Method for improving salt and alkali tolerance of rape seed

Abstract

This invention relates to the field of rapeseed treatment technology, specifically a method for improving the salt and alkali tolerance of rapeseed seeds. The method involves immersing the rapeseed seeds in an initiation solution after disinfection and washing. During the initiation solution preparation stage, the ortho-hydroxyl groups of CMC form reversible dynamic bonds with phenylboronic acid, inhibiting particle aggregation and sedimentation. When the seeds are immersed in the initiation solution, the localized hypertonic environment causes conformational contraction of the CMC chain segments, driving effective particle enrichment on the seed coat surface. The contraction and compression of CMC accelerates Zn... 2+ It penetrates into the inner side of the seed coat. Zn 2+ Activates SOD activity and catalyzes O2 ‑ Disproportionation to H₂O₂ promotes proline accumulation. As the H₂O₂ concentration increases, the thioacetate structure undergoes oxidative breakage, triggering the pre-existing Mg in the solution to... 2+ Competition with PEI for adsorption sites on the TiO2 surface significantly reduces the integrity of the coating layer, allowing TiO2 nanoparticles to be released freely from the network.

Description

Technical Field

[0001] This invention belongs to the field of rapeseed treatment technology, specifically a method for improving the salt and alkali tolerance of rapeseed seeds. Background Technology

[0002] Rapeseed is an oilseed crop and a moderately salt-tolerant crop. Cultivating rapeseed in saline-alkali land for seed harvesting or use as feed and vegetables presents a promising development prospect. Studies have shown that TiO2 nanoparticle-induced treatment can significantly improve the germination rate and seedling vigor of rapeseed and other crops under salt stress by regulating the expression of genes related to ion transport in rapeseed. ZnO nanoparticle-induced treatment can preferentially respond to osmotic stress during the germination initiation period by upregulating the accumulation of osmotic regulators (proline, soluble sugars, etc.) and the activity of antioxidant enzymes, effectively alleviating the inhibition of seed water absorption by high salt osmotic pressure.

[0003] However, existing technologies for mixing TiO2 and ZnO nanoparticles for seed initiation treatment face the following technical problems: First, during the preparation of the initiation solution, the two types of nanoparticles agglomerate and settle rapidly due to strong van der Waals forces, resulting in uneven coating of the seed coat and low utilization of active substances. Second, TiO2 and ZnO nanoparticles belong to different salt tolerance regulatory pathways. The former dominates the regulation of ion transport genes, while the latter dominates osmotic regulation and antioxidant enzyme activation. The two types of particles play independent roles and lack cross-activation and signal integration mechanisms between pathways. Finally, the germination process of rapeseed seeds is divided into a germination initiation period (0-18h, mainly driven by osmotic stress response) and a radicle elongation period (18-48h, mainly driven by ion transport regulation). The demand for active substances in these two stages has significant temporal differences, and existing formulations cannot achieve phased and on-demand release. Summary of the Invention

[0004] (1) Technical problems to be solved The purpose of this invention is to provide a method for improving the salt and alkali tolerance of rapeseed, in order to solve the problems of aggregation of TiO2 nanoparticles and ZnO nanoparticles when used for seed initiation treatment and the timing of their action.

[0005] (2) Technical solution To achieve the above objectives, the present invention provides a method for improving the salt and alkali tolerance of rapeseed seeds, comprising the following steps: S1. Preparation of stock solution and stock solution: Dissolve Tris·HCl in deionized water, adjust pH with NaOH, bring to volume, filter and sterilize to obtain Tris·HCl buffer; dissolve MgCl2·6H2O in deionized water, bring to volume, filter and sterilize to obtain MgCl2·6H2O. 2+For the stock solution, the modified ZnO was diluted to a concentration of 1 mg / mL with Tris·HCl buffer and then ultrasonically dispersed to obtain the modified ZnO stock solution; the modified TiO2 was diluted to a concentration of 2 mg / mL with Tris·HCl buffer and then ultrasonically dispersed to obtain the modified TiO2 stock solution. S2. Preparation of the initiating solution: Mg 2+ Add the stock solution to Tris·HCl buffer and stir well. Slowly add the modified ZnO stock solution while stirring. Then slowly add the modified TiO2 stock solution. Make up the volume with Tris·HCl buffer and stir magnetically to obtain the initiation solution. S3. Initiation: Add rapeseed seeds to NaClO solution, soak and disinfect, filter, wash with distilled water, absorb surface moisture with sterile filter paper, immerse in initiation solution, and incubate at a constant temperature in the dark. S4. Post-treatment: After initiation, the rapeseed seeds are blotted with filter paper to remove surface liquid, spread evenly on filter paper, and placed in a clean environment to dry again, thus obtaining salt- and alkali-tolerant rapeseed seeds. The modified TiO2 is TiO2 nanoparticles coated with polyethyleneimine and grafted with thioacetal and phenylboronic acid; The modified ZnO is a carboxymethyl cellulose-grafted ZnO nanoparticle microgel.

