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Full Research Article

Screening of Gamma-Irradiated Soybean Mutants for Drought Tolerance through In vitro PEG Selection and Field Performance

Y
Yuliasti1
N
Nilahayati2,*
T
Tiffani3
L
Lilik Harsanti1
1Research Center for Food Crops, Research Organization Food and Agriculture, National Research and Innovation Agency, Jl. Raya Jakarta-Bogor KM 46, Cibinong, Jawa Barat Indonesia, 16911.
2Department of Agroecotechnology, Faculty of Agriculture, Universitas Malikussaleh, Jl. Cot Tgk. Ni Reuleut Timu, Aceh Utara, Indonesia, 24355.
3Graduate Student of the Agriculture Faculty, Universitas Gajah Mada, Jl. Flora, Bulaksumur, Yogyakarta, Indonesia, 55281.

ABSTRACT

Background: Drought is one of the most severe abiotic stresses limiting soybean productivity in marginal areas. Gamma irradiation has been used to induce mutations and identify drought-tolerant soybean lines. This study aimed to screen gamma-ray-induced soybean mutant lines for drought tolerance using in vitro polyethylene glycol (PEG 6000) selection and evaluate their field performance under dryland conditions.

Methods: Four M5 mutant lines derived from the Argomulyo variety irradiated at 200 Gy, 300 Gy, 400 Gy and 500 Gy were tested together with two control varieties: Argomulyo (parent) and Dering (drought-tolerant check). Mature embryos were cultured on liquid MS medium supplemented with 3% sucrose and growth regulators (2,4-Diclorophenylaceticacid/2,4 D 0.5 mg L-1, Naphthaleneacetic acid/NAA 0.5 mg L-1 and Thidiazuron/TDZ 0.5 mg L-1) containing PEG 0% and 20% for 4 weeks.

Result: Significant genotypic variation was observed under PEG-induced osmotic stress. Mutant line A3 exhibited the highest somatic embryo induction (74.42%) and plantlet regeneration (29.67%) under 20% PEG, outperforming Argomulyo (6.67%) and Dering (10.67%). Under dryland field conditions in Gunung Kidul and Bantul, mutant lines A3 and A5 showed superior yield performance (2.47 and 2.40 t ha-1, respectively) compared to Argomulyo (1.85 t ha-1) and Dering (2.18 t ha-1). The integration of in vitro PEG selection with field evaluation effectively identified drought-tolerant mutant soybean lines that could contribute to breeding programs for marginal lands in Indonesia.

INTRODUCTION

Soybean (Glycine max L. Merrill) is a crucial legume crop and a rich source of high-quality plant-based protein, oil and essential amino acids for humans and animals. Their high protein content and significant oil production make them vital for global nutrition and food security. In Indonesia, domestic production has stagnated or declined, meeting only 20-35% of national demand in recent years. At the same time, consumption continues to rise due to population growth and the food industry’s needs, especially for tofu and tempeh (Harsono et al., 2021).
       
Among the major constraints, drought is the most severe abiotic stress affecting soybean growth and yield stability. Drought stress disrupts plant physiology, leading to reduced growth and crop yields. The primary physiological disturbances include stomatal closure, impaired photosynthesis and oxidative damage, all of which contribute to yield loss (Qiao et al., 2024; Wahab et al., 2022). Drought tolerance in soybean is a complex, quantitative trait controlled by multiple genes and heavily influenced by environmental factors, making selection slow and unpredictable (Zhao et al., 2020). Traditional breeding requires extensive multi-generational screening and is often confounded by low heritability and environmental (Rasheed et al., 2022). Therefore, alternative breeding approaches such as induced mutagenesis are being explored to accelerate genetic variability creation for drought-tolerant genotypes.
       
Mutation breeding using gamma rays has been proven effective in inducing heritable genetic variations that enhance stress tolerance and yield-related traits. In soybeans, gamma irradiation produced mutant lines with higher oil and protein content (Mohsen et al., 2023), early maturity and high yield potential (Nilahayati et al., 2018; Nilahayati et al., 2021; Nilahayati et al., 2022; Nilahayati, 2025). Similarly, mutants of other crops such as grasspea (Singh et al., 2018), Sorghum (Wanga et al., 2023), urdbean (Vanniarajan et al., 2022) and groundnut (Nilahayati et al., 2024) have shown various beneficial traits, ranging from disease resistance to drought tolerance.
       
In addition to induced mutation, in vitro selection, particularly with stress agents like polyethylene glycol (PEG), is a widely used and effective strategy for rapidly screening large plant populations for enhanced tolerance to abiotic stresses, such as drought. This approach complements induced mutation by enabling the early, controlled and high-throughput identification of tolerant genotypes at the cellular or tissue level, before field validation. PEG is commonly used in tissue culture media to simulate drought by reducing water availability without being toxic or absorbed by plant tissues. This creates osmotic stress, mimicking field drought conditions and enabling selection of tolerant cells or tissues (Mehmandar et al., 2023). This method has been successfully applied in the selection of drought-tolerant genotypes in rice (Goraguddi et al., 2023), sorghum (Tsago et al., 2014), durum wheat (Benlioğlu and Ozgen, 2021) and soybean (Mishra et al., 2021).
       
