Application of nano-cobalt hydroxide in improving seed germination rate of rape under salt stress

Abstract

The application of nano cobalt hydroxide in improving the seed germination rate of rape under salt stress is disclosed.The application is to apply a solution containing nano cobalt hydroxide to rape seeds;in the solution, the concentration of nano cobalt hydroxide is 0.008-0.012 g / L.After the seed treatment of the above specific concentration of nano cobalt hydroxide on the rape seeds under salt stress, the germination rate, germination potential and germination index of the rape seeds can be significantly improved, and the seed germination rate of rape is improved.

Description

Technical Field

[0001] This invention belongs to the technical field of agricultural applications, and more specifically, relates to the application of nano-cobalt hydroxide in improving the germination rate of rapeseed seeds under salt stress. Background Technology

[0002] Saline-alkali land accounts for approximately 7% of the world's total land area and about 20% of irrigated land. Soil salinization, as one of the most destructive abiotic stresses, has posed a significant threat to the sustainable development of global agriculture. In-depth research into the physiological and ecological response mechanisms of plants under salt stress, elucidating the complex interactions between plants and salt-stressed environments, and improving plant salt tolerance through modern cultivation techniques are of significant strategic importance.

[0003] Rapeseed (Brassica napus L.) seeds, with an oil content of 33%-50%, rank among the top major oilseed crops internationally, and its cultivation covers the globe. Rapeseed oil is rich in monounsaturated and polyunsaturated fatty acids, which can synergistically regulate lipid metabolism and reduce the risk of cardiovascular disease. Furthermore, rapeseed oil is rich in vitamins, phytosterols, and polyphenols, which can effectively alleviate chronic inflammatory responses by reducing free radical accumulation and regulating the expression of inflammation-related genes. Although rapeseed is considered a moderately salt-tolerant crop, its physiological functions and agronomic traits are still significantly affected by salt stress. Therefore, a deeper understanding of the response mechanism of rapeseed to salt stress and the development of effective strategies to enhance its salt tolerance are of significant theoretical and practical importance for achieving high and stable yields and improving the quality of rapeseed in saline-alkali areas.

[0004] Salt stress delays rapeseed seed germination, specifically manifested as a decrease in germination rate and germination potential, and significant inhibition of radicle and plumule elongation. Different salt concentrations have varying effects on seed germination. Low salt concentrations promote seed germination by activating endosperm storage substances through osmotic regulation signals, increasing energy metabolism efficiency, and enhancing seed germination rate and radicle growth. Medium salt concentrations inhibit seed germination (generally, concentrations above 50 mM inhibit germination). With increasing salt concentration and duration of exposure, ion toxicity disrupts cellular physiological activities, hinders imbibition, and decreases germination parameters (germination potential, vigor index). High salt concentrations completely inhibit seed germination. When the salt concentration exceeds a critical value, the cytoplasmic ion homeostasis of the seed is completely disrupted, cell development ceases, and ultimately, germination fails completely.

[0005] Improving salt tolerance in crop seeds and seedlings involves fundamentally different physiological and molecular mechanisms. Seed salt tolerance primarily concerns the tolerance during the germination stage, with its core mechanisms lying in maintaining embryonic viability, regulating dormancy and germination signaling pathways (such as ABA / GA balance), and enhancing the physical and biochemical shielding of the seed coat or embryonic tissue against osmotic stress and ion toxicity. In contrast, seedling salt tolerance involves the entire plant's adaptive response after establishment, a more complex systemic process. Its mechanisms mainly include the selective absorption and compartmentalization of ions (such as the selective absorption and compartmentalization of Na+ ions). + (It is retained in the roots or vacuoles), synthesizes osmotic regulators (such as proline and betaine), and enhances the reactive oxygen species scavenging system to maintain metabolic homeostasis.

[0006] Existing research has confirmed the specific salt tolerance of crops at different stages (seeds and seedlings). For example, exogenous application of betaine or certain polyamines can significantly improve the photosynthetic efficiency and membrane stability of seedlings under salt stress, but it does not significantly improve the germination rate of most crop seeds, and may even inhibit germination by altering the osmotic environment. This highlights that salt tolerance intervention strategies must be precisely designed for specific developmental stages of crops. Seed treatment aims at "protection and initiation," while seedling management focuses on "regulation and adaptation." Therefore, improving the germination rate of rapeseed seeds under salt stress has become an important research direction. Summary of the Invention

[0007] To address the aforementioned problems in the existing technology, the primary objective of this invention is to provide an application of nano-cobalt hydroxide in improving the germination rate of rapeseed seeds under salt stress.

[0008] The second objective of this invention is to provide a method for improving the germination rate of rapeseed seeds under salt stress.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: This invention seeks protection for the application of nano-cobalt hydroxide in improving the germination rate of rapeseed seeds under salt stress, wherein a solution containing nano-cobalt hydroxide is applied to rapeseed seeds; wherein the concentration of nano-cobalt hydroxide in the solution is 0.008-0.012 g / L.

[0010] This invention, through research, has found that soaking rapeseed seeds under salt stress with a specific concentration of nano-cobalt hydroxide significantly improves the germination rate, germination potential, and germination index, thereby enhancing the germination rate. Rapeseed seeds treated with salt stress also exhibit better growth in subsequent stages, with increased fresh and dry weights, showing significant differences compared to the control group. However, excessively high concentrations of nano-cobalt hydroxide may inhibit rapeseed germination and growth through toxic effects and physiological metabolic disorders.

[0011] Specifically, the concentration of the nano-cobalt hydroxide in the solution can be 0.0082 g / L, 0.0085 g / L, 0.0088 g / L, 0.009 g / L, 0.0092 g / L, 0.0095 g / L, 0.0098 g / L, 0.01 g / L, 0.011 g / L, etc., or any range formed by the above values, such as 0.009-0.011 g / L, 0.0095-0.0105 g / L, and the present invention is not limited thereto.

[0012] Preferably, the hydration kinetic diameter of the nano-cobalt hydroxide is 1.4-2.0 nm.

[0013] Preferably, the surface charge of the nano-cobalt hydroxide is -2.8mV to -4.0mV.

[0014] Preferably, the nano-cobalt hydroxide is at least one of levorotatory nano-cobalt hydroxide and dextrorotatory nano-cobalt hydroxide.

[0015] Preferably, the rapeseed is Brassica napus seed.

[0016] Preferably, the mass ratio of the nano-cobalt hydroxide to the rapeseed seeds is 0.001-0.002:1.

[0017] Preferably, the salt stress is performed using a sodium chloride solution with a concentration of 50-300 mM. More preferably, the salt stress is performed using a sodium chloride solution with a concentration of 200-300 mM.

[0018] Preferably, the preparation method of the nano-cobalt hydroxide includes the following steps: mixing solvent, cobalt source, aspartic acid or glutamic acid evenly, then adding alkaline solution to mix and react, centrifuging and precipitating to obtain the nano-cobalt hydroxide.

[0019] The cobalt source is at least one of cobalt chloride hexahydrate and cobalt sulfate hexahydrate; and / or the alkaline solution is at least one of sodium hydroxide and potassium hydroxide.

[0020] Preferably, the solvent is water.

[0021] Preferably, the concentration of the alkaline solution is 5-8M.

[0022] Preferably, the reactants after the reaction with alkali solution are mixed with an alcohol solution, centrifuged and precipitated to prepare the nano-cobalt hydroxide.

[0023] Preferably, the alcohol solution includes, but is not limited to, isopropanol.

[0024] Preferably, the molar mass ratio of the cobalt source to aspartic acid or glutamic acid is 1-2:1.