[0006] Furthermore, the final concentration of the modified ZnO stock solution in S2 is 50~100 mg / L, and the final concentration of the modified TiO2 stock solution is 100~200 mg / L.

[0007] Furthermore, the initiation time in S3 is 12~14h.

[0008] Furthermore, the preparation method of the modified TiO2 includes the following steps: The preparation method of modified TiO2 includes the following steps: S11. Anhydrous ZnCl2 was dispersed in anhydrous toluene, and acetylpropionic acid and 1,2-ethylenedithiol were added sequentially. Under nitrogen protection, the mixture was heated and refluxed. The reaction solution was washed sequentially with saturated NaHCO3 solution and deionized water. The organic phase was collected, dried with anhydrous Na2SO4, filtered, and rotary evaporated to obtain the first compound. S12. Dissolve 4-aminophenylboronic acid salt in anhydrous DMF, add N,N-diisopropylethylamine, stir at room temperature to obtain an aminophenylboronic acid solution; dissolve the first compound in anhydrous DMF, add EDC·HCl and NHS, activate by stirring in an ice bath, slowly add aminophenylboronic acid solution, heat, stir reaction, evaporate the resulting reaction solution by rotary evaporation, dissolve the product in ethyl acetate, wash successively with citric acid aqueous solution, saturated NaHCO3 solution, and saturated NaCl solution, collect the organic phase, dry with anhydrous Na2SO4, filter, evaporate by rotary evaporation, and purify by silica gel column chromatography to obtain the second compound; S13. Dissolve the second compound in MES buffer, add EDC·HCl and NHS, stir to activate, and obtain the third compound; S14. Disperse TiO2 nanoparticles ultrasonically in deionized water, add polyethyleneimine, continue ultrasonic dispersion, collect the solid by centrifugation, wash with deionized water, resuspend in deionized water, add the third compound, add MES buffer, adjust pH with NaOH, stir the reaction, collect the solid by centrifugation of the obtained reaction solution, wash with deionized water, and obtain modified TiO2.

[0009] Furthermore, the preparation method of the modified ZnO includes the following steps: S21. Dissolve Zn(NO3)2·6H2O and NaOH in deionized water respectively, mix the two solutions, perform hydrothermal reaction, cool and centrifuge, collect the precipitate, wash alternately with deionized water and anhydrous ethanol, and vacuum dry to obtain ZnO nanoparticles. S22. ZnO nanoparticles were ultrasonically dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane was added, and the reaction was refluxed under nitrogen protection. The precipitate was collected by centrifugation, washed with anhydrous ethanol and deionized water, and dried under vacuum to obtain NH2-ZnO nanoparticles. S23. Dissolve sodium carboxymethyl cellulose in PBS buffer with stirring, add EDC·HCl and NHS, stir to activate, and obtain an activated solution; disperse NH2-ZnO nanoparticles in PBS buffer with ultrasonication, slowly add them dropwise to the activated solution, stir to react, and dialysis to purify the resulting reaction solution to obtain CMC-g-ZnO solution; S24. Dissolve anhydrous CaCl2 in deionized water and slowly add it dropwise to CMC-g-ZnO solution while stirring. Allow it to stand for crosslinking, centrifuge to collect the precipitate, and wash it with deionized water to obtain modified ZnO.

