Open Access

ARTICLE

Responses of Osmotic Adjustment and Antioxidant Enzyme Activities in Alfalfa (Medicago sativa L.) Seedlings to Biochar Amendment under Salt Stress

Jianghan Ouyang^1,#, Yuxin Zhang^1,#, Xiaotong Ma^1,2, Xiangjian Tu^1,2, Pengxin Hou^1,2, Wei Cao³, Shangzhi Zhong^1,2,*, Lulu Sun^4,*
1 College of Grassland Science, Qingdao Agricultural University, Qingdao, China
2 Shandong Key Laboratory for Germplasm Innovation of Saline-alkaline Tolerant Grasses and Trees, Qingdao Agricultural University, Qingdao, China
3 Forestry and Grassland Science Research Institute of Hulunbuir, Hulunbuir, China
4 Laboratory Management Center, Qingdao Agricultural University, Qingdao, China
* Corresponding Author: Shangzhi Zhong. Email: ; Lulu Sun. Email:
# These authors contributed equally to this work
(This article belongs to the Special Issue: Plant Responses to Stress Factors)

Phyton-International Journal of Experimental Botany https://doi.org/10.32604/phyton.2026.079919

Received 30 January 2026; Accepted 25 February 2026; Published online 10 March 2026

Abstract

Salt stress has been demonstrated to trigger the overaccumulation of reactive oxygen species (ROS) within plant tissues. This process, in turn, has been shown to induce oxidative damage and further impair the physiological and metabolic processes. As a promising organic soil conditioner, biochar has proven effective in mitigating the adverse effects of salt stress in crops. Nevertheless, the synergistic and interactive effects of salt stress and biochar application on the osmotic adjustment system and antioxidant defense system of alfalfa (Medicago sativa L.) have not been fully elucidated. This study employed a completely randomized design, comprising four NaCl-induced salt stress levels (0, 3, 6, and 9 dS m⁻¹) and three biochar application rates (0, 30, and 60 g kg⁻¹). To evaluate the role and underlying mechanism of biochar in alleviating oxidative damage caused by salt stress in alfalfa, we determined the growth indicators (shoot biomass [SB], root biomass [RB], and total biomass [TB]), physiological indicators (total chlorophyll content and relative water content), and biochemical indicators (osmotic adjustment substances, antioxidant enzyme activities, and oxidative stress-related substances). Results indicated that salt stress significantly suppressed alfalfa biomass accumulation by 16%–84%. With increasing salt stress intensity, the contents of proline (Pro), glycine betaine (GB), soluble sugars (SS), and soluble proteins (SP), along with the activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and polyphenol oxidase (PPO), significantly increased in alfalfa leaves; yet, these defensive responses failed to prevent the decrease in leaf relative water content and the significant accumulation of superoxide anion (O₂^•−), hydrogen peroxide (H₂O₂), and malondialdehyde (MDA) contents. These results indicated that the endogenous defense system alone is insufficient to counteract salt stress-induced oxidative damage. In contrast, compared with the non-biochar control, biochar application significantly increased alfalfa chlorophyll content (by ~40%) and biomass accumulation (by ~12%). Notably, under the 30 g kg⁻¹ biochar treatment, the antioxidant enzyme activities and osmotic adjustment substance contents of alfalfa seedlings significantly decreased, accompanied by a marked reduction in O₂^•− (by ~13%), H₂O₂ (by ~21%), and MDA (by ~30%) contents. These results indicated that biochar could maintain the dynamic equilibrium of osmotic regulation and antioxidant enzyme systems in alfalfa leaves, and promote the synthesis of photosynthetic pigments and photosynthates, thereby enhancing alfalfa biomass accumulation by 54%–68%. However, under severe salt stress (9 dS m⁻¹), excessive biochar application rate (60 g kg⁻¹) failed to exert a positive effect on alfalfa growth, with only approximately a 1% increase in total biomass of alfalfa seedlings. Collectively, our findings demonstrated that appropriate biochar application can effectively alleviate salinity-induced oxidative damage in alfalfa and thus promote its growth in saline soils. To alleviate salt stress-induced phytotoxicity and optimize the efficacy of soil amendments, it is imperative to rationally regulate the application amount of biochar in saline soils.

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Keywords

Salt stress; biochar amendment; Medicago sativa L.; osmotic adjustment substances; antioxidant enzyme activities

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