دانشگاه اصفهان علوم زیستی گیاهی 3041-9603 18 2 2026 08 23 Comparative Effects of Selenium and Selenium Nanoparticles on the Physiological Responses of Saffron Callus (Crocus sativus L.) مطالعه مقایسه‌ای اثر سلنیوم و نانوذرات سلنیوم بر شاخص‌های فیزیولوژیکی کالوس زعفران مزروعی (Crocus sativus L.) 23 42 30748 10.22108/ijpb.2026.148729.1444 FA مهسا السادات خرسندی مقدم گروه زیست‌شناسی، دانشکده علوم پایه، دانشگاه بوعلی سینا، همدان، ایران رویا کرمیان گروه زیست‌شناسی، دانشکده علوم پایه، دانشگاه بوعلی سینا، همدان، ایران Journal Article 2026 04 25 Selenium (Se) can regulate plant growth and antioxidant metabolism, though its effects depend strongly on the form and concentration used. This study compared the effects of conventional selenium (sodium selenate) and selenium nanoparticles (SeNPs) on the physiological responses of saffron (Crocus sativus L.) callus cultured in vitro. Uniform calli were treated with 5 mg L-1 sodium selenate or SeNPs, while untreated calli served as the control. Both Se forms increased callus biomass, compatible solutes, phenolic compounds, total protein, and antioxidant enzyme activities. Sodium selenate reduced hydrogen peroxide and malondialdehyde contents, indicating improved redox regulation and reduced membrane lipid peroxidation. Conversely, SeNPs increased both oxidative-stress markers despite inducing stronger antioxidant responses, suggesting that the activated defense system was insufficient to prevent oxidative damage. Both treatments reduced photosynthetic pigment contents, with a more pronounced decline under SeNP exposure. Overall, sodium selenate produced a more favorable physiological response, whereas SeNPs at 5 mg L-1 induced both metabolic stimulation and oxidative toxicity. These findings emphasize the need to optimize the concentration of each Se form independently before its application in saffron tissue culture. Introduction Saffron (Crocus sativus L.) is a sterile, vegetatively propagated geophyte of considerable economic and medicinal importance. Propagation through corms is slow and may transmit pathogens; therefore, tissue culture is valuable for rapid clonal propagation, production of pathogen-free material, and controlled production of bioactive metabolites. Its success depends on plant growth regulators and the mineral composition of the culture medium. Selenium (Se), although not essential for higher plants, can exert beneficial effects at low concentrations by promoting growth and stimulating antioxidant systems. At excessive concentrations, however, Se may become pro-oxidant and impair cellular functions. Selenium nanoparticles (SeNPs) have attracted attention because their distinctive physicochemical properties may alter cellular uptake and biological activity. Nevertheless, plant responses depend on the form, concentration, species, and culture conditions of Se, and comparative information for saffron callus remains limited. This study compared the effects of sodium selenate and SeNPs, applied at the same nominal concentration, on growth, metabolites, oxidative-stress markers, and antioxidant enzymes in saffron callus cultured in vitro. Materials and Methods Healthy saffron corms were washed under running water and surface-disinfected sequentially with 70% ethanol for 5 min and 0.1% mercuric chloride for 10 min, followed by three rinses with sterile distilled water. Corm-derived explants were cultured on Murashige and Skoog (MS) medium containing 3% sucrose and 1 mg L⁻¹ each of 1-naphthaleneacetic acid (NAA) and 6-benzylaminopurine (BAP) to induce callus formation. After five subcultures, morphologically uniform calli were transferred to MS media with the same plant-growth-regulator composition and assigned to three treatments: an untreated control, 5 mg L-1 conventional Se supplied as sodium selenate, and 5 mg L-1 SeNPs. Cultures were maintained in darkness under controlled conditions in a culture room. At the end of the treatment period, calli were harvested, and samples intended for biochemical analyses