[0010] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are: 1. The ortho-hydroxyl groups on the CMC backbone of modified ZnO form reversible dynamic bonds with the phenylboronic acid in modified TiO2, causing both types of particles to self-assemble using the CMC chain as a backbone, thereby inhibiting the aggregation and sedimentation of both types of particles. The area around the seed coat is affected by Na...+ The concentration gradient creates a local hyperpermeable environment, causing the CMC chain segments to undergo conformational shrinkage. This drives the modified ZnO, along with the modified TiO2 it carries, to be effectively enriched on the seed coat surface, achieving high-salt-triggered site-specific deposition of active particles.

[0011] 2. CMC shrinkage extrusion makes the modified ZnO more tightly bonded to the seed coat surface, Zn 2+ Driven by a concentration gradient, Zn penetrates into the inner side of the seed coat. 2+ Synergistic endogenous Cu 2+ Maintaining the catalytic function of SOD enzymes and promoting O2 - Disproportionation to H₂O₂ simultaneously activates P5CS, a key enzyme in proline synthesis, promoting proline accumulation. As the H₂O₂ concentration increases, the thioketal structure undergoes oxidative breakage, and the boronic ester bonds dissociate, significantly reducing the integrity of the PEI coating layer and triggering the release of pre-existing Mg in the solution. 2+ It can partially shield the electrostatic interaction between TiO2 and PEI, further promoting the dissociation of the PEI coating layer, thereby allowing TiO2 nanoparticles to be released freely from the network. The released TiO2 penetrates the seed coat and enters the radicle region, improving Na+ by regulating reactive oxygen species homeostasis and ion transport gene expression. + / K + balance.

[0012] 3. The modified TiO2 is coated with PEI and remains in a closed state during the germination initiation period, inhibiting photocatalytic activity and reducing the risk of close-range oxidative damage to seed coat cells. Release is triggered only after the oxidative microenvironment is activated. The introduction of thioacetate into the modified TiO2 lowers the H2O2 response threshold compared to phenylboronic acid alone, ensuring that TiO2 release is triggered in the early stages of germination. Attached Figure Description

[0013] Figure 1 This is a comparison diagram of the germination of rapeseed seeds under mild and moderate salt stress according to the present invention. Detailed Implementation

[0014] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0015] Example 1: This example discloses a method for improving the salt and alkali tolerance of rapeseed seeds, including the following steps: S1. Preparation of stock solution and stock solution: Dissolve 7.88g Tris·HCl in 800mL deionized water, adjust pH to 8.0 with 1M NaOH, bring volume to 1000mL with deionized water, filter and sterilize through a 0.22μm filter membrane to obtain Tris·HCl buffer; Dissolve 20.33g MgCl2·6H2O in deionized water, bring volume to 100mL, filter and sterilize through a 0.22μm filter membrane to obtain Mg 2+ For the stock solution, the modified ZnO was diluted to a concentration of 1 mg / mL with Tris·HCl buffer and ultrasonically dispersed for 5 min to obtain the modified ZnO stock solution; the modified TiO2 was diluted to a concentration of 2 mg / mL with Tris·HCl buffer and ultrasonically dispersed for 5 min to obtain the modified TiO2 stock solution. S2. Preparation of the initiation solution: Add 5 mL of Mg 2+ Add the stock solution to 800 mL Tris·HCl buffer and stir well. Slowly add 75 mL modified ZnO stock solution while stirring. Then slowly add 75 mL modified TiO2 stock solution. Adjust the volume to 1000 mL with Tris·HCl buffer and stir magnetically at room temperature for 1 h to obtain the initiation solution. S3. Initiation: Add 100g of rapeseed seeds to 0.1% NaClO solution, soak and disinfect for 5min, filter, wash with distilled water, absorb the surface moisture of the disinfected seeds with sterile filter paper, immerse in the initiation solution, and incubate at 20℃ for 14h in the dark. S4. Post-treatment: After initiation, the rapeseed seeds are blotted with filter paper to remove surface liquid, spread evenly on filter paper, and placed in a clean environment at 20℃ and 45% relative humidity for 24~48 hours to re-dry, thus obtaining salt-tolerant rapeseed seeds. The modified TiO2 is TiO2 nanoparticles coated with polyethyleneimine and grafted with thioacetal and phenylboronic acid; The modified ZnO is a carboxymethyl cellulose-grafted ZnO nanoparticle microgel.

