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Background <p>Soil salinity severely hampers early establishment of alfalfa (<i>Medicago sativa</i> L.) by inhibiting growth; impairing root system architecture (RSA) and aggravating oxidative, osmotic and membrane damage. This study aimed to investigate the individual and combined effects of two plant growth regulators—melatonin (MT) and 24-epibrassinolide (EBL)—on salt stress tolerance in alfalfa seedlings grown in coastal saline soil.</p> Results <p>When applied individually, EBL primarily restored establishment-related structural traits, with the best EBL dose (300&#xa0;µg L<sup>− 1</sup>) increasing plant height by 41.09%, total biomass by 37.56% and root biomass by 55.33% relative to the S treatment (salt-stress control). Moreover, 300&#xa0;µg L<sup>− 1</sup> EBL markedly improved RSA parameters (primary root length by 28.43%, total root length by 60.35%, root surface area by 56.20% and root tip number by 62.04%). Meanwhile, MT more effectively alleviated stress injury and strengthened antioxidant defences, with 150 µmol L<sup>− 1</sup> MT reducing root/leaf malondialdehyde content by 51.86%/44.22%, root/leaf membrane permeability by 44.63%/44.78% and root/leaf proline content by 43.31%/46.78%. Moreover, 100 µmol L<sup>− 1</sup> MT enhanced root superoxide dismutase activity by 43.16%, and 150 µmol L<sup>− 1</sup> MT enhanced root catalase activity by 53.03%. The effects of both MT and EBL generally exhibited dose-dependent trends, but the recovery of certain traits peaked at intermediate doses. Co-application of MT and EBL produced the strongest overall recovery across growth, RSA, antioxidant, osmotic and membrane-stability traits. Multivariate analysis identified that M100E300 treatment (100 µmol L<sup>− 1</sup> MT and 300&#xa0;µg L<sup>− 1</sup> EBL) resulted in plants that were most similar to control plants. Two-way factorial ANOVA confirmed significant main effects of MT and EBL across all measured traits, while MT × EBL interactions were significant only for selected biomass, root architectural, antioxidant, osmotic and membrane-injury traits.</p> Conclusions <p>MT and EBL jointly improved salt tolerance in alfalfa seedlings, but their main effects differed. EBL mainly supported growth recovery and root architectural rebuilding, whereas MT contributed more to redox balance, osmotic adjustment and membrane stability. Only traits with significant MT × EBL terms were treated as interaction-dependent responses; other combined improvements were interpreted as additive or complementary. These findings suggest that combined MT and EBL application may help improve alfalfa seedling establishment in salt-affected soils.</p> Graphical Abstract <p></p>

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Synergistic effects of melatonin and 24-epibrassinolide on salt stress tolerance in Medicago sativa (Alfalfa) seedlings

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月6日

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  • Ruojun Sun,
  • Xinyu Hou,
  • Fei Han,
  • Hui Wang,
  • Hong Pan,
  • Quangang Yang,
  • Zhongchen Yang,
  • Yanhong Lou,
  • Yuping Zhuge

摘要

Background

Soil salinity severely hampers early establishment of alfalfa (Medicago sativa L.) by inhibiting growth; impairing root system architecture (RSA) and aggravating oxidative, osmotic and membrane damage. This study aimed to investigate the individual and combined effects of two plant growth regulators—melatonin (MT) and 24-epibrassinolide (EBL)—on salt stress tolerance in alfalfa seedlings grown in coastal saline soil.

Results

When applied individually, EBL primarily restored establishment-related structural traits, with the best EBL dose (300 µg L− 1) increasing plant height by 41.09%, total biomass by 37.56% and root biomass by 55.33% relative to the S treatment (salt-stress control). Moreover, 300 µg L− 1 EBL markedly improved RSA parameters (primary root length by 28.43%, total root length by 60.35%, root surface area by 56.20% and root tip number by 62.04%). Meanwhile, MT more effectively alleviated stress injury and strengthened antioxidant defences, with 150 µmol L− 1 MT reducing root/leaf malondialdehyde content by 51.86%/44.22%, root/leaf membrane permeability by 44.63%/44.78% and root/leaf proline content by 43.31%/46.78%. Moreover, 100 µmol L− 1 MT enhanced root superoxide dismutase activity by 43.16%, and 150 µmol L− 1 MT enhanced root catalase activity by 53.03%. The effects of both MT and EBL generally exhibited dose-dependent trends, but the recovery of certain traits peaked at intermediate doses. Co-application of MT and EBL produced the strongest overall recovery across growth, RSA, antioxidant, osmotic and membrane-stability traits. Multivariate analysis identified that M100E300 treatment (100 µmol L− 1 MT and 300 µg L− 1 EBL) resulted in plants that were most similar to control plants. Two-way factorial ANOVA confirmed significant main effects of MT and EBL across all measured traits, while MT × EBL interactions were significant only for selected biomass, root architectural, antioxidant, osmotic and membrane-injury traits.

Conclusions

MT and EBL jointly improved salt tolerance in alfalfa seedlings, but their main effects differed. EBL mainly supported growth recovery and root architectural rebuilding, whereas MT contributed more to redox balance, osmotic adjustment and membrane stability. Only traits with significant MT × EBL terms were treated as interaction-dependent responses; other combined improvements were interpreted as additive or complementary. These findings suggest that combined MT and EBL application may help improve alfalfa seedling establishment in salt-affected soils.

Graphical Abstract