Method for relieving cadmium stress of cardamine violifolia and increasing biomass based on nano-selenium

Abstract

The invention belongs to the technical field of agricultural environment restoration and functional crop cultivation, and discloses a method for relieving cadmium stress of cardamine violifolia and increasing biomass based on nano-selenium. According to the method, aiming at the problems that growth of cardamine violifolia is inhibited and cadmium accumulation exceeds the standard due to soil cadmium pollution in a selenium-enriched area, through a standardized process of seedling raising, domestication and precise nano-selenium treatment, after seedlings in the 5-6-leaf stage of cardamine violifolia are subjected to water culture domestication for 45 days, nano-selenium with the final concentration being 50 mg / L is applied to a cultivation system, and stress of cadmium smaller than or equal to 80 mg / L can be effectively relieved. According to the method, a peroxidase anti-oxidation system is activated through nano-selenium, stomatal movement is adjusted to protect a photosynthetic mechanism, finally, the cadmium accumulation amount of cardamine violifolia is reduced by 73.9%, the underground biomass is increased by 62.4%, and the net photosynthetic rate is increased by 56.2%. The method can be used for safe production of cardamine violifolia in a selenium-cadmium co-polluted environment, and provides technical support for sustainable development of functional agriculture.

Description

Technical Field

[0001] This invention belongs to the field of agricultural environmental remediation and functional crop cultivation technology, specifically targeting the mitigation of cadmium stress and biomass enhancement of the selenium hyperaccumulating plant *Violetia violacea* in selenium-cadmium co-contaminated soil. It is applicable to the safe production of *Violetia violacea* and its industrial application in selenium enrichment. Background Technology

[0002] Violet Leaf Broken Rice ( Cardamine violifolia This plant is a selenium hyperaccumulator unique to China, mainly distributed in selenium-rich areas such as Enshi, Hubei Province. Its selenium accumulation capacity can reach 10-100 times that of ordinary plants, and the selenium exists primarily in organic forms (selenocysteine ​​and selenomethionine), exhibiting high bioavailability. In 2021, it was listed as a new type of food ingredient by the National Health Commission of China. Furthermore, this plant can be used for the phytoremediation of selenium-contaminated soil, possessing both nutritional and environmental remediation value, making it a core crop in selenium-enriching functional agriculture.

[0003] However, soils in selenium-rich areas are generally accompanied by cadmium pollution. Cadmium can enter *Cephalotaxus fortunei* through the selenium transport pathway, leading to inhibited plant growth (impaired root development, reduced biomass), decreased photosynthetic efficiency, and excessive cadmium content in products, severely restricting the development of the selenium-rich industry. In existing technologies, traditional inorganic selenium (selenate, selenite) has limited effectiveness in reducing cadmium in *Cephalotaxus fortunei* and easily induces excessive selenium inhibition. While biochar, proline, and other amendments can partially fix cadmium, they cannot simultaneously increase plant selenium accumulation and biomass. Nano-selenium (SeNPs) show potential in alleviating cadmium stress in crops due to their large specific surface area and high biological activity; however, the optimal concentration, treatment time, and mechanism of action of nano-selenium for *Cephalotaxus fortunei* remain unclear, especially lacking an understanding of the "selenium-cadmium" interaction mechanism, making existing technologies insufficient to meet actual production needs.

[0004] Based on this, we propose a method based on nano-selenium to alleviate cadmium stress and increase biomass in Viola yedoensis, hoping to address the shortcomings of existing technologies. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of existing technologies and solve the following core problems: ① Determine the optimal concentration of nano-selenium for alleviating cadmium stress in Viola yedoensis: avoid excessively high concentrations (e.g., 100 mg / L) that inhibit growth or excessively low concentrations (e.g., 25 mg / L) that fail to effectively alleviate cadmium stress; ② Simultaneously achieve "cadmium reduction, selenium enhancement, and biomass preservation", providing a method for alleviating cadmium stress and enhancing biomass in Viola yedoensis based on nano-selenium.