[0016] The final concentration of the S2-modified ZnO stock solution is 75 mg / L, and the final concentration of the modified TiO2 stock solution is 150 mg / L.

[0017] The initiation time in S3 is 14 hours.

[0018] The preparation method of the modified TiO2 includes the following steps: S11. Disperse 7 mg of anhydrous ZnCl2 in 30 mL of anhydrous toluene, add 1.61 g of levulinic acid and 0.94 g of 1,2-ethylenedithiol sequentially, purge with nitrogen, heat to 110 °C, reflux for 12 h, and wash the reaction solution sequentially with saturated NaHCO3 solution and deionized water. Collect the organic phase, dry with anhydrous Na2SO4, filter, and rotary evaporate to obtain the first compound. S12. Dissolve 1.90 g of 4-aminophenylboronic acid salt in 15 mL of anhydrous DMF, add 1.42 g of N,N-diisopropylethylamine, and stir at room temperature for 5 min to obtain an aminophenylboronic acid solution; dissolve 1.76 g of the first compound in 10 mL of anhydrous DMF, add 2.11 g of EDC·HCl and 1.27 g of NHS, stir in an ice bath for 30 min to activate, slowly add the aminophenylboronic acid solution, heat to room temperature, stir and react for 24 h, evaporate the resulting reaction solution by rotary evaporation, dissolve the product in 50 mL of ethyl acetate, wash successively with 5% citric acid aqueous solution, saturated NaHCO3 solution, and saturated NaCl solution, collect the organic phase, dry with anhydrous Na2SO4, filter, evaporate by rotary evaporation, and purify by silica gel column chromatography to obtain the second compound; S13. Dissolve 590 mg of the second compound in 15 mL of LME buffer, add 460 mg of EDC·HCl and 276 mg of NHS, and activate at room temperature for 2 h to obtain the third compound; S14. 200 mg TiO2 nanoparticles were ultrasonically dispersed in 20 mL of deionized water. 100 mg of polyethyleneimine was added, and ultrasonic dispersion was continued for 30 min. The solid was collected by centrifugation, washed with deionized water, and resuspended in 10 mL of deionized water. The third compound was added, and MES buffer was added to a total volume of 20 mL. The pH was adjusted to 7.4 with 0.1 M NaOH, and the reaction was stirred at room temperature for 12 h. The solid obtained from the reaction solution was collected by centrifugation and washed with deionized water to obtain modified TiO2.

[0019] The method for preparing the modified ZnO includes the following steps: S21. Dissolve 2.97g Zn(NO3)2·6H2O and 0.8g NaOH in 20mL of deionized water, respectively. Mix the two solutions and react hydrothermally at 150℃ for 12h. After cooling, centrifuge, collect the precipitate, wash it alternately with deionized water and anhydrous ethanol, and dry it under vacuum at 60℃ for 12h to obtain ZnO nanoparticles. S22. 100 mg ZnO nanoparticles were ultrasonically dispersed in 30 mL of anhydrous ethanol, and 200 μL of 3-aminopropyltriethoxysilane was added. The mixture was refluxed at 60 °C for 12 h under nitrogen protection. The precipitate was collected by centrifugation, washed with anhydrous ethanol and deionized water, and dried under vacuum at 60 °C for 6 h to obtain NH2-ZnO nanoparticles. S23. Dissolve 500 mg sodium carboxymethyl cellulose in 25 mL PBS buffer, add 192 mg EDC·HCl and 115 mg NHS, and activate at room temperature for 30 min to obtain the activation solution; disperse 50 mg NH2-ZnO nanoparticles in 5 mL PBS buffer by ultrasonication, slowly add them dropwise to the activation solution, stir at room temperature for 4 h, and purify the reaction solution by dialyzing for 48 h to obtain CMC-g-ZnO solution; S24. Dissolve 20 mg of anhydrous CaCl2 in 1 mL of deionized water, and slowly add it dropwise to CMC-g-ZnO solution while stirring. Let it stand at 4 °C for 12 h for crosslinking, collect the precipitate by centrifugation, and wash it with deionized water to obtain modified ZnO.