The present study aimed to evaluate callus and plantlet responses to PEG-induced osmotic stress in vitro and to assess the yield performance of selected mutant lines under dryland field conditions. This approach is expected to identify promising genotypes adaptable to water-limited environments, supporting sustainable soybean production in Indonesia’s marginal areas.

MATERIALS AND METHODS

Plant materials
 
The plant material used in this study was the soybean variety Argomulyo, which was irradiated using a Cobalt-60 gamma source at the Center for Isotopes and Radiation Application (BATAN), Jakarta. Radiation doses of 200, 300, 400 and 500 Gy were applied to generate genetic variation. The irradiated seeds (M1 generation) were advanced through self-pollination up to the M5 generation. Four mutant lines, designated as A2, A3, A4 and A5 were selected in the previous generation. Two varieties were used as controls: Argomulyo (parent) and Dering (drought-tolerant variety).
 
In vitro selection using peg 6000
 
This study was conducted at the Plant Breeding Laboratory, Center for the Application of Isotopes and Radiation, National Nuclear Energy Agency of Indonesia, from July to December 2022.
       
Mature seeds from six soybean genotypes, four mutant lines derived from gamma-ray irradiation at doses of 200 Gy (A1), 300 Gy (A2), 400 Gy (A3) and 500 Gy (A4) and two check varieties were used as explants. Seeds were surface-sterilized with 70% (v/v) ethanol for 1 min, followed by 40% sodium hypochlorite (NaOCl) for 5 min and rinsed three times with sterile distilled water.
       
Mature embryos were cultured in liquid Murashige and Skoog (MS) medium with B5 vitamins for somatic embryo induction and drought selection. Drought stress was imposed using PEG 6000 at concentrations of 0% and 20%. The medium contained 3% sucrose and growth regulators (0.5 mg L-1 2,4-D, 0.5 mg L-1 NAA and 0.5 mg L-1 TDZ; L-MSG medium). Cultures were incubated at 24-25°C under a 16 h photoperiod with 1000 lux light intensity for four weeks.
       
The experiment was arranged in a completely randomized design with 10 replications, using PEG concentration and genotype as factors. Each replication consisted of seven explant clumps. Observations included the survival percentage of embryogenic callus and the relative growth rate of shoots after four weeks of culture.
 
Field evaluation under dryland conditions
 
Field evaluations were conducted in the Gunung Kidul and Bantul districts, Yogyakarta, Indonesia, from May to July 2023. The experiment followed a Randomized Complete Block Design with three replications. Each plot measured 3 × 5 m with a spacing of 40 × 20 cm. Standard agronomic practices were applied following local recommendations. The observed variable was grain yield (t ha-1).
 
Data analysis
 
Data were analyzed using analysis of variance. Significant differences among treatments were further tested using Duncan’s Multiple Range Test at the 5% significance level. The Drought Sensitivity Index (DSI) was calculated following the formula by Fischer and Maurer (1978), based on yield reduction under drought relative to non-stress conditions.

RESULTS AND DISCUSSION

The results showed that PEG stress and genotype significantly influenced the formation of embryogenic callus. Increasing PEG concentration from 0% to 20% led to a decrease in callus formation across all genotypes. The percentage of embryogenic callus formation of soybean mutant lines on media containing 0% and 20% PEG 6000 is presented in Table 1.

Table 1: The Percentage of embryonic callus soybean mutant lines on media PEG 6000.


       
Table 1 showed that the percentage of embryogenic callus decreased under PEG 20% compared to PEG 0%. Among the tested genotypes, the mutant line A3 and A5 maintained the highest callus formation under PEG 20% (74.4% and 70.2%), followed by the A4 and A2 mutant lines and the drought-tolerant check Dering (64%). In contrast, the parental variety Argomulyo exhibited the lowest percentage of callus formation (49.8%) under stress conditions. These findings are consistent with Saepudin et al. (2017) who found that PEG 6000 at 15-20% concentration effectively differentiated tolerant and sensitive soybean lines. Similarly, Hartati et al. (2018) observed a significant decline in callus formation in sugarcane with increasing PEG concentration, yet tolerant genotypes exhibited better callus survival.
       
The reduction of water potential in the culture medium caused by PEG not only affected callus growth but also limited the ability of callus cells to form embryogenic tissues capable of somatic embryo differentiation. Similar observations were reported by Tereso et al. (2007), who found that reduced osmotic potential inhibited cellular differentiation in embryogenic callus cultures. The inhibitory effects of PEG may also be associated with decreased levels of endogenous polyamines in explant tissues under osmotic stress.
       
