| Summary: | Selenium (Se) uptake by plants depends on its form and salicylic acid (SA) can increase the efficiency of plant biofortification in Se. This study investigated the effects of selenite (Na<sub>2</sub>SeO<sub>3</sub>) and selenomethionine (SeMet) applied individually or together with SA on a total content of Se, Se speciation forms, yield and content of sugars and ascorbic acid of lettuce, as well as activity of selenocysteine methyltransferase (<i>smt</i>) and methionine methyltransferase (<i>mmt</i>) genes of the Se metabolic pathway. Lettuce was grown in the nutrient film technique (NFT) system. Se and SA were used at doses of 0.5 and 10.0 mg dm<sup>−3</sup> of the nutrient solution, respectively. The treatments were: 1. control, 2. Na<sub>2</sub>SeO<sub>3</sub>, 3. Na<sub>2</sub>SeO<sub>3</sub> + SA, 4. SeMet, 5. SeMet + SA, 6. SA. Se was accumulated more in the roots than the leaves. SeMet was more effective in biofortification than Na<sub>2</sub>SeO<sub>3</sub>. SA enhanced Se uptake, especially organic Se. Plants supplied with SeMet alone or SeMet + SA accumulated in their leaves mainly SeMet and methylselenocysteine (MeSeCys), while those treated with Na<sub>2</sub>SeO<sub>3</sub> or Na<sub>2</sub>SeO<sub>3</sub> + SA accumulated MeSeCys and selenite (SeO<sub>3</sub><sup>−2</sup>). The roots showed no expression of <i>smt</i>, while the expression of two <i>mmt</i> genes was independent of either Se or SA. The leaves of plants supplied with Na<sub>2</sub>SeO<sub>3</sub> demonstrated the strongest expression of <i>mmt</i> and <i>smt</i>.
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