[0020] Example 2: This example is based on Example 1, but differs from Example 1 in that the final concentration of the modified ZnO stock solution in S2 is 50 mg / L and the final concentration of the modified TiO2 stock solution is 100 mg / L.

[0021] The other components and preparation methods are the same as in Example 1.

[0022] Example 3: This example is based on Example 1, but differs from Example 1 in that the final concentration of the modified ZnO stock solution in S2 is 100 mg / L and the final concentration of the modified TiO2 stock solution is 200 mg / L.

[0023] The other components and preparation methods are the same as in Example 1.

[0024] Example 4: This example is based on Example 1, but differs from Example 1 in that the initiation time in S3 of this example is 12 hours.

[0025] The other components and preparation methods are the same as in Example 1.

[0026] Comparative Example 1: This comparative example is based on Example 1, but differs from Example 1 in that the modified TiO2 in this comparative example does not contain polyethyleneimine.

[0027] The preparation method of the modified TiO2 includes the following steps: S11. Disperse 7 mg of anhydrous ZnCl2 in 30 mL of anhydrous toluene, add 1.61 g of levulinic acid and 0.94 g of 1,2-ethylenedithiol sequentially, purge with nitrogen, heat to 110 °C, reflux for 12 h, and wash the reaction solution sequentially with saturated NaHCO3 solution and deionized water. Collect the organic phase, dry with anhydrous Na2SO4, filter, and rotary evaporate to obtain the first compound. S12. Dissolve 1.90 g of 4-aminophenylboronic acid salt in 15 mL of anhydrous DMF, add 1.42 g of N,N-diisopropylethylamine, and stir at room temperature for 5 min to obtain an aminophenylboronic acid solution; dissolve 1.76 g of the first compound in 10 mL of anhydrous DMF, add 2.11 g of EDC·HCl and 1.27 g of NHS, stir in an ice bath for 30 min to activate, slowly add the aminophenylboronic acid solution, heat to room temperature, stir and react for 24 h, evaporate the resulting reaction solution by rotary evaporation, dissolve the product in 50 mL of ethyl acetate, wash successively with 5% citric acid aqueous solution, saturated NaHCO3 solution, and saturated NaCl solution, collect the organic phase, dry with anhydrous Na2SO4, filter, evaporate by rotary evaporation, and purify by silica gel column chromatography to obtain the second compound; S13. Dissolve 590 mg of the second compound in 15 mL of LME buffer, add 460 mg of EDC·HCl and 276 mg of NHS, and activate at room temperature for 2 h to obtain the third compound; S14. 200 mg TiO2 nanoparticles were ultrasonically dispersed in 10 mL of deionized water, the third compound was added, and MES buffer was added to bring the total volume to 20 mL. The pH was adjusted to 7.4 with 0.1 M NaOH, and the reaction was stirred at room temperature for 12 h. The resulting reaction solution was centrifuged to collect the solid, which was then washed with deionized water to obtain modified TiO2.

[0028] The other components and preparation methods are the same as in Example 1.

[0029] Comparative Example 2: This comparative example is based on Example 1, but differs from Example 1 in that the modified TiO2 described in this comparative example does not contain thioacetal.

[0030] The preparation method of the modified TiO2 includes the following steps: S11. Dissolve 332 mg of 4-carboxyphenylboronic acid in MES buffer, add 460 mg of EDC·HCl and 276 mg of NHS, and activate at room temperature for 2 h to obtain the fourth compound; S12. 200 mg TiO2 nanoparticles were ultrasonically dispersed in 20 mL of deionized water, 100 mg polyethyleneimine was added, and ultrasonic dispersion was continued for 30 min. The solid was collected by centrifugation, washed with deionized water, resuspended in 10 mL of deionized water, the fourth compound was added, and MES buffer was added to a total volume of 20 mL. The pH was adjusted to 7.4 with 0.1 M NaOH, and the reaction was stirred at room temperature for 12 h. The solid obtained from the reaction solution was collected by centrifugation and washed with deionized water to obtain modified TiO2.