[0025] Furthermore, this invention claims protection for a method to improve the germination rate of rapeseed seeds under salt stress, which involves applying a solution containing nano-cobalt hydroxide to rapeseed seeds; wherein the concentration of nano-cobalt hydroxide in the solution is 0.008-0.012 g / L.

[0026] Compared with the prior art, the present invention has the following beneficial effects: This invention has found that soaking rapeseed seeds under salt stress with a specific concentration of nano-cobalt hydroxide can significantly improve the germination rate, germination potential, and germination index of rapeseed seeds, thereby increasing the germination rate of rapeseed seeds. Attached Figure Description

[0027] Figure 1 Characterization of chiral cobalt hydroxide nanoparticles. Among them, Figure 1 Figures A and B in the image are transmission electron microscopy (TEM) images of L-ACH and D-ACH, respectively. Figure 1 Figure C shows the characteristic UV absorption spectra of L-ACH and D-ACH; Figure 1 Figure D in the figure shows the hydrodynamic diameters of L-ACH and D-ACH; Figure 1 Figure E in the figure shows the Zeta potentials of L-ACH and D-ACH; Figure 1 Figure F in the figure shows the circular dichroism chromatograms of L-ACH and D-ACH.

[0028] Figure 2 The effects of L-ACH and D-ACH soaking treatments on rapeseed seed germination under 250 mM salt stress were investigated. Figure 2 Figure A shows the growth of rapeseed seedlings after 7 days of treatment with different concentrations of materials under salt stress. Figure 2 Figures B and C show the germination rates of rapeseed after soaking in different concentrations of L-ACH and D-ACH. Figure 2 Figure D in the figure shows the fresh weight of rapeseed seedlings.

[0029] Figure 3 These are confocal microscopy images showing the co-localization of DiI-L-ACH and DiI-D-ACH with rapeseed seeds; the green fluorescence represents DiI-L / D-ACH fluorescence. Figure 2 Figures A and B show the imaging and quantitative analysis of fluorescence intensity of DiI-L-ACH and DiI-D-ACH in rapeseed seed coat; Figure 2 Figures C and D show the imaging and quantitative analysis of fluorescence intensity of DiI-L-ACH and DiI-D-ACH in the radicle of rapeseed.

[0030] Figure 4 The effects of L-ACH and D-ACH soaking treatments on reactive oxygen species (ROS) content in rapeseed seedlings under salt stress were investigated. Figure 4Figure A in the figure shows the superoxide anion content; Figure 4 Figure B in the figure shows the hydrogen peroxide content; Figure 4 Figure C shows the hydroxyl radical content.

[0031] Figure 5 The effects of L-ACH and D-ACH soaking treatments on the antioxidant enzyme activity of rapeseed seedlings under salt stress were investigated. Figure 5 Figure A shows the activity of superoxide dismutase (SOD); Figure 5 Figure B shows the peroxidase (POD) activity; Figure 5 Figure C shows the catalase (CAT) activity.

[0032] Figure 6 The effects of L-ACH and D-ACH soaking treatments on the content of osmotic regulators in rapeseed seedlings under salt stress were investigated. Figure 6 Figure A in the figure shows the soluble protein content; Figure 6 Figure B shows the proline content; Figure 6 Figure C shows the soluble sugar content.

[0033] Figure 7 The effects of L-ACH and D-ACH soaking treatments on starch metabolism in rapeseed seedlings under salt stress were investigated. Figure 7 Figure A in the figure shows the starch content; Figure 7 Figure B shows α-amylase activity.

[0034] Figure 8 The effects of L-ACH and D-ACH soaking treatments on malondialdehyde content and electrolyte permeability in rapeseed seedlings under salt stress were investigated. Figure 8 Figure A shows the malondialdehyde content; Figure 8 Figure B in the figure shows the electrolyte permeability.

[0035] Figure 9 The effects of L-ACH and D-ACH soaking treatments on the nitrogen assimilation enzyme activity of rapeseed seedlings under salt stress were investigated. Figure 9 Figure A shows the nitrate reductase (NR) activity; Figure 9 Figure B shows the activity of glutamine synthase (GS); Figure 9 Figure C shows the activity of glutamate synthase (GOGAT). Detailed Implementation

[0036] The present invention will be further described below with reference to the specification and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0037] In this specific implementation, the test case data are presented as Mean ± SE, where n = biological replicates. The Least significant difference (LSD) test and a two-tailed t-test were used to compare differences between treatments. These represent p < 0.05, p < 0.01, and p < 0.001, respectively. Different lowercase letters represent p < 0.05, while the same lowercase letters represent p ≥ 0.05.

[0038] Example 1: Synthesis of L-ACH Nanoparticles 0.2380 g of cobalt chloride hexahydrate (CoCl2·6H2O, Sinopharm, AR) and 0.1330 g of L-aspartic acid (L-Asp, Sinopharm, AR) were weighed into an Erlenmeyer flask and added to 16 mL of ultrapure water. The mixture was stirred at 600 r / min for 2 min at room temperature to dissolve the cobalt chloride. Then, 350 μL of 6 M NaOH solution was added dropwise, and the mixture was stirred at 600 r / min for 2 h until the solution color changed from pink to orange-red. The L-ACH stock solution was then mixed with isopropanol at a volume ratio of 1:4 and centrifuged at 8000 r / min for 10 min to obtain a pink precipitate. This precipitate was then placed in a fume hood overnight to obtain L-aspartic acid nanoparticles (L-ACH).

[0039] Example 2 Synthesis of D-ACH Nanoparticles The difference between this embodiment and Embodiment 1 is that: L-aspartic acid is replaced with dextrorotatory aspartic acid (D-Asp, Sinopharm, AR).

[0040] Example 3 Synthesis of L-GCH nanoparticles (L-GCH) The difference between this embodiment and Embodiment 1 is that L-aspartic acid is replaced with L-glutamic acid (D-Glu, Sinopharm, AR).

[0041] Example 4 Synthesis of L-Cobalt Hydroxide Nanoparticles (L-ACH) The difference between this embodiment and Embodiment 1 is that cobalt chloride hexahydrate is replaced with cobalt sulfate hexahydrate (Sinopharm, AR).

[0042] Example 5 Synthesis of L-Cobalt Hydroxide Nanoparticles (L-ACH) The difference between this embodiment and Embodiment 1 is that sodium hydroxide is replaced with potassium hydroxide (Sinopharm, AR).

[0043] Test Example 1: Characterization of Chiral Cobalt Hydroxide Nanoparticles (1) Test method The size and morphology of the L-ACH and D-ACH synthesized in Examples 1 and 2 were characterized using a transmission electron microscope (TEM) (H-7650, Hitachi); their hydration kinetic diameter and Zeta potential were determined using a nanoparticle size analyzer (NanoBrook 173plus); their ultraviolet absorption was determined using a UV-Vis spectrophotometer (UV-1800 PC, Aoyi, China); and their optical rotation was determined using a circular dichroism chromatograph (J-1500, Japan Spectrophotometer).

[0044] (2) Test results The morphology of L-ACH and D-ACH can be observed by transmission electron microscopy (TEM). Both are granular and uniformly dispersed. Figure 1 (Figures A and B in the text).

[0045] Figure 1 Figure C shows the characteristic UV absorption spectra of L-ACH and D-ACH. The results show that both L-ACH and D-ACH have characteristic absorption peaks at 510 nm, with the peak value of D-ACH at 510 nm being slightly lower than that of D-ACH.

[0046] Figure 1 Figure D shows the results of hydration particle size determination. The hydration dynamic diameter of L-ACH is 1.70±0.13 nm, and the hydration dynamic diameter of D-ACH is 1.73±0.25 nm, with no significant difference.