In this study, mutant lines A2-A5 produced a higher number of somatic embryos at 20% PEG compared with the parent variety Argomulyo (Table 1). However, a significant reduction in the growth rate of embryogenic callus was observed, particularly in mutant A4, when exposed to 20% PEG. The addition of PEG to the medium reduces the free water content, leading to osmotic stress that interferes with nutrient and water uptake by the cells (Li and Zhang, 2012). At higher PEG concentrations, callus development, proliferation and embryoid survival were strongly inhibited. This may be explained by the ability of PEG’s ethylene oxide subunits to retain water molecules through hydrogen bonding, thereby reducing the availability of free water in the medium (Sayar et al., 2010).
       
Fig 1 presents the general appearance of callus formation from soybean mutant lines and control varieties cultured under different PEG treatments. Callus induction occurred in all genotypes, both under normal and PEG-supplemented media. However, visible differences in callus proliferation were observed between genotypes and PEG concentrations. Under 0% PEG, callus proliferation appeared more extensive, while at 20% PEG, the growth was visibly reduced, reflecting the osmotic stress effect of PEG on tissue development. These visual observations correspond with quantitative data showing a decline in embryogenic callus formation under PEG treatment (Table 1).

Fig 1: General appearance of callus formation in soybean mutant lines cultured on MS medium with 0% and 20% PEG 6000.


       
The fresh weight of embryogenic callus decreased significantly with increasing PEG concentration (Table 2). All genotypes exhibited reduced callus biomass under PEG 20% compared to PEG 0%, indicating that osmotic stress limited cell expansion and water uptake. However, mutant lines A3 and A4 maintained relatively high fresh weights (0.66-0.63 g), comparable to the drought-tolerant check Dering (0.64 g), whereas the parental variety Argomulyo showed the lowest value (0.45 g).

Table 2: Fresh weight (g) of callus of soybean six mutant lines and two check varieties cultures on MS medium containing PEG 6000.


       
This reduction in fresh weight under PEG stress reflects the inhibition of cell turgor and the consequent decline in tissue hydration, as PEG decreases the water potential of the culture medium (Li and Zhang, 2012). The relatively high biomass in mutants A3 and A4 suggests improved physiological adaptation to osmotic stress, possibly through the accumulation of compatible solutes such as proline and soluble sugars that help maintain water balance. This finding supports the use of in vitro PEG selection as a reliable approach to identify drought-tolerant soybean genotypes before field evaluation.
       
Fresh and actively proliferating embryogenic calli of soybean mutant lines derived from gamma irradiation showed varying responses to PEG stress. Under control conditions (0% PEG), the calli exhibited compact, yellowish and friable structures with high proliferation rates, whereas under 20% PEG, the calli became brownish and exhibited reduced growth and water content. These morphological differences clearly indicate the effect of osmotic stress induced by PEG on callus viability and proliferation (Fig 2 and 3).

Fig 2: Embryogenic callus of soybean mutant lines derived from gamma irradiation under PEG-induced osmotic stress.



Fig 3: Regeneration of soybean mutant plantlets derived from embryogenic callus after PEG stress selection.


       
Table 3 showed that both fresh and dry weights of embryogenic callus were significantly affected by PEG treatment. A consistent decrease was observed as PEG concentration increased from 0% to 20%. Under stress conditions, mutant A3 recorded the highest callus fresh weight (0.66 g), followed by A5 (0.63 g) and Dering (0.64 g), while Argomulyo and A2 showed the lowest values. The same pattern was observed for dry weight, where a 25-40% reduction occurred under PEG stress compared with the control treatment.

Table 3: Dry weight of embryogenic callus of six soybean mutant lines and two check varieties cultured on MS medium containing PEG 6000.


       
PEG stress reduces cell turgor and limits the uptake of water, thereby decreasing biomass accumulation. The ability of certain lines (A3, A5 and Dering) to maintain higher fresh and dry weights under osmotic stress indicates better osmotic adjustment capacity. According to Jasmine et al. (2022), under osmotic stress, tolerant plants accumulate proline, which helps balance osmotic potential between the cytosol, vacuole and outside medium, thereby reducing water loss and protecting cells. These results suggest that callus biomass can be used as an early indicator of drought tolerance in soybean.
       
PEG application significantly reduced the percentage of plantlet regeneration in all genotypes. However, mutants A3 and A5 exhibited higher regeneration percentages (29.67% and 26.45%, respectively) compared to the control varieties. These lines also produced longer roots (2.88-3.02 cm) under PEG stress, indicating better root growth and adaptation to low water potential (Table 4 and Table 5). Reduction in regeneration capacity under PEG stress is commonly associated with limited water availability and hormonal imbalance in cultured tissues. Nevertheless, the high regeneration response of mutants A3 and A5 indicates their ability to maintain active cell division and organogenesis even under osmotic stress. These findings align with the report of Hartati et al. (2018) that drought-tolerant genotypes tend to maintain morphogenic activity due to improved turgor regulation.