were stored at −80 °C. Fresh and dry weights were recorded as indices of biomass accumulation. Photosynthetic pigments, including chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids, were determined spectrophotometrically. Soluble sugars and free proline were measured as indicators of carbon status and osmotic adjustment. Total phenolics, flavonoids, and anthocyanins were assayed to evaluate non-enzymatic antioxidant and secondary-metabolic responses. Malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) were quantified as indicators of lipid peroxidation and oxidative status, respectively. Total soluble protein was determined using the Bradford assay. The activities of peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) were also measured spectrophotometrically. Data were analyzed using SPSS, and treatment means were compared using Duncan’s multiple range test at P ≤ 0.05. Results and Discussion Both Se treatments significantly affected most of the evaluated physiological and biochemical traits, but the magnitude and direction of the responses differed between sodium selenate and SeNPs. Application of either Se form increased callus fresh and dry weights relative to the control, indicating that Se at the tested nominal concentration could promote biomass accumulation under the present culture conditions. This response may reflect improved cellular redox regulation and metabolic activity. Nevertheless, the growth-promoting effect should be interpreted alongside the oxidative-stress responses, because increased biomass does not necessarily imply the absence of cellular stress. The highest concentrations of chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids were observed in untreated calli. Both sodium selenate and SeNPs reduced pigment contents, with SeNPs generally exerting the stronger negative effect, particularly on carotenoids. Because the cultures were maintained in darkness and callus is a largely undifferentiated tissue, pigment values should be regarded as biochemical characteristics of the callus rather than direct measures of photosynthetic performance. The decline may be associated with inhibition of pigment biosynthesis or reduced pigment stability under Se-induced changes in cellular redox status. The stronger response to SeNPs is consistent with their potentially greater interaction with cellular surfaces; however, nanoparticle uptake and intracellular localization were not directly measured in this study and require further investigation. Both Se forms enhanced the accumulation of phenolics, flavonoids, and anthocyanins, and the highest phenolic levels were generally recorded in SeNP-treated calli. These compounds contribute to non-enzymatic antioxidant capacity and may accumulate when secondary-metabolic pathways are stimulated by moderate redox perturbation. Selenium may influence phenylpropanoid metabolism, including processes associated with phenylalanine ammonia-lyase activity, whereas the pronounced response to SeNPs may reflect a stronger elicitation signal. Proline and soluble sugars also increased following both treatments, with the greatest accumulation under SeNP exposure. In cultured tissues, these compatible solutes may support osmotic balance, stabilize macromolecules, store carbon, and buffer redox. Their accumulation, however, may represent either improved protective capacity or a response to treatment-induced stress; thus, it should not be interpreted solely as evidence of enhanced tolerance. The clearest distinction between the two Se forms was observed in oxidative-stress markers. Sodium selenate reduced both H₂O₂ and MDA contents relative to the control, suggesting improved control of reactive oxygen species and lower membrane lipid peroxidation. In contrast, SeNPs at the same nominal concentration markedly increased H₂O₂ and MDA levels, indicating increased oxidative stress and membrane damage. These findings show that equal mass concentrations of conventional and nanoparticulate Se are not necessarily