[0031] The other components and preparation methods are the same as in Example 1.

[0032] Comparative Example 3: This comparative example is based on Example 1, but differs from Example 1 in that the modified TiO2 described in this comparative example does not contain phenylboronic acid.

[0033] The preparation method of the modified TiO2 includes the following steps: S11. Disperse 7 mg of anhydrous ZnCl2 in 30 mL of anhydrous toluene, add 1.61 g of levulinic acid and 0.94 g of 1,2-ethylenedithiol sequentially, purge with nitrogen, heat to 110 °C, reflux for 12 h, and wash the reaction solution sequentially with saturated NaHCO3 solution and deionized water. Collect the organic phase, dry with anhydrous Na2SO4, filter, and rotary evaporate to obtain the first compound. S12. Dissolve 352 mg of the first compound in 15 mL of MES buffer containing 10% DMSO, add 460 mg EDC·HCl and 276 mg NHS, and stir at room temperature for 2 h to obtain the fifth compound; S13. 200 mg TiO2 nanoparticles were ultrasonically dispersed in 20 mL of deionized water, 100 mg polyethyleneimine was added, and ultrasonic dispersion was continued for 30 min. The solid was collected by centrifugation, washed with deionized water, resuspended in 10 mL of deionized water, compound 5 was added, and MES buffer was added to a total volume of 20 mL. The pH was adjusted to 7.4 with 0.1 M NaOH, and the reaction was stirred at room temperature for 12 h. The solid obtained from the reaction solution was collected by centrifugation and washed with deionized water to obtain modified TiO2.

[0034] The other components and preparation methods are the same as in Example 1.

[0035] Comparative Example 4: This comparative example is based on Example 1, but differs from Example 1 in that the modified ZnO in this comparative example is not modified, that is, ZnO nanoparticles are used instead of modified ZnO.

[0036] The other components and preparation methods are the same as in Example 1.

[0037] Comparative Example 5: This comparative example differs from Example 1 in that Mg is not added to the initiating solution. 2+ .

[0038] The other components and preparation methods are the same as in Example 1.

[0039] Comparative Example 6: This comparative example is based on Example 1, but unlike Example 1, no modified TiO2 is added to the initiation solution of this comparative example.

[0040] The other components and preparation methods are the same as in Example 1.

[0041] Comparative Example 7: This comparative example is based on Example 1, but unlike Example 1, no modified ZnO is added to the initiating solution of this comparative example.

[0042] The other components and preparation methods are the same as in Example 1.

[0043] Comparative Example 8: This comparative example is based on Example 1, but differs from Example 1 in that TiO2 nanoparticles and ZnO nanoparticles are used instead of modified TiO2 and modified ZnO in the initiation solution of this comparative example.

[0044] The other components and preparation methods are the same as in Example 1.

[0045] Comparative Example 9: This comparative example is based on Example 1, but unlike Example 1, this comparative example does not add modified TiO2 and modified ZnO.

[0046] This comparative example discloses a method for improving the salt and alkali tolerance of rapeseed seeds, including the following steps: S1. Preparation of initiation solution: Dissolve 7.88g Tris·HCl monohydrate in 800mL of deionized water, adjust the pH to 8.0 with 1M NaOH, bring the volume to 1000mL with deionized water, filter and sterilize with a 0.22μm filter membrane to obtain the initiation solution; S3. Initiation: Add 100g of rapeseed seeds to 0.1% NaClO solution, soak and disinfect for 5min, filter, wash with distilled water, absorb the surface moisture of the disinfected seeds with sterile filter paper, immerse in the initiation solution, and incubate at 20℃ for 14h in the dark. S4. Post-treatment: After initiation, the rapeseed seeds are blotted with filter paper to remove surface liquid, spread evenly on filter paper, and placed in a clean environment at 20℃ and 45% relative humidity for 24~48 hours to re-dry, thus obtaining salt-tolerant rapeseed seeds.