[0006] This invention is achieved through the following technical solution: A method for alleviating cadmium stress and increasing biomass in *Violet spp.* based on nano-selenium includes the following steps: (1) Seedling raising: Select Viola leaf fragments ( Cardamine violifolia Seeds of the variety “Se-Run” were sown in the substrate and germinated at 25±1℃ until the seedlings had 5-6 leaves. (2) Domestication: The seedlings obtained in step (1) were transplanted into a hydroponic tank containing modified Hogland nutrient solution and acclimatized for 45 days under specific conditions. (3) Nano-selenium treatment: After domestication, nano-selenium and cadmium at different concentration gradients were added to the modified Hogrange nutrient solution in the hydroponic tank, and a fully crossover experimental design was adopted. (4) Harvest: Once the plants have grown to the target growth period, they are harvested and their biomass and selenium and cadmium content are tested.

[0007] Furthermore, the matrix described in step (1) is peat and perlite in a volume ratio of 2.5:1.

[0008] Furthermore, the hydroponic trough described in step (2) has a size of 200 × 30 × 18 cm and is equipped with a floating cultivation board (96 holes / trough, one seedling per hole). The modified Hogland nutrient solution is changed every 7 days during the acclimatization period.

[0009] Furthermore, the acclimatization under a specific environment mentioned in step (2) specifically refers to acclimatization under an environment with a temperature of 25±1℃ and a relative humidity of 60%.

[0010] Furthermore, during the acclimatization period in step (2), the nutrient solution is aerated twice a day for 30 minutes each time.

[0011] Furthermore, the different concentration gradients of nano-selenium mentioned in step (3) are specifically 25, 50, and 100 mg / L.

[0012] Furthermore, the cadmium mentioned in step (3) is CdCl2·2.5 H2O, and its concentration gradient is 20, 40, and 80 mg / L.

[0013] The present invention has the following advantages over the prior art: 1. This invention provides a method for alleviating cadmium stress and increasing biomass in *Corydalis violaceus* based on nano-selenium (SeNPs). This method addresses the problem of cadmium pollution in selenium-rich soils leading to inhibited growth and excessive cadmium accumulation in *Corydalis violaceus*. Through a standardized process of "seedling cultivation-acclimatization-precise nano-selenium treatment," after 45 days of hydroponic acclimatization of 5-6 leaf stage seedlings, nano-selenium with a final concentration of 50 mg / L is applied to the cultivation system, effectively alleviating cadmium stress ≤ 80 mg / L (calculated as CdCl2·2.5H2O). This invention activates the peroxidase (POD) antioxidant system and regulates stomatal movement to protect photosynthetic structures (PSII reaction centers, Rubisco enzyme activity) through nano-selenium, ultimately reducing cadmium accumulation in *Corydalis violaceus* by 73.9%, increasing underground biomass by 62.4%, and improving net photosynthetic rate by 56.2%. This method can be used for the safe production of *Corydalis yanhusuo* in selenium-cadmium co-polluted environments, providing technical support for the sustainable development of functional agriculture.

[0014] 2. Under 80 mg / L cadmium stress, treatment with 50 mg / L nano-selenium reduced cadmium accumulation in *Corydalis violaceus* by 73.9%, far superior to traditional inorganic selenium. Nano-selenium treatment increased selenium accumulation in plants by 95 times, underground biomass increased by 32.2% compared to the cadmium-only stress group, and total biomass increased by 16.3%. Under nano-selenium treatment, POD activity increased by 65% ​​compared to the cadmium-only stress group, MDA content decreased by 18.8%, net photosynthetic rate increased by 56.2%, and PSII function recovered to near cadmium-free stress levels. It is also compatible with hydroponic and soil cultivation systems and can be used in soils with varying degrees of selenium-cadmium co-contamination, balancing safe production of *Corydalis violaceus* with soil remediation. Attached Figure Description

[0015] Figure 1 Highly reducing bacteria and fungi isolated from the soil in which Viola yedoensis is grown, provided for this invention; Figure 2 The effect of nano-selenium and cadmium treatment on the growth and element accumulation of Viola yedoensis provided by this invention is shown in the figure. Figure 3 The effects of nano-selenium provided by this invention on the photosynthetic physiological parameters of *Corydalis yanhusuo* (a type of wild rice). Figure 4 This invention provides the effects of selenium, cadmium, and changes in the antioxidant properties of plants under cadmium stress relief treatment. Figure 5 The cell structure and root morphology of *Violet spp.* under nano-selenium treatment provided by this invention are shown in the diagram. Detailed Implementation

[0016] To further explain the present invention, the following specific embodiments are described.