Table 4: Percentage of regenerated plantlets from embryogenic callus of soybean mutant lines and two check varieties cultured on MS medium containing PEG 6000.



Table 5: Root length of plantlet soybean genotypes (cm).


       
The enhanced regeneration ability observed in mutant A3 may also be related to gamma-ray-induced modifications in gene expression related to stress adaptation and antioxidative defense, as suggested by Kadhimi et al. (2016). Thus, the combination of gamma irradiation and PEG-based in vitro selection is effective in identifying mutant lines with superior regenerative capacity under stress conditions.
       
The values presented in Table 6 represent the drought sensitivity index (S), which was calculated according to Fischer and Maurer (1978). This index quantifies the relative reduction in a genotype’s performance under drought compared to normal conditions. Based on the classification, genotypes with S < 0.5 are considered highly tolerant, those with 0.5 ≤ S < 1.0 are moderately tolerant and those with S > 1.0 are sensitive to drought stress.

Table 6: Sensitivity Index of Genotype soybean.


       
The results showed that mutant lines A2 and A3 exhibited the lowest S values across all parameters, including callus fresh and dry weight, plantlet height and root length, indicating that these lines were the most tolerant to drought stress. In particular, mutant line A3 (300 Gy) consistently displayed low S values (0.37-0.80), indicating strong osmotic adjustment and stable growth under PEG-induced osmotic stress. In contrast, the parental Argomulyo variety showed the highest S index (S > 1.0) for all traits, confirming its sensitivity to drought. The drought-tolerant check Dering showed moderate tolerance (S = 0.71-1.01), consistent with its known field performance under water-limited conditions.
       
Mutant lines A4 and A5 showed intermediate responses, with some parameters (particularly root length and dry weight) approaching the sensitivity threshold (S ≈ 1.0). These results indicate that the gamma irradiation treatment, especially at 300 Gy, effectively induced beneficial genetic variability associated with improved drought tolerance. The enhanced tolerance observed in A2 and A3 mutant lines may be attributed to their ability to maintain water balance, cell turgor and growth stability under osmotic stress.
       
Analysis of variance for yield under drought conditions is shown in Table 7. The data indicated significant differences among the mutant lines and control varieties grown under dry land conditions. The mutant soybean lines showed higher yield potential than the Argomulyo and Dering variety. Among all genotypes tested, the mutant line A3 recorded the highest mean yield of 2.47 t ha-1, followed by A5 with 2.40 t ha-1 and A4  with 2.30 t ha-1. The lowest yield was observed in the parental variety Argomulyo (1.85 t ha-1).

Table 7: Yield performance of soybean mutant lines under the dry land condition.


       
These results demonstrated that the selected mutant lines, particularly A3 and A5, maintained higher yield stability under water-limited conditions. The improvement in yield could be associated with the better physiological adaptation of these mutant lines, as reflected by their lower drought sensitivity index values (Table 6). This suggests that the mutation treatment effectively produced genotypes capable of maintaining productivity under drought stress.
       
A similar increase in grain yield under drought conditions due to mutation induction has also been reported in other crops (Kadhimi et al., 2016). The consistent yield performance of mutant lines A3 and A5 in both dry land locations indicates their stability and adaptability to marginal environments. These findings confirm that mutation breeding combined with in vitro selection can be an effective approach to developing drought-tolerant soybean lines suitable for cultivation in dry areas.
       
The results from both in vitro and field evaluations clearly demonstrated the positive impact of gamma-ray-induced mutation on improving drought tolerance and yield performance in soybean. The in vitro selection using PEG 6000 as an osmotic agent effectively differentiated tolerant genotypes and the same lines (A2 and A3) also performed well under field conditions. Therefore, the in vitro screening technique developed in this study could serve as a reliable preliminary method for identifying drought-tolerant soybean genotypes. The consistent performance of mutant lines A3 and A5 across various environments further supports the potential use of mutation breeding combined with in vitro selection as an effective approach to developing drought-tolerant soybean varieties suitable for dryland cultivation in Indonesia.

CONCLUSION

In vitro selection using PEG 6000 effectively identified drought-tolerant soybean lines. Mutant lines A3 and A5 showed the best tolerance to osmotic stress and the highest yields under dry land conditions. The consistency between in vitro and field results confirms that in vitro selection is a reliable method for early screening of drought-tolerant soybean genotypes suitable for cultivation in water-limited areas

ACKNOWLEDGEMENT

The authors gratefully acknowledge the financial support provided by the Center for Isotopes and Radiation Application (BATAN). Sincere appreciation is also extended to the Ministry of Agriculture of Indonesia for supporting the national soybean research grant program that made this study possible.

CONFLICT OF INTEREST

On behalf of all co-authors, I hereby confirm that there are no conflicts of interest.