[0047] Experimental verification: Experiment 1: The initiating solution of Example 1 and Comparative Example 8 (unmodified nanoparticle mixture) were prepared at the same concentration and allowed to stand at room temperature. The supernatant was collected at 0, 1, 2, 4, 8, and 12 hours and the absorbance (OD) was measured at 600 nm. 600 Each group was set up with 3 parallel repeated experiments.

[0048] Table 1: Results of the comparative test on particle dispersion stability:

[0049] Table 1 shows the results of the particle dispersion stability comparison test. After standing for 12 hours, the OD of the initiating liquid in Example 1... 600The retention rate was greater than 90%, while that of control 8 was less than 20%, indicating that the dynamic bond network has a significant inhibitory effect on the aggregation and sedimentation of nanoparticles. The initiating solution can maintain stable dispersion within the 12-14h initiation window, ensuring uniform coating on the seed coat surface.

[0050] Experiment 2: Seeds treated in each group were placed in petri dishes (9 cm in diameter) lined with double-layered filter paper. 5 mL of 50 mM NaCl solution (simulating mild salt stress) and 5 mL of 100 mM NaCl solution (simulating moderate salt stress) were added, respectively. The dishes were incubated in a 20°C light incubator with a 12-hour / 12-hour light / dark cycle, with the solution replenished daily to the initial volume. Germination was defined as the radicle breaking through the seed coat by 1 mm. The number of germinations was recorded for 7 consecutive days, with each group having three replicates.

[0051] Table 2: Germination rate of rapeseed seeds under mild and moderate salt stress:

[0052] Table 2 shows the germination rates of rapeseed seeds under mild and moderate salt stress. Example 1 showed the highest germination rate, significantly better than all comparative examples. Comparative example 8 (unmodified mixed group) had a lower germination rate than comparative examples 6 and 7 (using modified particles alone) due to poor aggregation and sedimentation. Comparative example 9, as a blank control, had the lowest germination rate. Figure 1 The figure shows a comparison of the germination rate of rapeseed seeds in Example 1 (a) and Comparative Example 9 (b) under mild salt stress. As can be seen from the figure, the germination rate of Example 1 is significantly higher than that of Comparative Example 9.

[0053] Experiment 3: After culturing seedlings under moderate salt stress in Experiment 2 for seven days, 20 seedlings from each group were randomly selected. Radicle length was measured, and root tissue was collected to determine SOD activity (nitrocyanate tetrazolium method), proline content (acidic ninhydrin method), and Na+ content. + / K + The ratio (atomic absorption spectrophotometry) was used, and three parallel replicates were set up for each group.

[0054] Table 3: Results of seedling morphological index determination:

[0055] Table 3 shows the results of seedling morphological index determination. The root length, SOD activity, and proline content of the example group were significantly higher than those of the control group. + / K + The ratio was significantly lower than that of the comparative example, with Example 1 showing the best results.

[0056] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, and improvements made by those skilled in the art 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 improving the salt and alkali tolerance of rapeseed seeds, characterized in that, Includes the following steps: S1. Preparation of stock solution and stock solution: Dissolve Tris·HCl in deionized water, adjust pH with NaOH, bring to volume, filter and sterilize to obtain Tris·HCl buffer; dissolve MgCl2·6H2O in deionized water, bring to volume, filter and sterilize to obtain MgCl2·6H2O. 2+ For the stock solution, the modified ZnO was diluted to a concentration of 1 mg / mL with Tris·HCl buffer and then ultrasonically dispersed to obtain the modified ZnO stock solution; the modified TiO2 was diluted to a concentration of 2 mg / mL with Tris·HCl buffer and then ultrasonically dispersed to obtain the modified TiO2 stock solution. S2. Preparation of the initiating solution: Mg 2+ Add the stock solution to Tris·HCl buffer and stir well. Slowly add the modified ZnO stock solution while stirring. Then slowly add the modified TiO2 stock solution. Make up the volume with Tris·HCl buffer and stir magnetically to obtain the initiation solution. S3. Initiation: Add rapeseed seeds to NaClO solution, soak and disinfect, filter, wash with distilled water, absorb surface moisture with sterile filter paper, immerse in initiation solution, and incubate at a constant temperature in the dark. S4. Post-treatment: After initiation, the rapeseed seeds are blotted with filter paper to remove surface liquid, spread evenly on filter paper, and placed in a clean environment to dry again, thus obtaining salt- and alkali-tolerant rapeseed seeds. The modified TiO2 is TiO2 nanoparticles coated with polyethyleneimine and grafted with thioacetal and phenylboronic acid; The modified ZnO is a carboxymethyl cellulose-grafted ZnO nanoparticle microgel.