[0017] Example 1: Plant material, growth conditions and exogenous treatment The *Violet-leaved Water Chestnut* used in this invention was provided by Enshi Deyuan Selenium Materials Engineering Technology Co., Ltd., and cultivated in a controlled hydroponic system at Yangtze University (Jingzhou, Hubei) from September to November 2024. Seeds germinated in a peat moss:perlite (volume ratio 2.5:1). When seedlings reached 5-6 leaves, they were transplanted into aerated hydroponic tanks (200×30×18 cm) containing modified Hoagland's solution (conductivity 500-800 μS / cm). After 45 days of acclimatization, a full-factor experiment was conducted to treat the plants with nano-selenium (concentration gradient: 25, 50, 100 mg / L) and cadmium (added as CdCl2·2.5H2O, concentration gradient: 20, 40, 80 mg / L). The nano-selenium used was biosynthesized nano-selenium (SeNPs), derived from highly reducing bacteria and fungi isolated from the *Violet-leaved Water Chestnut* planting soil. Figure 1 The CdCl2·2.5H2O was purchased from Sinopharm Group. The hydroponic system was equipped with floating boards (90 holes per tank), and aerated twice daily for 30 minutes each time. To further determine the optimal experimental concentration of selenium for alleviating cadmium stress, this invention set up four treatment groups: a control group, a 50 mg / L nano-selenium treatment group, an 80 mg / L CdCl2 treatment group, and a combined treatment group of 50 mg / L nano-selenium and 80 mg / L CdCl2, to comprehensively explore the individual effects and interactions of each treatment. The effects of three concentration gradients of selenium (25, 50, 100 mg / L) and cadmium (20, 40, 80 mg / L) on plant biomass were analyzed.

[0018] Note: Unless otherwise specified, the raw materials used in this invention are all from commercially available conventional products.

[0019] Example 2: Determination of plant biological indicators and selenium-cadmium content 1.1 Measurement of biological indicators Plant height was measured using a calibrated ruler, accurate to ±0.1 cm (measurement range: vertical distance from the substrate surface to the tip of the highest leaf). For root analysis, plants were carefully removed, and roots were rinsed with distilled water to remove attached particles. Taproot length was measured using a Mitutoyo digital vernier caliper (accuracy ±0.01 cm). After morphological index determination, plants were divided into aboveground (stem and leaf) and underground (root) parts. Enzyme activity was terminated by blanching at 105℃ for 30 min, followed by drying at 80℃ to constant weight in a Memmert oven (Germany). Dry weight was then determined using an analytical balance (accuracy ±0.0001 g). Total biomass was the sum of the aboveground and underground dry weights. All measurements were performed under standardized environmental conditions (25 ± 1℃, relative humidity 60%), with 10 biological replicates for each treatment.

[0020] The results show that... Figure 2 As shown in Figure a, nano-selenium treatment promoted plant growth in a concentration-dependent manner, with the 50 mg / L (Se2) treatment showing the most significant promoting effect, while higher concentrations (100 mg / L) exhibited an inhibitory effect. Conversely, the inhibitory effect of cadmium treatment on plant growth increased with increasing concentration, with the 80 mg / L (Cd3) treatment showing the strongest inhibitory effect. The four treatment groups exhibited significantly different biological effects, as shown in Figure a. Figure 2 b shows that cadmium stress significantly reduced plant height (by 62.1%), while selenium treatment partially restored growth (relief rate 32.2%). Nano-selenium increased root length (by 40.2% compared to the control) and alleviated cadmium-induced root inhibition (recovery rate 15.0%). Regarding biomass, selenium treatment significantly promoted underground dry weight accumulation (by 62.4%) and offset the inhibitory effect of cadmium on underground growth (relief rate 32.2%), but both selenium and cadmium reduced aboveground dry weight (by 8.2% for selenium treatment and 32.4% for cadmium treatment). Overall, selenium treatment increased total biomass (by 16.3%) and offset the inhibitory effect of cadmium on total biomass (relief rate 16.0%), indicating that selenium has a selective promoting effect on underground growth.

[0021] Figure 2 (a) Screening results of nano-selenium (25 / 50 / 100 mg / L) and cadmium (20 / 40 / 80 mg / L) concentrations; (b) Changes in plant height, root length and dry weight of different treatment groups; (c) Comparison of cadmium and selenium content in plants.