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    Full Research Article

    Screening of Gamma-Irradiated Soybean Mutants for Drought Tolerance through In vitro PEG Selection and Field Performance

    Y
    Yuliasti1
    N
    Nilahayati2,*
    T
    Tiffani3
    L
    Lilik Harsanti1
    1Research Center for Food Crops, Research Organization Food and Agriculture, National Research and Innovation Agency, Jl. Raya Jakarta-Bogor KM 46, Cibinong, Jawa Barat Indonesia, 16911.
    2Department of Agroecotechnology, Faculty of Agriculture, Universitas Malikussaleh, Jl. Cot Tgk. Ni Reuleut Timu, Aceh Utara, Indonesia, 24355.
    3Graduate Student of the Agriculture Faculty, Universitas Gajah Mada, Jl. Flora, Bulaksumur, Yogyakarta, Indonesia, 55281.

    ABSTRACT

    Background: Drought is one of the most severe abiotic stresses limiting soybean productivity in marginal areas. Gamma irradiation has been used to induce mutations and identify drought-tolerant soybean lines. This study aimed to screen gamma-ray-induced soybean mutant lines for drought tolerance using in vitro polyethylene glycol (PEG 6000) selection and evaluate their field performance under dryland conditions.

    Methods: Four M5 mutant lines derived from the Argomulyo variety irradiated at 200 Gy, 300 Gy, 400 Gy and 500 Gy were tested together with two control varieties: Argomulyo (parent) and Dering (drought-tolerant check). Mature embryos were cultured on liquid MS medium supplemented with 3% sucrose and growth regulators (2,4-Diclorophenylaceticacid/2,4 D 0.5 mg L-1, Naphthaleneacetic acid/NAA 0.5 mg L-1 and Thidiazuron/TDZ 0.5 mg L-1) containing PEG 0% and 20% for 4 weeks.

    Result: Significant genotypic variation was observed under PEG-induced osmotic stress. Mutant line A3 exhibited the highest somatic embryo induction (74.42%) and plantlet regeneration (29.67%) under 20% PEG, outperforming Argomulyo (6.67%) and Dering (10.67%). Under dryland field conditions in Gunung Kidul and Bantul, mutant lines A3 and A5 showed superior yield performance (2.47 and 2.40 t ha-1, respectively) compared to Argomulyo (1.85 t ha-1) and Dering (2.18 t ha-1). The integration of in vitro PEG selection with field evaluation effectively identified drought-tolerant mutant soybean lines that could contribute to breeding programs for marginal lands in Indonesia.

    INTRODUCTION

    Soybean (Glycine max L. Merrill) is a crucial legume crop and a rich source of high-quality plant-based protein, oil and essential amino acids for humans and animals. Their high protein content and significant oil production make them vital for global nutrition and food security. In Indonesia, domestic production has stagnated or declined, meeting only 20-35% of national demand in recent years. At the same time, consumption continues to rise due to population growth and the food industry’s needs, especially for tofu and tempeh (Harsono et al., 2021).
           
    Among the major constraints, drought is the most severe abiotic stress affecting soybean growth and yield stability. Drought stress disrupts plant physiology, leading to reduced growth and crop yields. The primary physiological disturbances include stomatal closure, impaired photosynthesis and oxidative damage, all of which contribute to yield loss (Qiao et al., 2024; Wahab et al., 2022). Drought tolerance in soybean is a complex, quantitative trait controlled by multiple genes and heavily influenced by environmental factors, making selection slow and unpredictable (Zhao et al., 2020). Traditional breeding requires extensive multi-generational screening and is often confounded by low heritability and environmental (Rasheed et al., 2022). Therefore, alternative breeding approaches such as induced mutagenesis are being explored to accelerate genetic variability creation for drought-tolerant genotypes.
           
    Mutation breeding using gamma rays has been proven effective in inducing heritable genetic variations that enhance stress tolerance and yield-related traits. In soybeans, gamma irradiation produced mutant lines with higher oil and protein content (Mohsen et al., 2023), early maturity and high yield potential (Nilahayati et al., 2018; Nilahayati et al., 2021; Nilahayati et al., 2022; Nilahayati, 2025). Similarly, mutants of other crops such as grasspea (Singh et al., 2018), Sorghum (Wanga et al., 2023), urdbean (Vanniarajan et al., 2022) and groundnut (Nilahayati et al., 2024) have shown various beneficial traits, ranging from disease resistance to drought tolerance.
           
    In addition to induced mutation, in vitro selection, particularly with stress agents like polyethylene glycol (PEG), is a widely used and effective strategy for rapidly screening large plant populations for enhanced tolerance to abiotic stresses, such as drought. This approach complements induced mutation by enabling the early, controlled and high-throughput identification of tolerant genotypes at the cellular or tissue level, before field validation. PEG is commonly used in tissue culture media to simulate drought by reducing water availability without being toxic or absorbed by plant tissues. This creates osmotic stress, mimicking field drought conditions and enabling selection of tolerant cells or tissues (Mehmandar et al., 2023). This method has been successfully applied in the selection of drought-tolerant genotypes in rice (Goraguddi et al., 2023), sorghum (Tsago et al., 2014), durum wheat (Benlioğlu and Ozgen, 2021) and soybean (Mishra et al., 2021).
           