[0047] Figure 1 Figure E shows the results of the Zeta potential measurement. The surface charge of L-ACH is -3.43±0.53 mV, and the surface charge of D-ACH is -3.37±0.32 mV. Both are electrically neutral and there is no significant difference in their charges.

[0048] Figure 1 Figure F in the figure shows the results of the circular dichroism chromatogram. The CD signals of L-ACH and D-ACH at 530 nm are +359.87 mdeg and -347.30 mdeg, respectively. The image shows strict mirror symmetry, indicating that L-ACH and D-ACH have mirror symmetric chiral structures with opposite optical rotations, which is consistent with the characteristics of chiral enantiomers.

[0049] Test Example 2: In vitro reactive oxygen species scavenging efficiency determination of L / D-ACH (1) Hydroxyl radical scavenging efficiency: The determination was performed according to the improved method of (Lu et al 2016). The specific preparation method was as follows: 3 mL of L-ACH and D-ACH (1 g / L) solutions synthesized in Examples 1 and 2 containing Tris-HCl (0.1 M, pH=4.7), methyl violet (24 μM), FeSO4 (1.08 mM), and H2O2 (0.1 M) were taken. The above solutions without nanoparticles were used as standard controls, and 3 mL of methyl violet (24 μM) solution was used as blank controls. Each test tube was placed in a constant temperature water bath at 37℃ for 30 min. After the reaction, the absorbance of each tube was measured at 582 nm, and the measurement data were recorded.

[0050] Calculate the hydroxyl radical scavenging rate of L-ACH or D-ACH using the following formula: In the formula: △α582 is A blank control - A determination, △A582 is A blank control - A standard control.

[0051] (2) Hydrogen peroxide removal efficiency: The efficiency of removing H2O2 from L-ACH and D-ACH was determined by spectrophotometry: The absorbance change of the mixed solution of L-ACH and D-ACH with H2O2 at 240 nm was directly measured by ultraviolet spectrophotometer.

[0052] (3) Superoxide anion scavenging efficiency: The “SOD detection kit (WST-1)” (A001-3-2, Nanjing Jiancheng Biotechnology Co., Ltd.) was used to generate a detectable water-soluble formazan dye (with maximum absorption at 450 nm) by reacting ·O2- generated by the reaction of xanthine and xanthine oxidase with WST-1. Specifically, L-ACH or D-ACH solution was added to the mixture of xanthine and xanthine oxidase (the final L-ACH or D-ACH concentration was 1 g / L), and then incubated at 37°C for 30 min. The absorbance at 450 nm was then measured by spectrophotometer.

[0053] (4) Test results Table 1

[0054] Figure 1Characterization of chiral cobalt hydroxide nanoparticles. The in vitro reactive oxygen species (ROS) scavenging capabilities of the synthesized L-ACH and D-ACH were determined, as shown in Table 1. The results showed that both L-ACH and D-ACH chiral materials exhibited good in vitro ROS scavenging efficiency at a concentration of 1 g / L. Regarding hydroxyl radical (·OH) scavenging, the scavenging rate of L-ACH was 23.54±1.63%, and that of D-ACH was 23.23±0.08%, with no significant difference. Regarding hydrogen peroxide (H2O2) scavenging capability, the scavenging rates of L-ACH and D-ACH were consistent (L-ACH: 31.03±1.45%; D-ACH: 31.03±2.02%), with no significant difference. Superoxide anion (·O2) scavenging capability was also assessed. - Superoxide anion (SOS) is one of the most harmful reactive oxygen species to cells, and its excessive accumulation can lead to lipid peroxidation of biological membranes and oxidative damage to proteins. The superoxide anion scavenging rates of L-ACH and D-ACH were as high as 84.66±0.78% and 85.95±0.90%, respectively. Overall, L-ACH and D-ACH showed consistent performance in the scavenging of the three reactive oxygen species, especially demonstrating a significant advantage in superoxide anion scavenging (scavenging rate >84%).

[0055] Test Example 3: Effects of different concentrations of L-ACH and D-ACH on rapeseed germination under salt stress (1) The variety used in this experiment was Zhongshuang 11 (ZS11), a semi-winter type of rapeseed. The growth temperature was 25℃±2℃, the humidity was 60%, and the light intensity was 200μmol / m². 2 s 1 PAR. The light cycle is 14 / 10 hours (day / night).

[0056] In this experiment, a 250 mM NaCl solution was selected to simulate salt stress. Three treatments were set up: a NaCl solution without nanomaterials (control group), a NaCl solution containing levorotatory cobalt hydroxide nanoparticles (synthesized in Example 1) (L-ACH treatment group), and a NaCl solution containing dextrorotatory cobalt hydroxide nanoparticles (synthesized in Example 2) (D-ACH treatment group). Each treatment was replicated in 4 places.

[0057] Seed disinfection: Select healthy, mature rapeseed seeds that are plump, uniform in size, and undamaged. Disinfect the surface of the seeds with a 1% sodium hypochlorite solution for 15 minutes, then rinse with deionized water for 4-6 minutes. Allow the disinfected seeds to air dry at room temperature.

[0058] Germination box setup: Add 15 mL of treatment solution to the germination box: Add 15 mL of 250 mM NaCl solution to the control treatment, add 15 mL of a mixed solution of L-ACH (0.0003 g) and 250 mM NaCl to the L-ACH treatment group, and add 15 mL of a mixed solution of D-ACH (0.0003 g) and 250 mM NaCl to the D-ACH treatment group. Then, lay three sheets of germination paper flat in the box.

[0059] Sowing: Sow 30 dried rapeseed seeds in each germination box (rapeseed seed: nano cobalt hydroxide mass ratio is 0.0015:1). Germination is considered to be when the length of the seed radicle is equal to the length of the seed.

[0060] (2) Germination index determination The germination rate (GR), germination index (GI), germination potential (GF), and average germination time (GMT) are calculated using the following formulas: Germination rate (GR) = N / S × 100%, where N and S are the number of germinated seeds and the total number of seeds, respectively.

[0061] Germination Index (GI) = ∑(Gt / Dt), where Gt is the number of germinated germinations on day t, and Dt is the corresponding germination day.

[0062] Germination potential (GF) = (Number of seeds germinated on day 3 / Total number of seeds tested) × 100%.

[0063] Average germination time Di represents the number of days since the seeds were placed in the germination box, and Gi represents the number of germinated seeds on each corresponding day.

[0064] Determination of the fresh and dry weight of rapeseed seeds after soaking: Rapeseed seeds treated with salt stress for 7 days were dried with absorbent paper and weighed using an electronic balance, which was recorded as fresh weight (FW). All samples were then placed in an oven, blanched at 105℃ for 30 minutes, and then dried at 80℃ to constant weight. After cooling to room temperature, the dry weight (DW) was measured.

[0065] (3) Experimental results Soaking experiments were conducted on rapeseed seeds at different concentrations of L-ACH and D-ACH to investigate their effects on seed germination under salt stress. Figure 2 The effects of L-ACH and D-ACH soaking treatments on rapeseed seed germination under 250 mM salt stress were investigated. The effects of different concentrations of L-ACH and D-ACH treatments on rapeseed seed germination after 7 days of 250 mM salt stress were shown in the figure. Figure 2Figures A to C in the table show the effects of different concentrations of L-ACH and D-ACH on germination rate, germination potential, germination index, and average germination time. The rapeseed seeds treated with 0.01 g / L L-ACH had the highest germination rate, reaching 61.11%, significantly higher than the control group (47.78%), with an increase of 13.33%. The corresponding D-ACH treatment group (0.01 g / L D-ACH) had a germination rate of 60%, also significantly better than the control group, with no statistically significant difference from the L-ACH treatment group. The germination rate of the low-concentration (0.001 g / L) L-ACH treatment group was 56.67%, with no significant difference from the control group; while the germination rates of the high-concentration (0.05 g / L) L-ACH and D-ACH treatment groups were 52.22% and 53.33%, respectively, with no significant difference compared to the control group.

