the roots to remove soil without causing damage.
     
    Assessment of root anatomy: TS of root
     
    Medium-sized, freshly harvested Glycine max roots were manually sectioned into extremely thin cross-sections, stained with Acetocarmine stain (45% w/v) for 30 to 90 seconds and images were taken under a compound light microscope (Rajan et al., 2025).
     
    Statistical analysis
     
    The data collected from the experiment were statistically analyzed using a completely randomized design, following the procedure described by Panse and Sukhatme, 1954; (Ramanadane et al., 2025).

    RESULTS AND DISCUSSION

    Impact of waterlogging on soybean growth
     
    Waterlogging reduces soil oxygen, negatively impacting soybean growth and yield. Tolerant genotypes adapt through increased root formation and aerenchyma (Shimamura et al., 2010), while early reproductive stages are particularly sensitive, affecting pod and seed development (Ploschuk et al., 2022).
     
    Aerenchyma formation in soybean roots
     
    Flood stress induced a notable enhancement of aerenchyma formation in soybean roots, indicating an adaptive response (Fig 1). Cross-sectional analysis showed significant aerenchyma in tolerant genotypes (MAUS 710, JS 20-76, KDS-726, NRC-257) and minimal to absent in susceptible ones (MAUS 725, MAUS 731, EC 250591, JS 97-52). Extending from cortex to medulla, aerenchyma supports oxygen transport under flooding (Shimamura et al., 2010). Ethylene accumulation during waterlogging restricts gas diffusion, increases ROS and triggers cell death, forming aerenchyma lacunae (Hayashi et al., 2013). This adaptation enhances flood tolerance in soybeans, supported by earlier studies (Hossain and Uddin, 2011; Hingane et al., 2015; Ryser et al., 2011; Visser et al., 1997; Sakazono et al., 2000; Hayashi et al., 2013; Solaiman et al., 2007).

    Fig 1: TS of root-Aerenchyma formation in roots.


     
    Root length responses to flood stress
     
    Significant reduction in root length was observed under flood stress compared to control across genotypes (Table 2). It highlights the impact of flood stress on soybean root length. Under control conditions, root lengths ranged from 37 cm (MAUS 725) to 49.1 cm (NRC 256), while waterlogging reduced them significantly-down to 10 cm in EC 457464 and EC 250591 and 22.6 cm in NRC 257. These findings align with earlier studies showing that flooding during the reproductive stage limits root growth due to anaerobic conditions (Jhorar et al., 1995; Sorte et al., 1996; Sallam and Scott, 1987, Jitsuyama, 2015; Figueroa-Bustos  et al., 2018). Similar reductions were reported by Solaiman et al., (2007) and Sakazono et al., (2014), with losses ranging from 2.7% to 83.1%.

    Table 2: Root morphological traits and yield performance of soybean genotypes under control and flood conditions.


     
    Root average diameter responses to flood stress
     
    Root average diameter varied among genotypes, with higher values recorded under flood stress relative to control (Table 2). It shows that root diameter varied significantly among soybean genotypes under control and flood conditions. Under control, diameters ranged from 0.22 mm (EC 457464) to 0.36 mm (NRC 256, NRC 257), while flooding increased them to 0.43-0.57 mm in NRC 186, NRC 257. These findings support Keeley (1979), who noted that flood-tolerant genotypes develop larger, more branched roots and align with reports that aerenchymatous roots have greater diameters (Visser et al., 2000a, b; Matsui and Tsuchiya, 2006; Grimoldi et al., 2005).
     
    Root surface area responses to flood stress
     
    Flood stress caused a notable decline in root surface area, reflecting restricted root development (Table 2). It shows that root surface area varied significantly between control and waterlogged conditions. Under control, it ranged from 27.63 cm2 (EC 457464) to 57.54 cm² (NRC 256), while flooding reduced it to 13.5 cm² (NRC 257) to 39.5 cm² (NRC 186).
     
    Number of seminal roots responses to flood stress
     
    Significant increase in the number of seminal roots was observed under flood stress compared to control across genotypes (Table 2). It shows that flood-tolerant soybean genotypes increased seminal root numbers under waterlogging (Fig 2). In control conditions, root counts ranged from 6 (JS 20-76, MAUS 71) to 12 (JS 20-116, KDS 726, NRC 256), while flooding increased them up to 19 in MAUS 710 and 18 in JS 20-76. This adaptive response aligns with studies reporting that waterlogging promotes adventitious root and aerenchyma formation for survival (Hossain and Uddin, 2011; Jhorar et al., 1995; Keeley, 1979). Ethylene plays a key role in this response by enhancing adventitious and lateral root development under low oxygen (Malik et al., 2001; Sauter, 2013).

    Fig 2: Comparison of number of seminal roots in control and flood condition.


     
    Average yield (g/row)
     
    Significant reduction in average yield was observed under flood stress compared to control across genotypes (Table 2). It highlights yield differences among soybean genotypes under control and flood conditions. Under control, NRC 256 had the highest yield (248 g/row) and MAUS 725 the lowest (61 g/row). Under flooding, MAUS 710 (112.5 g/row), KDS-726 (108.5 g/row) and JS 20-76 (106 g/row) performed best, while MAUS 731 (32.5 g/row) and MAUS 725 (38.5 g/row) showed the lowest yields. These findings confirm genotype-dependent yield variation under flooding, with tolerant genotypes experiencing less yield loss (Kumar et al., 2013; Ren et al., 2014; Zhou et al., 2021).

    CONCLUSION

    Waterlogging significantly affects soybean root traits and yield performance. Tolerant genotypes showed well-developed aerenchyma, supporting internal oxygen transport. These genotypes also maintained better root length under stress compared to susceptible ones. Flooding led to a notable increase in root diameter, especially in tolerant lines. Root surface area decreased under stress, but tolerant genotypes retained relatively higher values. Seminal root number increased in tolerant genotypes, indicating adaptive root proliferation. Ethylene-induced responses played a role in enhancing root development under waterlogging. Tolerant genotypes also produced higher yields under flood conditions. Yield losses were more pronounced in susceptible genotypes with poor root traits. Root morphological traits proved critical in determining flood tolerance. These findings provide a valuable basis for future breeding programs aimed at developing flood-resilient soybean cultivars.

    ACKNOWLEDGEMENT

    The present study was supported by Department of Plant Physiology, VNMKV, Parbhani.
           
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
     
    Informed consent
     
    All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling techniques were approved by the University of Animal Care Committee.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.

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