    The present study aimed to evaluate callus and plantlet responses to PEG-induced osmotic stress in vitro and to assess the yield performance of selected mutant lines under dryland field conditions. This approach is expected to identify promising genotypes adaptable to water-limited environments, supporting sustainable soybean production in Indonesia’s marginal areas.

    MATERIALS AND METHODS

    Plant materials
     
    The plant material used in this study was the soybean variety Argomulyo, which was irradiated using a Cobalt-60 gamma source at the Center for Isotopes and Radiation Application (BATAN), Jakarta. Radiation doses of 200, 300, 400 and 500 Gy were applied to generate genetic variation. The irradiated seeds (M1 generation) were advanced through self-pollination up to the M5 generation. Four mutant lines, designated as A2, A3, A4 and A5 were selected in the previous generation. Two varieties were used as controls: Argomulyo (parent) and Dering (drought-tolerant variety).
     
    In vitro selection using peg 6000
     
    This study was conducted at the Plant Breeding Laboratory, Center for the Application of Isotopes and Radiation, National Nuclear Energy Agency of Indonesia, from July to December 2022.
           
    Mature seeds from six soybean genotypes, four mutant lines derived from gamma-ray irradiation at doses of 200 Gy (A1), 300 Gy (A2), 400 Gy (A3) and 500 Gy (A4) and two check varieties were used as explants. Seeds were surface-sterilized with 70% (v/v) ethanol for 1 min, followed by 40% sodium hypochlorite (NaOCl) for 5 min and rinsed three times with sterile distilled water.
           
    Mature embryos were cultured in liquid Murashige and Skoog (MS) medium with B5 vitamins for somatic embryo induction and drought selection. Drought stress was imposed using PEG 6000 at concentrations of 0% and 20%. The medium contained 3% sucrose and growth regulators (0.5 mg L-1 2,4-D, 0.5 mg L-1 NAA and 0.5 mg L-1 TDZ; L-MSG medium). Cultures were incubated at 24-25°C under a 16 h photoperiod with 1000 lux light intensity for four weeks.
           
    The experiment was arranged in a completely randomized design with 10 replications, using PEG concentration and genotype as factors. Each replication consisted of seven explant clumps. Observations included the survival percentage of embryogenic callus and the relative growth rate of shoots after four weeks of culture.
     
    Field evaluation under dryland conditions
     
    Field evaluations were conducted in the Gunung Kidul and Bantul districts, Yogyakarta, Indonesia, from May to July 2023. The experiment followed a Randomized Complete Block Design with three replications. Each plot measured 3 × 5 m with a spacing of 40 × 20 cm. Standard agronomic practices were applied following local recommendations. The observed variable was grain yield (t ha-1).
     
    Data analysis
     
    Data were analyzed using analysis of variance. Significant differences among treatments were further tested using Duncan’s Multiple Range Test at the 5% significance level. The Drought Sensitivity Index (DSI) was calculated following the formula by Fischer and Maurer (1978), based on yield reduction under drought relative to non-stress conditions.

    RESULTS AND DISCUSSION

    The results showed that PEG stress and genotype significantly influenced the formation of embryogenic callus. Increasing PEG concentration from 0% to 20% led to a decrease in callus formation across all genotypes. The percentage of embryogenic callus formation of soybean mutant lines on media containing 0% and 20% PEG 6000 is presented in Table 1.

    Table 1: The Percentage of embryonic callus soybean mutant lines on media PEG 6000.


           
    Table 1 showed that the percentage of embryogenic callus decreased under PEG 20% compared to PEG 0%. Among the tested genotypes, the mutant line A3 and A5 maintained the highest callus formation under PEG 20% (74.4% and 70.2%), followed by the A4 and A2 mutant lines and the drought-tolerant check Dering (64%). In contrast, the parental variety Argomulyo exhibited the lowest percentage of callus formation (49.8%) under stress conditions. These findings are consistent with Saepudin et al. (2017) who found that PEG 6000 at 15-20% concentration effectively differentiated tolerant and sensitive soybean lines. Similarly, Hartati et al. (2018) observed a significant decline in callus formation in sugarcane with increasing PEG concentration, yet tolerant genotypes exhibited better callus survival.
           
    The reduction of water potential in the culture medium caused by PEG not only affected callus growth but also limited the ability of callus cells to form embryogenic tissues capable of somatic embryo differentiation. Similar observations were reported by Tereso et al. (2007), who found that reduced osmotic potential inhibited cellular differentiation in embryogenic callus cultures. The inhibitory effects of PEG may also be associated with decreased levels of endogenous polyamines in explant tissues under osmotic stress.
           