[0066] Table 2

[0067] like Figure 2 As shown in Figure D, after 7 days of 250 mM salt stress, the germinated rapeseed seedlings were harvested and weighed, and the fresh weight was recorded. The fresh weight of the seedlings in the 0.01 g / L L-ACH treatment group reached 0.72 g, an increase of 60.56% compared with the control group (0.45 g), showing a significant advantage. The corresponding fresh weight of the D-ACH treatment group (0.01 g / L) was 0.71 g, an increase of 58.72%, which was also significant. In contrast, the fresh weights of the low concentration (0.001 g / L) L-ACH and D-ACH treatment groups were 0.56 g and 0.52 g, respectively; the fresh weights of the high concentration (0.05 g / L) L-ACH and D-ACH treatment groups were 0.51 g and 0.48 g, respectively, with no significant difference between the two treatments and the control group. The data above indicate that the L-ACH and D-ACH treatments at a concentration of 0.01 g / L significantly improved rapeseed seed germination and seedling biomass accumulation under salt stress, and the effect levels of the two materials were similar with no significant difference.

[0068] Test Example 4: Co-localization of L-ACH and D-ACH with rapeseed seeds (1) Synthesis of DiI-L-ACH: L-ACH was labeled with DiI (1,1'-octacoyl-3,3,3',3'-tetramethylindole carbocyanine perchlorate, a fluorescent group used to demonstrate the co-localization of nanomaterials in rapeseed seeds). 12 μL of DiI staining solution (5 mg / mL) was added to 188 μL of DMSO solution to dissolve and obtain DiI staining solution (0.6 mg / mL). The above DiI staining solution was added dropwise to 4 mL of 1 g / L L-ACH and stirred at 500 r / min for 2 min at room temperature on a magnetic stirrer. The mixed solution was then transferred to a dialysis bag with a molecular cutoff of 10 kDa and dialyzed in ultrapure water for 24 h, with the water changed every 12 h, to finally obtain the DiI-L-ACH solution, which was stored at 4℃ for later use. Synthesis of DiI-D-ACH: L-ACH was replaced with D-ACH, and the rest was the same as above.

[0069] Rapeseeds were immersed in synthesized DiI-L-ACH and DiI-D-ACH and treated in the dark for 8 h. Slice preparation: Seeds were washed with running deionized water, dried with absorbent paper, and then cut into seed coat and radicle sections using a blade. Specifically: In the dark, seeds were held with tweezers and cut into seed coat and radicle sections (approximately 200 µm in size) using a blade. The slices were immediately mounted on glass slides, and one drop of perfluoronaphthylamine (PFD) was added to each slice. The samples were then covered with coverslips, ensuring no air bubbles. The slices were observed under a Leica laser scanning confocal microscope (LASM SP8). The LASM parameters were set as follows: 40x objective lens (add one drop of ultrapure water before observation), 514 nm excitation, laser intensity 30%; PMT1: 550nm-615nm (fluorescence of DiI-ACH); bright field on, 4-6 repetitions, and the fluorescence intensity of DiI-ACH was analyzed using LAS (Leica Application Suite) software.

[0070] (2) In order to investigate the distribution of different chiral materials in rapeseed, L-ACH and D-ACH were labeled with DiI fluorescent dye, and their distribution in the seed coat and radicle of rapeseed was observed by laser confocal imaging. Figure 3 These are confocal microscopy images showing the co-localization of DiI-L-ACH and DiI-D-ACH with rapeseed seeds. Figure 3 As shown in Figures A and C, obvious fluorescence signals were observed in the DiI-L-ACH and DiI-D-ACH soaking treatments, while no fluorescence was observed in the control group. Figure 3In the figures, B and D represent the results of quantitative fluorescence analysis. The fluorescence signal intensities of DiI-L-ACH and DiI-D-ACH in the seed coat were 10.05 and 9.18, respectively, with no significant difference. In the radicle, the fluorescence signal intensities of DiI-L-ACH and DiI-D-ACH were 3.50 and 3.60, respectively, again with no significant difference. This indicates that both L-ACH and D-ACH can enter the seed coat and radicle tissues of rapeseed, and their translocation levels are similar, showing no significant difference.

[0071] Test Example 5: Effects of L-ACH and D-ACH on reactive oxygen species in rapeseed seeds under salt stress (1) Experimental method: The rapeseed seeds were treated according to the experimental method in Test Example 3.

[0072] (2) Test method: The reactive oxygen species were measured in rapeseed seedlings grown from treated rapeseed seeds.

[0073] Superoxide anion content determination: The "Superoxide Anion Content Determination Kit" (BC1290, Solarbio Biotechnology Co., Ltd.) was used according to the instructions. 0.05 g of sample (seedlings after 7 days of germination under different treatments) was taken, and 0.1 mL of superoxide anion extraction buffer was added. The sample was ground using a Tissuelyser-32L grinder (Jingxin) for 180 seconds (65 Hz). The mixture was then centrifuged at 10000 r / min for 20 minutes at 4℃ in a low-temperature refrigerated centrifuge (D3024R, Seloz). The supernatant was collected for later use. The standard solutions were diluted to 0.125 μmol / mL, 0.0625 μmol / mL, 0.03125 μmol / mL, 0.015625 μmol / mL, 0.0078125 μmol / mL, and 0.0039 μmol / mL to construct a standard curve. Zero the instrument with a blank sample and measure the absorbance at 530 nm using a microplate reader. ΔA = Atest tube - Ablank tube. Plot the standard curve: y = kx + b, with the standard solution concentration as the x-axis and ΔAstandard as the y-axis.

[0074] The superoxide anion content is calculated using the following formula: Superoxide anion content (umol / g FW) = 2x / m, where m is the fresh weight (g).

[0075] Hydrogen peroxide (H2O2) content determination: The "Hydrogen Peroxide Content Determination Kit" (A064-1-1, Nanjing Jiancheng Biotechnology Co., Ltd.) was used, following the instructions. Accurately weigh 0.1 g of sample (seedlings after germination for 7 days under different treatments), add 1 mL of phosphate buffer solution, and grind using a grinder for 180 seconds (65 Hz). Then, centrifuge the extract at 12000 r / min, 4℃ for 20 minutes, and collect the supernatant. Add the sample according to the instructions, mix well, and measure the absorbance at a wavelength of 405 nm, a light path of 1 cm, and zero with deionized water.

[0076] Calculate the hydrogen peroxide content using the following formula: Hydrogen peroxide content in tissue (mmol / g FW) = .

[0077] Hydroxyl radical inhibition rate determination: The "Hydroxy radical scavenging ability test kit" (BC1325, Solarbio Biotechnology Co., Ltd.) was used, and the procedure was performed according to the instructions. Accurately weigh 0.1 g of sample, add 1 mL of extraction buffer, and grind using a grinder for 180 seconds (65 Hz). Then, centrifuge the extraction buffer at 10000 r / min at 4℃ for 10 minutes, and collect the supernatant for later use. Add the sample according to the instructions, mix well, and incubate in a 37℃ water bath for 60 min. Subsequently, centrifuge at 10000 r / min at room temperature for 10 min, and measure the absorbance at a wavelength of 405 nm, recording the values ​​as Ablank, Apair, and Ameasured, respectively.