2. The method for improving the salt and alkali tolerance of rapeseed according to claim 1, characterized in that, The final concentration of the modified ZnO stock solution in S2 is 50~100 mg / L, and the final concentration of the modified TiO2 stock solution is 100~200 mg / L.

3. The method for improving the salt and alkali tolerance of rapeseed according to claim 1, characterized in that, The initiation time in S3 is 12-14 hours.

4. The method for improving the salt and alkali tolerance of rapeseed according to claim 1, characterized in that, The preparation method of the modified TiO2 includes the following steps: S11. Anhydrous ZnCl2 was dispersed in anhydrous toluene, and acetylpropionic acid and 1,2-ethylenedithiol were added sequentially. Under nitrogen protection, the mixture was heated and refluxed. The reaction solution was washed sequentially with saturated NaHCO3 solution and deionized water. The organic phase was collected, dried with anhydrous Na2SO4, filtered, and rotary evaporated to obtain the first compound. S12. Dissolve 4-aminophenylboronic acid salt in anhydrous DMF, add N,N-diisopropylethylamine, stir at room temperature to obtain an aminophenylboronic acid solution; dissolve the first compound in anhydrous DMF, add EDC·HCl and NHS, activate by stirring in an ice bath, slowly add aminophenylboronic acid solution, heat, stir reaction, evaporate the resulting reaction solution by rotary evaporation, dissolve the product in ethyl acetate, wash successively with citric acid aqueous solution, saturated NaHCO3 solution, and saturated NaCl solution, collect the organic phase, dry with anhydrous Na2SO4, filter, evaporate by rotary evaporation, and purify by silica gel column chromatography to obtain the second compound; S13. Dissolve the second compound in MES buffer, add EDC·HCl and NHS, stir to activate, and obtain the third compound; S14. Disperse TiO2 nanoparticles ultrasonically in deionized water, add polyethyleneimine, continue ultrasonic dispersion, collect the solid by centrifugation, wash with deionized water, resuspend in deionized water, add the third compound, add MES buffer, adjust pH with NaOH, stir the reaction, collect the solid by centrifugation of the obtained reaction solution, wash with deionized water, and obtain modified TiO2.

5. The method for improving the salt and alkali tolerance of rapeseed according to claim 1, characterized in that, The method for preparing the modified ZnO includes the following steps: S21. Dissolve Zn(NO3)2·6H2O and NaOH in deionized water respectively, mix the two solutions, perform hydrothermal reaction, cool and centrifuge, collect the precipitate, wash alternately with deionized water and anhydrous ethanol, and vacuum dry to obtain ZnO nanoparticles. S22. ZnO nanoparticles were ultrasonically dispersed in anhydrous ethanol, 3-aminopropyltriethoxysilane was added, and the reaction was refluxed under nitrogen protection. The precipitate was collected by centrifugation, washed with anhydrous ethanol and deionized water, and dried under vacuum to obtain NH2-ZnO nanoparticles. S23. Dissolve sodium carboxymethyl cellulose in PBS buffer with stirring, add EDC·HCl and NHS, stir to activate, and obtain an activated solution; disperse NH2-ZnO nanoparticles in PBS buffer with ultrasonication, slowly add them dropwise to the activated solution, stir to react, and dialysis to purify the resulting reaction solution to obtain CMC-g-ZnO solution; S24. Dissolve anhydrous CaCl2 in deionized water and slowly add it dropwise to CMC-g-ZnO solution while stirring. Allow it to stand for crosslinking, centrifuge to collect the precipitate, and wash it with deionized water to obtain modified ZnO.

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