Selenium (Se) is indispensable to animals. The aims of this study were to explore the threshold dose of Se application in soil and reveal the mechanism of selenite effect on alfalfa growth, which is important for producing Se-enriched forage. Pot experiments were carried out in a greenhouse to explore the physiological mechanisms that are related to the response of forage crop to Se application. Alfalfa plants were treated with different Se levels (Se as Na₂SeO₃ dissolved in water was evenly sprinkled on the soil) to determine the effects of selenite supply on antioxidant activity, photosynthetic parameters, root activities, and the absorption of essential elements of alfalfa. Compared with the control plants, those treated with 5 and 10 mg kg⁻¹ Se exhibited increased shoot Se concentrations by 4.5- and 8.1-fold, respectively. The Se partitioning factors for alfalfa ranged from 0.47 to 0.89. The dry weights of roots and whole plants reached maximum values after applying 1 mg kg⁻¹ Se. At < 20 mg kg⁻¹ Se, plant growth was not inhibited in contrast to the control plants, and the activities of antioxidants superoxide dismutase (SOD), peroxidase (POD), and glutathione peroxidase (GSH-Px) increased, whereas the MDA content decreased significantly. With low-dose Se, the photosynthetic parameters (except for intercellular CO₂ concentration), leaf pigment content, and root activities were significantly enhanced, and the absorption of micronutrient elements (Fe and Mn) was promoted. However, at > 20 mg kg⁻¹ Se, the plant growth, antioxidant enzyme activity, photosynthetic parameters, root activity, and P and K absorption were inhibited. Alfalfa growth was not negatively affected by Se application less than 20 mg kg⁻¹. Low-dose selenite (0.5 and 1 mg kg⁻¹ Se) stimulated plant growth, mainly due to the stimulation of root growth by enhancing root activities and therefore promoting the absorption of P, K, Ca, Fe, and Mn. The increase in antioxidant enzyme activity and net photosynthetic rate partially resulted from the increased carotenoid content, which also contributed to the increasing dry matter at low-dose selenite levels. Given the relatively high transport efficiency of Se from root to overground part, alfalfa can be potentially used in the production of Se-enriched forage, and 10 mg kg⁻¹ Se is the optimum reference dose of Se application in soil.
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