[0078] The hydroxyl radical inhibition rate is calculated using the following formula: Hydroxyl radical inhibition rate (%) = (Atest - Apair) / (Ablank - Apair) · 100%.

[0079] (3) Experimental results The effects of L-ACH and D-ACH soaking treatments on reactive oxygen species (ROS) content in rapeseed seedlings under 250 mM salt stress are as follows: Figure 4 As shown. Among them, Figure 4 Figure A shows the results of superoxide anion determination. The contents of L-ACH and D-ACH treatment groups were reduced by 36.62% and 44.37% respectively compared with the control group (1.35 μmol / g FW), with the scavenging effect of D-ACH treatment group being more obvious. Figure 4 Figure B shows the results of hydrogen peroxide determination. The average content of L-ACH and D-ACH treatment groups was 0.15 μmol / g FW and 0.16 μmol / g FW, respectively, which were significantly lower than those of the control group (0.19 μmol / g FW), with reductions of 21.96% and 15.62%, respectively.

[0080] Figure 4 Figure C shows the hydroxyl radical scavenging capacity. The L-ACH treatment group had the highest scavenging efficiency (62.20%), which was significantly higher than the control group (51.41%), with an increase of 21.0%. The D-ACH treatment group had a scavenging rate of 59.98%, which was 16.7% higher than the control group.

[0081] In summary, both L-ACH and D-ACH treatments effectively reduced the accumulation of reactive oxygen species in rapeseed seedlings under salt stress, while enhancing antioxidant defense capabilities and improving the salt tolerance of rapeseed seeds. Although the L-ACH treatment group performed slightly better in some indicators, there was no significant difference between it and the D-ACH treatment group.

[0082] Test Example 6: Effects of L-ACH and D-ACH on the activity of antioxidant enzymes in rapeseed seeds under salt stress (1) Experimental method: The rapeseed seeds were treated according to the experimental method in Test Example 3.

[0083] (2) Test method: The antioxidant enzyme activity of rapeseed seedlings grown from treated rapeseed seeds was tested.

[0084] Weigh 0.1 g of sample (seedlings after germination for 7 days under different treatments) and add 1 mL of 0.05 mol / L sodium phosphate buffer (PBS). Grind for 180 seconds (65 Hz) using a grinder, then centrifuge at 12000 r / min and 4℃ for 20 min. The supernatant is the enzyme extract.

[0085] Superoxide dismutase (SOD) assay: In a 5 mL test tube, add 1.5 mL of 0.05 mol / L PBS, 0.3 mL of 0.13 mol / L methionine solution, 0.3 mL of 0.75 mol / L NBT solution, 0.3 mL of EDTA-Na2 solution, and 0.3 mL of 0.02 mol / L riboflavin solution. Add 0.05 mL of enzyme extraction buffer (use buffer solution instead of enzyme solution in the control tube), and finally add 0.25 mL of distilled water to make a total volume of 3 mL. After mixing, place the control tube in the dark, and react the other tubes under 4000 lx sunlight for 20 min. Measure the absorbance of each tube at 560 nm.

[0086] SOD activity can be calculated using the following formula: ; In the formula: ACK is the absorbance of the control tube, AE is the absorbance of the sample tube, V is the total volume of the sample (mL), VT is the amount of sample used during the determination (mL), and m is the fresh weight of the sample (g).

[0087] Peroxidase (POD) assay: Prepare the reaction mixture: Take 200 mL of sodium phosphate buffer (PBS, 0.2 mol / L, pH=6.0), add 0.076 mL of guaiacol stock solution (2-methoxyphenol), heat and stir to dissolve, cool, and then add 0.112 mL of 30% H2O2 solution. Enzyme activity assay: Take 3 mL of the above reaction mixture, add 50 µL of enzyme extraction solution, mix well, and zero the sample using PBS as a control. Measure the absorbance of the mixture at 470 nm. The measurement lasts for 180 seconds, with readings every 30 seconds. Calculate the peroxidase activity using the following formula: ; In the formula: △A470 is the average absorbance during the reaction time, m is the fresh sample mass (g), T is the reaction time, V is the enzyme solution volume taken during the determination (mL), and VT is the total volume of the extracted enzyme solution (mL).

[0088] Catalase (CAT) assay: Prepare the reaction mixture: Take 200 mL of sodium phosphate buffer (0.15 mol / L, pH=7.0), add 0.3092 mL of 30% H2O2 solution, shake well, and measure immediately. Enzyme activity assay: Take 3 mL of the above reaction mixture, add 20 µL of enzyme extraction solution, zero the sample using sodium phosphate buffer as a control, and measure the absorbance of the mixture at 240 nm. The measurement lasts for 180 seconds, with readings every 30 seconds. One unit of enzyme activity (U) is defined as a decrease in absorbance of 0.01 μL per minute.

[0089] CAT activity is calculated using the following formula: ; In the formula: △A240 is the average absorbance during the reaction time, m is the fresh sample mass (g), T is the reaction time, V is the volume of enzyme solution taken during the determination (mL), and VT is the total volume of enzyme extract (mL).

[0090] (3) Experimental results Figure 5 This study investigated the effects of L-ACH and D-ACH seed soaking treatments on the antioxidant enzyme activities of rapeseed seedlings under salt stress. The superoxide dismutase (SOD) activity in the L-ACH treatment group was 315.99 U / g FW, a 20.05% increase compared to the control group (263.23 U / g FW); the activity in the D-ACH treatment group was 299.72 U / g FW, an increase of 13.86%. Both were significantly higher than the control group, showing statistically significant differences.

[0091] Peroxidase (POD) activity assay results showed that the activity in the L-ACH treatment group was 980.58 U·min -1 ·g -1 FW) compared to the control group (568.33 U·min) -1 ·g -1 The FW group saw a 72.56% improvement, while the D-ACH treatment group (830.58 U·min) achieved a 72.56% improvement. -1 ·g - 1 The FW (Fairness and Weakness) increased by 46.15%, which is a significant difference.

[0092] Furthermore, catalase (CAT) activity data showed that the L-ACH treatment group (1740.07 U·min) -1 ·g -1 FW) and D-ACH treatment group (1652.90 U·min) -1 ·g -1 FW) was significantly different from the control group (1160.41 U·min) -1 ·g -1 The FW (free trades) increased by 49.95% and 42.45%, respectively, showing a significant difference.

[0093] In summary, L-ACH and D-ACH soaking treatments significantly enhanced the activities of SOD, POD, and CAT in seedlings under salt stress. The increase in SOD activity was the lowest among the three (L-ACH: +20.05%; D-ACH: +13.86%). In vitro scavenging efficiency results showed that L-ACH and D-ACH achieved in vitro scavenging efficiencies of 84.66% and 85.95% for superoxide anions, respectively. This result indicates that the materials themselves possess extremely strong superoxide anion scavenging capabilities, potentially directly reducing the accumulation of superoxide anions under salt stress and thus reducing dependence on SOD activity enhancement. Combined with the overall trend of enhanced antioxidant enzyme activity, it can be inferred that the materials synergistically alleviate reactive oxygen species damage by directly scavenging and activating antioxidant enzyme activity, maintaining reactive oxygen species homeostasis in rapeseed seedlings under salt stress, and improving the salt tolerance of rapeseed.

[0094] Test Example 7: Effects of L-ACH and D-ACH on Osmotic Regulation Substances in Rapeseeds under Salt Stress (1) Experimental method: The rapeseed seeds were treated according to the experimental method in Test Example 3.