[0022] 1.2 Determination of selenium and cadmium content in plants After washing the plant tissues (roots, stems, and leaves), they were first blanched at 105℃ for 30 min, then dried at 70℃ to constant weight, and ground through a 100-mesh sieve. 0.2 g of sample was taken and digested using microwave-assisted digestion (digestion solution: HNO3:H2O2 = 8:2 mL, program: 180℃ / 15 min to 200℃ / 20 min). After evaporation at 150℃, the volume was adjusted to 25 mL and filtered through a 0.45 μm filter membrane. The cadmium content was determined using graphite furnace atomic absorption spectrometry (GFAAS): detection wavelength 228.8 nm, ashing temperature 600℃, atomization temperature 1800℃, calibrated with 0-10 μg / L standard solution. Selenium content was determined using hydride generation-atomic fluorescence spectrometry (HG-AFS): detection wavelength 196.0 nm, current carrier 5% HCl, reducing agent 0.5% KBH4, calibrated with 0-50 μg / L Se(IV) standard solution. Method validation included setting up blank controls and standard reference materials, requiring spiked recoveries of 85%-105% for cadmium and 80%-110% for selenium, with a relative standard deviation (RSD) <5%. The limits of detection were 0.01 μg / g for cadmium and 0.05 μg / g for selenium.

[0023] The formula for calculating element content is: Content (μg / g dry weight, DW) = (C × V × D) / m Where C is the concentration to be measured (μg / L), V is the final volume (mL), D is the dilution factor, and m is the sample mass (g). This closed digestion protocol conforms to the principles of green chemistry and can reduce acid consumption.

[0024] Elemental analysis results as follows Figure 2 c shows that cadmium treatment increased plant cadmium content by 46 times, while selenium co-treatment reduced cadmium accumulation by 73.9%; selenium treatment increased plant selenium content by 95 times, but the presence of cadmium slightly reduced selenium accumulation (the selenium content in the co-treatment group was 94.4% of that in the selenium-only treatment group). These results indicate that selenium has a dual role: both mitigating cadmium absorption and maintaining its own bioaccumulation capacity, although cadmium slightly interferes with selenium accumulation.

[0025] Example 3: Measurement of photosynthetic parameters and antioxidant capacity of plant leaves 1.1 Measurement of photosynthetic parameters A systematic evaluation of photosynthetic performance was conducted using a combination of gas exchange, chlorophyll fluorescence, and biochemical analysis. Using a LI-6400XT portable photosynthesis system, under controlled conditions (25±1℃, CO2 concentration 400 μmol·mol⁻¹), the following was performed: -1 Light intensity 1200 μmol·m -2 ·s -1Net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and stomatal limitation (Ls) were measured under these conditions. The photosynthetic analysis results showed that ( Figure 3 Cadmium stress significantly inhibited net photosynthetic rate (a decrease of 56.8%), while nano-selenium treatment increased net photosynthetic rate (an increase of 27.0%) and alleviated cadmium-induced photosynthetic inhibition (alleviation rate of 56.2%). Stomatal factors are the main regulators of photosynthetic response: cadmium mainly inhibits photosynthesis through stomatal limitation (confirmed by the enhanced correlation between net photosynthetic rate and stomatal conductance / CO2 parameter), while selenium optimizes stomatal regulation and significantly improves gas exchange efficiency. In the combined treatment group, selenium counteracted the inhibitory effect of cadmium by regulating transpiration rate, intercellular CO2 concentration, and stomatal conductance, indicating that selenium plays a key role in maintaining photosynthetic efficiency under stress conditions. These results confirm that selenium, by optimizing stomatal function, is the core mechanism by which plants are protected from photosynthetic inhibition caused by cadmium toxicity.

[0026] Figure 3 In the study: (a) the trend of net photosynthetic rate in different treatment groups, (b) the trend parameters of photosynthetic physiological changes, and (c) the correlation analysis between photosynthetic physiological parameters and net photosynthetic rate.