    In this study, mutant lines A2-A5 produced a higher number of somatic embryos at 20% PEG compared with the parent variety Argomulyo (Table 1). However, a significant reduction in the growth rate of embryogenic callus was observed, particularly in mutant A4, when exposed to 20% PEG. The addition of PEG to the medium reduces the free water content, leading to osmotic stress that interferes with nutrient and water uptake by the cells (Li and Zhang, 2012). At higher PEG concentrations, callus development, proliferation and embryoid survival were strongly inhibited. This may be explained by the ability of PEG’s ethylene oxide subunits to retain water molecules through hydrogen bonding, thereby reducing the availability of free water in the medium (Sayar et al., 2010).
           
    Fig 1 presents the general appearance of callus formation from soybean mutant lines and control varieties cultured under different PEG treatments. Callus induction occurred in all genotypes, both under normal and PEG-supplemented media. However, visible differences in callus proliferation were observed between genotypes and PEG concentrations. Under 0% PEG, callus proliferation appeared more extensive, while at 20% PEG, the growth was visibly reduced, reflecting the osmotic stress effect of PEG on tissue development. These visual observations correspond with quantitative data showing a decline in embryogenic callus formation under PEG treatment (Table 1).

    Fig 1: General appearance of callus formation in soybean mutant lines cultured on MS medium with 0% and 20% PEG 6000.


           
    The fresh weight of embryogenic callus decreased significantly with increasing PEG concentration (Table 2). All genotypes exhibited reduced callus biomass under PEG 20% compared to PEG 0%, indicating that osmotic stress limited cell expansion and water uptake. However, mutant lines A3 and A4 maintained relatively high fresh weights (0.66-0.63 g), comparable to the drought-tolerant check Dering (0.64 g), whereas the parental variety Argomulyo showed the lowest value (0.45 g).

    Table 2: Fresh weight (g) of callus of soybean six mutant lines and two check varieties cultures on MS medium containing PEG 6000.


           
    This reduction in fresh weight under PEG stress reflects the inhibition of cell turgor and the consequent decline in tissue hydration, as PEG decreases the water potential of the culture medium (Li and Zhang, 2012). The relatively high biomass in mutants A3 and A4 suggests improved physiological adaptation to osmotic stress, possibly through the accumulation of compatible solutes such as proline and soluble sugars that help maintain water balance. This finding supports the use of in vitro PEG selection as a reliable approach to identify drought-tolerant soybean genotypes before field evaluation.
           
    Fresh and actively proliferating embryogenic calli of soybean mutant lines derived from gamma irradiation showed varying responses to PEG stress. Under control conditions (0% PEG), the calli exhibited compact, yellowish and friable structures with high proliferation rates, whereas under 20% PEG, the calli became brownish and exhibited reduced growth and water content. These morphological differences clearly indicate the effect of osmotic stress induced by PEG on callus viability and proliferation (Fig 2 and 3).

    Fig 2: Embryogenic callus of soybean mutant lines derived from gamma irradiation under PEG-induced osmotic stress.



    Fig 3: Regeneration of soybean mutant plantlets derived from embryogenic callus after PEG stress selection.


           
    Table 3 showed that both fresh and dry weights of embryogenic callus were significantly affected by PEG treatment. A consistent decrease was observed as PEG concentration increased from 0% to 20%. Under stress conditions, mutant A3 recorded the highest callus fresh weight (0.66 g), followed by A5 (0.63 g) and Dering (0.64 g), while Argomulyo and A2 showed the lowest values. The same pattern was observed for dry weight, where a 25-40% reduction occurred under PEG stress compared with the control treatment.

    Table 3: Dry weight of embryogenic callus of six soybean mutant lines and two check varieties cultured on MS medium containing PEG 6000.


           
    PEG stress reduces cell turgor and limits the uptake of water, thereby decreasing biomass accumulation. The ability of certain lines (A3, A5 and Dering) to maintain higher fresh and dry weights under osmotic stress indicates better osmotic adjustment capacity. According to Jasmine et al. (2022), under osmotic stress, tolerant plants accumulate proline, which helps balance osmotic potential between the cytosol, vacuole and outside medium, thereby reducing water loss and protecting cells. These results suggest that callus biomass can be used as an early indicator of drought tolerance in soybean.
           
    PEG application significantly reduced the percentage of plantlet regeneration in all genotypes. However, mutants A3 and A5 exhibited higher regeneration percentages (29.67% and 26.45%, respectively) compared to the control varieties. These lines also produced longer roots (2.88-3.02 cm) under PEG stress, indicating better root growth and adaptation to low water potential (Table 4 and Table 5). Reduction in regeneration capacity under PEG stress is commonly associated with limited water availability and hormonal imbalance in cultured tissues. Nevertheless, the high regeneration response of mutants A3 and A5 indicates their ability to maintain active cell division and organogenesis even under osmotic stress. These findings align with the report of Hartati et al. (2018) that drought-tolerant genotypes tend to maintain morphogenic activity due to improved turgor regulation.