[0095] (2) Test method: The content of osmotic conditioning substances in rapeseed seedlings grown from treated rapeseed seeds was determined as follows.

[0096] Soluble protein content determination: A protein quantification (TP) assay kit (A045-2, Nanjing Jiancheng Biotechnology Co., Ltd.) was used, following the instructions. Accurately weigh 0.1 g of the sample leaf, add 0.9 mL of physiological saline, grind using a grinder for 180 seconds (65 Hz), centrifuge at 12000 r / min at 4℃ for 20 min, and collect the supernatant. Add the sample according to the instructions, mix well, let stand for 10 min, and measure the absorbance at 595 nm. Calculate the soluble protein content using the formula provided in the instructions.

[0097] Proline content determination: Weigh 0.2 g of sample and add 5 mL of 3% sulfosalicylic acid solution. Extract proline by reacting in a boiling water bath for 10 min. Transfer 2 mL of the extract to a 10 mL test tube and add 2 mL of glacial acetic acid and 2 mL of acidic ninhydrin. Mix well and heat the mixture in a boiling water bath for 30 min. After cooling, extract the reaction mixture with 4 mL of toluene. Centrifuge and collect the supernatant of the reaction mixture. Using toluene as a blank control, measure the absorbance at 520 nm.

[0098] Calculate using the following formula: ; In the formula: Vt is the volume of the extract (mL), Vs is the volume of the sample taken during the determination (mL), and m is the sample mass (g).

[0099] Soluble sugar content determination: The "Plant Soluble Sugar Content Detection Kit" (BC0030, Solarbio Biotechnology Co., Ltd.) was used, following the instructions. Take 0.05 g of sample (seedlings after 7 days of germination under different treatments), add 0.5 mL of distilled water, and grind using a grinder (Tissuelyser-32L, Jingxin) for 180 seconds (65 Hz). Pour into a capped centrifuge tube and incubate in a boiling water bath for 10 min (tightly capped to prevent moisture loss). After cooling, centrifuge at 10000 r / min in a low-temperature refrigerated centrifuge (D3024R, Seloz) at room temperature for 20 minutes. Collect the supernatant in a 5 mL test tube, bring the volume to 10 mL with distilled water, and mix well. Dilute the standard solution with distilled water to prepare concentrations of 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 mg / mL for later use. Add the sample according to the instructions, mix well, and place in a 95℃ water bath for 10 min (tightly cover to prevent moisture loss). After cooling to room temperature, zero the instrument with a blank control and measure the absorbance of the sample at 620 nm using an ELISA reader. Record the values ​​as A blank tube, A test tube, and A standard tube, and calculate the results. A = A measurement - A blank, Astandard = Astandard - Ablank. Standard curve Plot a standard curve y = kx + b, with standard A as the x-axis and the concentration of the standard solution as the y-axis. Based on the standard curve, Substitute A into the formula to calculate the sample concentration y (mg / mL).

[0100] The soluble sugar content can be calculated using the following formula: Soluble sugar content (mg / g FW) = 10y / m; where m is the fresh weight (g).

[0101] (3) Experimental results Figure 6 This study investigated the effects of L-ACH and D-ACH seed soaking treatments on the content of osmotic regulators in rapeseed seedlings under salt stress. The soluble protein content in the L-ACH and D-ACH treated groups was 11.12 mg / g FW and 10.84 mg / g FW, respectively, both significantly higher than the control group (9.42 mg / g FW), with increases of 18.05% and 15.07%, respectively. This indicates that L-ACH and D-ACH seed soaking treatments significantly enhanced protein accumulation in rapeseed seedlings and could alleviate salt stress damage by regulating osmotic balance.

[0102] Rapeseed has a very low proline content under normal conditions, but its proline content increases significantly when subjected to stress. Figure 6 Figure B shows the proline content. The proline levels in the L-ACH and D-ACH treatment groups were 0.40% and 0.38%, respectively, which were 14.89% and 19.15% lower than the control group (0.47%), showing a significant difference. This indicates that L-ACH and D-ACH soaking treatments significantly reduced salt stress damage in rapeseed seeds.

[0103] Figure 6 Figure C shows the soluble sugar content of the L-ACH and D-ACH treatment groups. The L-ACH and D-ACH treatment groups had soluble sugar contents of 9.87 mg / g FW and 10.18 mg / g FW, respectively, which were significantly higher than the control group (6.11 mg / g FW), with increases of 61.54% and 66.45%. High sugar accumulation can alleviate the negative effects of salt stress on rapeseed seedlings by maintaining cellular osmotic balance and energy supply.

[0104] In summary, L-ACH and D-ACH treatments significantly increased the soluble protein and soluble sugar content of rapeseed seedlings while reducing proline accumulation, thereby enhancing their resistance to osmotic stress in high-salt environments, reducing water loss, and increasing biomass.

[0105] Test Example 8: Effects of L-ACH and D-ACH on starch metabolism in rapeseed seeds under salt stress (1) Experimental method: The rapeseed seeds were treated according to the experimental method in Test Example 3.

[0106] (2) Test method: The starch metabolism index of rapeseed seedlings grown from treated rapeseed seeds was determined.

[0107] α-Amylase activity assay: The "α-Amylase Activity Assay Kit" (BC0610, Solarbio Biotechnology Co., Ltd.) was used, and the procedure was performed according to the instructions. Take 0.05 g of sample, add 0.4 mL of extraction buffer, grind using a grinder for 180 seconds (65 Hz), transfer to a centrifuge tube, centrifuge at 8000 r / min at 4℃ for 10 min, and then extract at room temperature for 15 min, shaking every 5 min to ensure complete extraction; centrifuge at 8000 r / min at room temperature for 10 min, collect the supernatant, and add distilled water to a final volume of 5 mL. This is the amylase stock solution. The standard solution was diluted with distilled water to prepare concentrations of 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, 0.0125 mg / mL, and 0.00625 mg / mL for later use. Add the sample according to the instructions, mix well, and then incubate in a boiling water bath for 10 min (tightly capped to prevent moisture loss). After cooling to room temperature, zero the sample using a blank control and measure the absorbance at 540 nm using an ELISA reader. Record these values ​​as A control, A blank tube, A test tube, and A standard tube, respectively, and calculate... A measurement = A measurement - A control A standard = A standard - A blank. Standard curve plotting: based on the concentration (x, mg / mL) and absorbance of the standard tube. A standard (y, (Standard A) Establish a standard curve, and based on the standard curve, ... Substituting A into the equation yields x (mg / mL).

[0108] α-Amylase activity is calculated using the following formula: α-Amylase activity of sample (U / g FW) = 2x / m; where m is the fresh weight (g).

[0109] Starch content determination: Use the "Starch Content Detection Kit" (BC0700, Solarbio Biotechnology Co., Ltd.) and follow the instructions. Take 0.05 g of sample, add 0.5 mL of extraction buffer, grind using a grinder for 180 seconds (65 Hz), transfer to a centrifuge tube, and extract in an 80℃ water bath for 30 min. After cooling, centrifuge at 8000 r / min in a low-temperature refrigerated centrifuge (D3024R, Seloz) at room temperature for 10 min, discard the supernatant, and keep the precipitate. Add 0.3 mL of distilled water to the precipitate and gelatinize in a boiling water bath for 15 min (tightly capped to prevent moisture loss). After cooling, add 0.6 mL of reagent II, extract in a boiling water bath for 15 min, and shake 3-5 times. After cooling, centrifuge at 8000 r / min at room temperature for 15 min, and collect the supernatant for testing. Dilute the standard solution with distilled water to prepare concentrations of 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.04 mg / mL, 0.03 mg / mL, 0.02 mg / mL, and 0.01 mg / mL for later use. Add the samples according to the manufacturer's instructions, mix well, and incubate in a 95°C water bath for 10 min (tightly capped to prevent moisture loss). After cooling to room temperature, zero the sample using a blank control and measure the absorbance at 620 nm using a microplate reader. Record these values ​​as A blank tube, A test tube, and A standard tube, respectively, and calculate the results. A = A standard - A blank A' = A_measured - A_blank. Standard curve plotting: Plot a standard curve y = kx + b, with standard A as the x-axis and the concentration of the standard solution as the y-axis. Based on the standard curve, Substitute A into the formula to calculate the sample concentration x (mg / mL).