[0027] 1.2 Antioxidant capacity determination Using established spectrophotometric methods, oxidative stress markers and antioxidant components were systematically quantified. (1) Malondialdehyde (MDA) content: Thiobarbituric acid (TBA) method, detection wavelength 532 / 600 nm; (2) Hydrogen peroxide (H2O2) content: iron ion reduction / oxidation (FOX) method, detection wavelength 560 nm; (3) Antioxidant enzyme activities: peroxidase (POD, guaiacol method, 470 nm), catalase (CAT, UV method, 240 nm), superoxide dismutase (SOD, nitroblue tetrazolium method, 560 nm), ascorbate peroxidase (APX, 290 nm), monodehydroascorbate reductase (MDHAR, 340 nm), dehydroascorbate reductase (DHAR, 340 nm), glutathione reductase (GR, 340 nm), glutathione peroxidase (GSH-Px, 340 nm); (4) Redox metabolites: ascorbic acid (AsA, 265 nm), dehydroascorbic acid (DHA, 2,4-dinitrophenylhydrazine method, 520 nm), reduced glutathione / oxidized glutathione (GSH / GSSG, 5,5'-dithiobis(2-nitrobenzoic acid) method, 412 nm), galactose-1-phosphate dehydrogenase (GalLDH, cytochrome c reduction method, 550 nm).

[0028] All analyses employed standardized enzyme cycling or colorimetric protocols, with specific detection wavelengths set for each parameter. All reagents used in the experiments were purchased from Solvay Company.

[0029] Analysis of oxidative stress markers and antioxidant systems showed that ( Figure 4 Cadmium stress significantly increased malondialdehyde (MDA) content in roots (+90.9%) and leaves (+50.0%) (indicating exacerbated membrane damage), while selenium treatment reduced MDA content in roots and leaves by 18.8% and 17.1%, respectively. The MDA content in the selenium-cadmium combined treatment group was lower than that in the cadmium-only treatment group, indicating that selenium has a protective effect on membrane structure. Although hydrogen peroxide (H2O2) content did not show a clear pattern, correlation analysis ( Figure 4 The study found that peroxidase (POD) is the primary antioxidant defense enzyme—POD activity was strongly correlated with MDA content in all treatment groups (R = 8.723–9.654). Selenium treatment specifically increased POD activity in roots and leaves, while cadmium treatment broadly activated multiple antioxidant enzymes. Notably, the combined treatment group showed the most significant activation of antioxidant enzymes, and selenium-associated glutathione peroxidase (GSH-Px) was activated only under cadmium stress, indicating that the effect of selenium is stress-dependent. These results suggest that selenium can alleviate cadmium stress in two ways: directly protecting membrane structures and selectively enhancing stress-responsive antioxidant activity (especially POD activation); while GSH-Px activation requires cadmium stress induction, demonstrating the environmentally dependent regulation of plant defense mechanisms by selenium.

[0030] Figure 4 In the study: (a) changes in the content of oxidative biomarkers; (b) changes in the antioxidant properties of enzyme systems; (c) the regulatory role of non-enzymatic antioxidant systems; and (d) correlation analysis of antioxidant systems under different treatments.

[0031] Example 4: Fluorescent labeling and scanning electron microscopy observation 1.1 Fluorescent labeling Fresh leaves were cut into 5×5 mm pieces and incubated at 25°C in 20 mM phosphate buffer (pH 7.4) for 30 min in the dark with either 10 μM dihydroetidium ether (DHE, for detecting superoxide anions) or CellROX® deep red fluorescent probe (for detecting total reactive oxygen species). After rinsing three times with buffer to remove excess probe, the samples were observed using a Zeiss LSM 880 laser confocal microscope: the excitation / emission wavelengths for DHE were 488 / 580-620 nm, and for CellROX®, 640 / 665-720 nm; 20× or 40× water immersion objectives were used, with a pinhole size of 1 Airy unit, and the laser power was controlled at 5%-10% to reduce photobleaching. Fluorescence intensity was quantified using ImageJ / Fiji software: the fluorescence value was calculated after subtracting the background signal from the region of interest (ROIs), and at least 30 images were analyzed for each treatment (3 technical replicates × 10 images each). Unstained leaves and hydrogen peroxide-treated leaves were used as controls to verify the reliability of the results. Fluorescent probe labeling results showed that cadmium stress significantly increased the accumulation of reactive oxygen species (ROS) in leaves, while selenium treatment both reduced basal ROS levels and alleviated cadmium-induced oxidative stress. Figure 5 a).