    Table 4: Percentage of regenerated plantlets from embryogenic callus of soybean mutant lines and two check varieties cultured on MS medium containing PEG 6000.



    Table 5: Root length of plantlet soybean genotypes (cm).


           
    The enhanced regeneration ability observed in mutant A3 may also be related to gamma-ray-induced modifications in gene expression related to stress adaptation and antioxidative defense, as suggested by Kadhimi et al. (2016). Thus, the combination of gamma irradiation and PEG-based in vitro selection is effective in identifying mutant lines with superior regenerative capacity under stress conditions.
           
    The values presented in Table 6 represent the drought sensitivity index (S), which was calculated according to Fischer and Maurer (1978). This index quantifies the relative reduction in a genotype’s performance under drought compared to normal conditions. Based on the classification, genotypes with S < 0.5 are considered highly tolerant, those with 0.5 ≤ S < 1.0 are moderately tolerant and those with S > 1.0 are sensitive to drought stress.

    Table 6: Sensitivity Index of Genotype soybean.


           
    The results showed that mutant lines A2 and A3 exhibited the lowest S values across all parameters, including callus fresh and dry weight, plantlet height and root length, indicating that these lines were the most tolerant to drought stress. In particular, mutant line A3 (300 Gy) consistently displayed low S values (0.37-0.80), indicating strong osmotic adjustment and stable growth under PEG-induced osmotic stress. In contrast, the parental Argomulyo variety showed the highest S index (S > 1.0) for all traits, confirming its sensitivity to drought. The drought-tolerant check Dering showed moderate tolerance (S = 0.71-1.01), consistent with its known field performance under water-limited conditions.
           
    Mutant lines A4 and A5 showed intermediate responses, with some parameters (particularly root length and dry weight) approaching the sensitivity threshold (S ≈ 1.0). These results indicate that the gamma irradiation treatment, especially at 300 Gy, effectively induced beneficial genetic variability associated with improved drought tolerance. The enhanced tolerance observed in A2 and A3 mutant lines may be attributed to their ability to maintain water balance, cell turgor and growth stability under osmotic stress.
           
    Analysis of variance for yield under drought conditions is shown in Table 7. The data indicated significant differences among the mutant lines and control varieties grown under dry land conditions. The mutant soybean lines showed higher yield potential than the Argomulyo and Dering variety. Among all genotypes tested, the mutant line A3 recorded the highest mean yield of 2.47 t ha-1, followed by A5 with 2.40 t ha-1 and A4  with 2.30 t ha-1. The lowest yield was observed in the parental variety Argomulyo (1.85 t ha-1).

    Table 7: Yield performance of soybean mutant lines under the dry land condition.


           
    These results demonstrated that the selected mutant lines, particularly A3 and A5, maintained higher yield stability under water-limited conditions. The improvement in yield could be associated with the better physiological adaptation of these mutant lines, as reflected by their lower drought sensitivity index values (Table 6). This suggests that the mutation treatment effectively produced genotypes capable of maintaining productivity under drought stress.
           
    A similar increase in grain yield under drought conditions due to mutation induction has also been reported in other crops (Kadhimi et al., 2016). The consistent yield performance of mutant lines A3 and A5 in both dry land locations indicates their stability and adaptability to marginal environments. These findings confirm that mutation breeding combined with in vitro selection can be an effective approach to developing drought-tolerant soybean lines suitable for cultivation in dry areas.
           
    The results from both in vitro and field evaluations clearly demonstrated the positive impact of gamma-ray-induced mutation on improving drought tolerance and yield performance in soybean. The in vitro selection using PEG 6000 as an osmotic agent effectively differentiated tolerant genotypes and the same lines (A2 and A3) also performed well under field conditions. Therefore, the in vitro screening technique developed in this study could serve as a reliable preliminary method for identifying drought-tolerant soybean genotypes. The consistent performance of mutant lines A3 and A5 across various environments further supports the potential use of mutation breeding combined with in vitro selection as an effective approach to developing drought-tolerant soybean varieties suitable for dryland cultivation in Indonesia.

    CONCLUSION

    In vitro selection using PEG 6000 effectively identified drought-tolerant soybean lines. Mutant lines A3 and A5 showed the best tolerance to osmotic stress and the highest yields under dry land conditions. The consistency between in vitro and field results confirms that in vitro selection is a reliable method for early screening of drought-tolerant soybean genotypes suitable for cultivation in water-limited areas

    ACKNOWLEDGEMENT

    The authors gratefully acknowledge the financial support provided by the Center for Isotopes and Radiation Application (BATAN). Sincere appreciation is also extended to the Ministry of Agriculture of Indonesia for supporting the national soybean research grant program that made this study possible.

    CONFLICT OF INTEREST

    On behalf of all co-authors, I hereby confirm that there are no conflicts of interest.

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