[0110] Starch content is calculated using the following formula: Starch content (mg / g FW) = 0.811x / m; where m is the fresh weight (g).

[0111] (3) Experimental results Figure 7 This study investigated the effects of L-ACH and D-ACH soaking treatments on starch metabolism in rapeseed seedlings under salt stress. The results showed that the starch contents of rapeseed seedlings in the L-ACH and D-ACH treatment groups were 5.85 mg / g FW and 5.93 mg / g FW, respectively, representing increases of 13.37% and 14.84% compared to the control group (5.16 mg / g FW), indicating significant differences. This suggests that the treatments significantly promoted starch accumulation in rapeseed seedlings under salt stress, potentially by increasing carbon source reserves to provide energy support for cells.

[0112] Figure 7Figure B shows the results of α-amylase activity assays. The enzyme activity in the L-ACH treatment group was 0.82 U / g FW, an increase of 95.24% compared to the control group (0.42 U / g FW); the activity in the D-ACH treatment group was 0.76 U / g FW, an increase of 81.90% compared to the control group. The significant enhancement of α-amylase activity may accelerate the decomposition of starch into soluble sugars, consistent with the increase in soluble sugar content. L-ACH and D-ACH seed soaking treatments significantly increased the activity of key enzymes in starch metabolism in rapeseed seedlings under salt stress, resulting in a significant increase in leaf starch content. The enhanced carbon source reserves not only provide an energy basis for cell membrane repair and osmotic regulation, but also maintain cellular osmotic balance through soluble sugar metabolism, thereby effectively alleviating the damage of salt ions to chloroplast structure.

[0113] In summary, L-ACH and D-ACH seed soaking treatments promote the conversion of starch to soluble sugars by increasing starch accumulation and α-amylase activity in rapeseed seedlings, thereby maintaining cell osmotic potential and enhancing energy supply. This metabolic regulatory mechanism is consistent with the trend of increased soluble sugar and protein content and decreased proline content, jointly alleviating the osmotic damage of salt stress to rapeseed seedlings.

[0114] Test Example 9: Effects of L-ACH and D-ACH on malondialdehyde content and electrolyte permeability in rapeseed seeds under salt stress (1) Experimental method: The rapeseed seeds were treated according to the experimental method in Test Example 3.

[0115] (2) Test method: The malondialdehyde content and electrolyte permeability of rapeseed seedlings grown from treated rapeseed seeds were determined.

[0116] Malondialdehyde (MDA) content determination: Weigh 0.15 g of sample, add 1.5 mL of 5% trichloroacetic acid (TCA), grind for 180 seconds (65 Hz) using a grinder, centrifuge at 12000 r / min in a low-temperature refrigerated centrifuge at 4℃ for 20 min, and collect the supernatant for later use. Take 1 mL of the supernatant, add 1 mL of 0.67% thiobarbituric acid (TBA), mix, boil in a water bath for 30 min, cool, and centrifuge. Using deionized water instead of the extract as a control, measure the absorbance of the supernatant at 450 nm, 532 nm, and 600 nm.

[0117] The malondialdehyde (MDA) content is calculated using the following formula: MDA content (μmol / g FW) = 6.452·(A532-A600)-0.559·A450·V / m; where m is the fresh sample mass (g) and V is the volume of MDA extract (mL).

[0118] Electrolyte permeability determination: Specifically, clean rapeseed leaves, weigh 0.1 g of leaf sample, place it in a 15 mL centrifuge tube, add 10 mL of deionized water to submerge the leaves, place the tube in a vacuum desiccator and evacuate for 30 min, then shake on a shaker for 2 h. After removing the centrifuge tube, let it stand for 10 min, and measure the initial conductivity S1 and the blank conductivity (deionized water) S0. Then, place the sample in a 100℃ boiling water bath for 15 min, remove it, cool it for 10 min, and measure the conductivity S2 of the boiled sample.

[0119] The relative electrolyte extravasation rate of the sample is calculated using the following formula: Relative electrolyte extravasation rate (%) = (S1-S0) / (S2-S0) * 100.

[0120] (3) Experimental results Figure 8 The effects of L-ACH and D-ACH soaking treatments on malondialdehyde content and electrolyte permeability in rapeseed seedlings under salt stress. Figure 8 Figure A shows the results of malondialdehyde (MDA) content determination. The MDA content in the L-ACH treatment group was 10.78 ± 0.44 nmol / g FW, and in the D-ACH treatment group it was 11.31 ± 0.53 nmol / g FW, which were significantly lower than the control group (13.95 ± 0.29 nmol / g FW) by 22.70% and 18.92%, respectively. There were significant differences between the L-ACH and D-ACH treatment groups and the control group. This indicates that both L-ACH and D-ACH treatments can effectively alleviate oxidative damage to the membrane system caused by salt stress by inhibiting membrane lipid peroxidation (MDA reduction of 18.92%-22.70%), and their effects are similar.

[0121] Figure 8 Figure B shows the electrolyte permeability measurement results. The permeability of the L-ACH treatment group was 62.36±3.39%, and that of the D-ACH treatment group was 65.27±2.43%, which were significantly lower than those of the control group (72.62±1.70%) by 14.12% and 10.12%, respectively. There were significant differences between the L-ACH and D-ACH treatment groups and the control group. This indicates that both L-ACH and D-ACH treatments can significantly maintain cell membrane integrity by reducing electrolyte extravasation (permeability reduction of 10.12%-14.12%).

[0122] Test Example 10: Effects of L-ACH and D-ACH on the activity of nitrogen assimilates in rapeseed seeds under salt stress (1) Experimental method: The rapeseed seeds were treated according to the experimental method in Test Example 3.

[0123] (2) Test method: Nitrogen assimilation index of rapeseed seedlings grown from treated rapeseed seeds was determined.

[0124] Nitrate reductase (NR) activity assay: Prepare a 1 μg / mL nitrite nitrogen standard solution using sodium nitrite (NaNO2), add the sample according to the manufacturer's instructions, shake well, and incubate at 25℃ for 30 min. Then measure the absorbance at 540 nm. Plot a standard curve or establish a regression equation with the nitrite nitrogen content (μg) in each tube as the x-axis and the absorbance value as the y-axis. Weigh 0.05 g of sample, add 400 μL of extraction buffer, grind using a grinder for 180 seconds (65 Hz), centrifuge at 6000 r / min at 4℃ for 15 min, and the supernatant is the crude enzyme extract. Add 40 μL of the crude enzyme extract to a test tube, then add 120 μL of 0.1 mol / L KNO3 phosphate buffer and 0.4 mL of NADH solution, mix well, and incubate accurately in a 25℃ water bath for 30 min. In the control tube, 0.4 mL of 0.1 mol / L pH 7.5 phosphate buffer was used instead of NADH solution. Immediately after incubation, 1 mL of 1 g / L sulfonamide solution was added to terminate the enzyme reaction. Subsequently, 1 mL of naphthylethyleneamine solution was added to each tube, and after color development for 15 min, the tubes were centrifuged at 6000 r / min for 5 min. The absorbance of the supernatant was measured at 540 nm. The nitrate reductase activity of the samples was calculated based on the amount of nitrite nitrogen (μg) produced in the reaction solution obtained from the standard curve or calculated from the regression equation.