[0032] 1.2 Observation of plant morphology and cellular microscopic level Root samples were processed as follows: rinsing with 0.1 M phosphate buffer (pH 7.2) → fixation with 2.5% glutaraldehyde at 4°C for 24 h → gradient dehydration with 30%-100% ethanol → critical point drying → gold sputtering (10 nm thickness). Imaging was performed using a Hitachi SU8010 scanning electron microscope at an accelerating voltage of 5-15 kV and a working distance of 8-10 mm, achieving a resolution of up to 3 nm. At least 10 fields of view were analyzed for each treatment (using ImageJ software) to quantify root hair density and cell morphology. Quality control measures included: setting up negative controls, maintaining an anti-static environment, and storing samples at low humidity (<10% RH) to ensure image fidelity.

[0033] Microscopic analysis revealed the protective effects of selenium at the tissue and cellular levels: in roots, cadmium causes cortical deformation and vascular tissue shrinkage, while selenium maintains normal root structure; even under cadmium stress, selenium can restore xylem / phloem morphology. Figure 5 b). Leaf observations showed that cadmium caused severe disruption of the vascular bundles, epidermis, and mesophyll tissue, while selenium treatment significantly alleviated these damages. Figure 5 d). Quantitative root analysis showed that selenium promoted root development (increasing root number, root tip number, and root diameter) and counteracted the inhibitory effects of cadmium on all root parameters (total root length, root surface area, and root volume). Figure 5(c, e). These results collectively confirm that selenium can protect the tissue integrity and physiological function of both above-ground and underground organs, thereby resisting cadmium stress.

[0034] Figure 5 (a) ROS fluorescent labeling showing ROS accumulation in leaves of the Se+Cd and Cd groups; (b) Root structure diagram from scanning electron microscopy; (c) Root morphology scan; (d) Comparison of leaf cross sections; (e) Changes in total root length, surface area, number of root tips, and other indicators.

[0035] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for alleviating cadmium stress and increasing biomass in *Violet violet* based on nano-selenium, characterized in that, Includes the following steps: (1) Seedling raising: Seeds of Viola yedoensis were selected, sown in the substrate, and germinated at 25±1℃ until the seedlings reached the 5-6 leaf stage. (2) Domestication: The seedlings obtained in step (1) were transplanted into a hydroponic tank containing modified Hogland nutrient solution and acclimatized for 45 days under specific conditions. (3) Nano-selenium treatment: After domestication, nano-selenium and cadmium at different concentration gradients were added to the modified Hogrange nutrient solution in the hydroponic tank, and a fully crossover experimental design was adopted. (4) Harvest: Once the plants have grown to the target growth period, they are harvested and their biomass and selenium and cadmium content are tested.

2. The method for alleviating cadmium stress and increasing biomass in *Violet violet* based on nano-selenium according to claim 1, characterized in that, The matrix mentioned in step (1) is peat and perlite in a volume ratio of 2.5:

1.

3. The method for alleviating cadmium stress and increasing biomass in *Violet violet* based on nano-selenium according to claim 1, characterized in that... The hydroponic trough described in step (2) has dimensions of 200 × 30 × 18 cm and is equipped with a floating cultivation board. The modified Hogland nutrient solution is changed every 7 days during the acclimatization period.

4. The method for alleviating cadmium stress and increasing biomass in *Violet violet* based on nano-selenium according to claim 1, characterized in that, The acclimatization under a specific environment mentioned in step (2) specifically refers to acclimatization under an environment with a temperature of 25±1℃ and a relative humidity of 60%.

5. The method for alleviating cadmium stress and increasing biomass in *Violet violet* based on nano-selenium according to claim 1, characterized in that, During the acclimatization period in step (2), the nutrient solution is aerated twice a day for 30 minutes each time.

6. The method for alleviating cadmium stress and increasing biomass in *Violet violet* based on nano-selenium according to claim 1, characterized in that, The different concentration gradients of nano-selenium mentioned in step (3) are specifically 25, 50, and 100 mg / L.

7. The method for alleviating cadmium stress and increasing biomass in *Violet violet* based on nano-selenium according to claim 1, characterized in that, The cadmium mentioned in step (3) is CdCl2·2.5 H2O, and its concentration gradient is 20, 40, and 80 mg / L.

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