physiologically equivalent. Differences in dissolution, surface reactivity, cellular contact, and effective availability could account for the contrasting responses, although these mechanisms were not directly assessed. Consequently, the increase in protective metabolites and antioxidant enzymes under SeNP treatment should be viewed as a compensatory defense response rather than unequivocal evidence of a beneficial effect. Total soluble protein increased in response to both Se forms. Moreover, POD, CAT, and SOD activities were enhanced, with the highest activities generally occurring in SeNP-treated calli. SOD provides an initial line of enzymatic defense by converting superoxide radicals to H₂O₂, whereas CAT and POD participate in H₂O₂ detoxification. The coordinated induction of these enzymes indicates activation of the antioxidant defense network. Under sodium selenate treatment, this activation coincided with reduced H₂O₂ and MDA, suggesting that antioxidant capacity was sufficient to constrain oxidative damage. Under SeNP treatment, however, elevated enzyme activities did not prevent H₂O₂ accumulation or lipid peroxidation, suggesting that reactive oxygen species production exceeded the tissue's detoxification capacity. Thus, the stronger biochemical response elicited by SeNPs included both defense stimulation and evidence of toxicity at 5 mg L-1. Conclusion Conventional Se and SeNPs both modified growth and metabolism in saffron callus, but they produced distinct redox outcomes. Sodium selenate promoted biomass, compatible-solute accumulation, phenolic metabolism, protein content, and antioxidant activity while reducing H₂O₂ and MDA. SeNPs also stimulated biomass and defense-related traits, often more strongly, but simultaneously decreased pigment contents and increased oxidative-stress markers. Therefore, the SeNP response at 5 mg L-1 cannot be regarded as uniformly beneficial. The results emphasize that Se form and concentration must be optimized independently before using Se-based treatments in saffron tissue culture. Dose-response experiments, physicochemical characterization of SeNPs in the culture medium, and direct assessment of Se uptake and localization are recommended to distinguish beneficial elicitation from nanoparticle-induced toxicity and to define a safe and effective application range. سلنیوم به‌عنوان یک عنصر ریزمغذی مفید، نقش مهمی در تنظیم فرایندهای فیزیولوژیکی گیاهان ایفا می‌کند و در سال‌های اخیر، استفاده از نانوذرات آن در سیستم‌های کشت بافت گیاهی مورد توجه قرار گرفتهاست. پژوهش حاضر با هدف مقایسه اثرات سلنیوم و نانوذرات سلنیوم بر برخی شاخصهای فیزیولوژیکی کالوس زعفران مزروعی (Crocus sativus L.) انجام شد. بدین منظور کالوس‌های ایجاد شده از بنه زعفران در محیط کشت MS حاوی یک میلیگرم در لیتر از هورمونهای BAP وNAA پس از 4 دوره واکشت، به همان محیط کشت شامل ۵ میلی‌گرم در لیتر سلنیوم یا نانو‌سلنیوم منتقل شدند. اثر این اشکال سلنیوم بر وزن کالوس، محتوای رنگیزه‌های فتوسنتزی، ترکیبات فنلی، قند محلول، پرولین، مالون‌دی‌آلدئید، پراکسید هیدروژن و پروتئین کل و نیز فعالیت آنزیم‌های آنتیاکسیدانی (پراکسیداز، کاتالاز و سوپراکسید دیسموتاز) مورد ارزیابی قرار گرفت. نتایج نشان داد که تیمارهای سلنیوم و نانو‌سلنیوم موجب افزایش وزن تر و وزن خشک کالوس و محتوای فنل، فلاونوئید و آنتوسیانین کل شدند، لیکن محتوای رنگیزه‌های فتوسنتزی را کاهش دادند. همچنین محتوای پرولین، قند محلول و پروتئین کل و نیز فعالیت آنزیم‌های آنتی‌اکسیدانی به‌ویژه کاتالاز تحت تاثیر هر دو شکل سلنیوم افزایش یافت. تیمار سلنیوم باعث کاهش محتوای پراکسید هیدروژن و مالوندیآلدئید شد، لیکن نانوسلنیوم در همین غلظت موجب افزایش آنها شد. در مجموع، نتایج نشان داد که اغلب شاخصهای فیزیولوژیکی تحت تأثیر هر دو شکل سلنیوم در غلظت ۵ میلی‌گرم در لیتر بهبود یافتند. لیکن در برخی موارد القاء تنش اکسیداتیو ناشی از نانوذرات سلنیوم، نشاندهنده حساسیت بیشتر کالوس زعفران به غلظت این شکل از سلنیوم به علّت سرعت جذب و نفوذ بالاتر آن است. بنابراین استفاده از سلنیوم به ویژه در شکل نانو در غلظت مناسب در شیشه، برای بهره‌برداری ایمن و مؤثر از این عنصر ضروری است.

https://ijpb.ui.ac.ir/article_30748_c8e2ba71c39a1ce3b421e95ea1525426.pdf