[0125] The nitrate reductase activity of the sample is calculated using the following formula: nitrate reductase activity of the sample [μg / (g·h)] = (x·VT) / (m·VS·t); where x is the content of nitrite nitrogen produced by enzyme catalysis in the reaction solution (μg), VT is the volume of buffer solution added during enzyme extraction (mL), VS is the volume of crude enzyme extract added during the enzyme reaction (mL), m is the sample mass (g), and t is the reaction time (h).

[0126] Assay for glutamine synthase (GS) activity: The "Glutamine Syntheticase (GS) Kit" (G0401W96, Gree Biotech Co., Ltd.) was used, following the manufacturer's instructions. 0.05 g of sample was weighed, 1 mL of extraction buffer was added, and the sample was ground for 180 seconds (65 Hz) using a grinder. The mixture was then transferred to a centrifuge tube. The tube was centrifuged at 12000 r / min and 4℃ for 10 min in a refrigerated centrifuge (D3024R, Cerro Czech). The supernatant was collected and placed on ice for analysis. The sample was added according to the manufacturer's instructions, mixed, and reacted for 2 min. Subsequently, the tube was centrifuged at 8000 r / min and 4℃ for 10 min. 200 μL of the supernatant was transferred to a 96-well plate, and the absorbance (A) was measured at 540 nm. A = A test tube - A control tube (each test tube must have a corresponding control tube).

[0127] One unit of enzyme activity is defined as a change of 0.005 in A540 absorbance per gram of tissue per minute. The glutamine synthase activity of a sample is calculated using the following formula: Glutamine synthase activity of sample (U / g FW) = ( A·V) / (m·V1·t·0.005)=66.7· A / m; where, A represents A determination - A control, V represents the total volume of crude enzyme solution (mL), V1 represents the volume of crude enzyme solution added to the reaction system (mL), m represents the sample mass (g), and t represents the reaction time (h).

[0128] Glutamate synthase (GOGAT) activity assay: The "Glutamate Synthase Activity Assay Kit" (BC0075, Solarbio Biotechnology Co., Ltd.) was used, following the instructions. Weigh 0.05 g of sample, add 1 mL of extraction buffer, and grind using a grinder for 180 seconds (65 Hz). Transfer to a centrifuge tube. Centrifuge at 12000 r / min and 4℃ for 10 min in a refrigerated centrifuge (D3024R, Cerro Czech). Collect the supernatant and place it on ice for testing. Add the sample according to the instructions, mixing thoroughly in a micro-volume quartz cuvette or a 96-well UV plate. Start timing simultaneously with sample addition. Record the initial absorbance A1 at 340 nm wavelength after 20 seconds. After colorimetric measurement, quickly place the micro-volume quartz cuvette and reaction solution into a 25℃ water bath or incubator for an accurate reaction time of 5 min. Quickly remove the quartz cuvette and dry it. Measure the absorbance A2 at 340 nm after 5 min and calculate... A = A1 - A2.

[0129] One unit of enzyme activity is defined as 1 nmol of NADH consumed per gram of tissue per minute. The glutamate synthase activity of a sample is calculated using the following formula: Glutamate synthase activity of sample (U / g FW) = ( A·V·V1) / (m·V2·ε·t;where: A = A1 - A2, V is the total volume of the reaction system, V1 is the volume of the added extract (mL), ε is the molar extinction coefficient of NADH, V2 is the volume of the added sample (mL), m is the sample mass (g), and t is the reaction time (h).

[0130] (3) Experimental results Figure 9 The effects of L-ACH and D-ACH soaking treatments on the nitrogen assimilation enzyme activity of rapeseed seedlings under salt stress. Figure 9Figure A shows the results of nitrate reductase (NR) activity assay. The activity in the L-ACH treatment group was 107.50 ± 5.31 U / g FW, and the activity in the D-ACH treatment group was 104.77 ± 5.41 U / g FW, which were significantly higher than those in the control group (80.92 ± 5.25 U / g FW) by 32.85% and 29.46%, respectively. There were significant differences between the L-ACH and D-ACH treatment groups and the control group.

[0131] Figure 9 Figure B shows the results of glutamine synthase (GS) activity assay. The activity in the L-ACH treatment group was 18.87 ± 1.13 U / g FW, and the activity in the D-ACH treatment group was 17.34 ± 1.63 U / g FW, which were significantly higher than those in the control group (10.37 ± 0.48 U / g FW) by 82.03% and 67.21%, respectively. There were significant differences between the L-ACH and D-ACH treatment groups and the control group.

[0132] Figure 9 Figure C shows the results of glutamate synthase (GOGAT) activity assay. The activity in the L-ACH treatment group was 95.28 ± 5.14 U / g FW, and the activity in the D-ACH treatment group was 92.28 ± 2.29 U / g FW, which were significantly higher than those in the control group (78.27 ± 6.56 U / g FW) by 21.73% and 17.91%, respectively. There were significant differences between the L-ACH and D-ACH treatment groups and the control group.

[0133] In summary, L-ACH and D-ACH soaking treatments significantly enhance the activities of nitrate reductase, glutamine synthase, and glutamate synthase, thereby promoting nitrate nitrogen assimilation and the conversion efficiency of glutamine to glutamate, thus accelerating nitrogen metabolism, alleviating the inhibition of nitrogen metabolism by salt stress, and enhancing the resistance of rapeseed to salt stress.

[0134] The foregoing examples are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are demonstrated by the applicant's actual experimental results. Therefore, the applicant intends that the appended claims are not limited by the selection of examples illustrating the features of the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. The application of nano-cobalt hydroxide to improve the germination rate of rapeseed seeds under salt stress, characterized in that, A solution containing nano-cobalt hydroxide was applied to rapeseed seeds; the concentration of nano-cobalt hydroxide in the solution was 0.008-0.012 g / L.

2. The application according to claim 1, characterized in that, The hydration kinetic diameter of the nano-cobalt hydroxide is 1.4-2.0 nm.

3. The application according to claim 1, characterized in that, The surface charge of the nano-cobalt hydroxide is -2.8mV to -4.0mV.

4. The application according to claim 1, characterized in that, The rapeseed seeds mentioned are Brassica napus seeds.

5. The application according to claim 1, characterized in that, The mass ratio of the nano-cobalt hydroxide to rapeseed seeds is 0.001-0.002:

1.

6. The application according to claim 1, characterized in that, The salt stress was achieved by using a sodium chloride solution with a concentration of 50-300 mM.

7. The application according to claim 1, characterized in that, The preparation method of the nano-cobalt hydroxide includes the following steps: mixing solvent, cobalt source, aspartic acid or glutamic acid evenly, then adding alkaline solution to mix and react, centrifuging and precipitating to obtain the nano-cobalt hydroxide.

8. The application according to claim 7, characterized in that, The cobalt source is at least one of cobalt chloride hexahydrate and cobalt sulfate hexahydrate; and / or the alkaline solution is at least one of sodium hydroxide and potassium hydroxide.

9. The application according to claim 7, characterized in that, The cobalt source has a molar mass ratio of 1-2:1 to aspartic acid or glutamic acid.

10. A method for improving the germination rate of rapeseed seeds under salt stress, characterized in that, A solution containing nano-cobalt hydroxide was applied to rapeseed seeds; the concentration of nano-cobalt hydroxide in the solution was 0.008-0.012 g / L.

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