A method for accelerating germination of pepper seeds

Abstract

The present application relates to the field of agricultural planting technology, and specifically relates to a pepper seed germination accelerating method, which comprises the following steps: S1. seed soaking liquid preparation: component A is added to deionized water; the component A comprises the following components in weight fractions: 55-65 parts of polyethylene glycol, 5-7 parts of potassium nitrate, 0.2-0.4 parts of zinc sulfate heptahydrate; S2. pretreatment: dry pepper seeds are poured into the prepared seed soaking liquid, and then soaked and drained; S3. coating agent preparation: component B is added to deionized water; the component B comprises the following components in weight fractions: 20-25 parts of modified sodium polyacrylate, 3-5 parts of modified glycine betaine, 3-5 parts of sodium alginate, and 2-4 parts of potassium fulvate; S4. coating treatment: after seed dressing, the coated pepper seeds are obtained after drying. The present application solves the problem of weak oxidative damage defense line of glycine betaine under the stress of soil salinization caused by dry climate.

Description

Technical Field

[0001] This invention relates to the field of agricultural planting technology, specifically a method for germinating chili seeds. Background Technology

[0002] Under the stress of soil salinization caused by drought, excessive sodium and chloride ions migrate into seed cells, disrupting ion homeostasis and directly inhibiting the activity of key metabolic enzymes. Simultaneously, these ions interfere with the electron transport chains of mitochondria and plasma membranes, inducing the generation of reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide, and hydroxyl radicals. During the early stages of germination, seeds transition from a dormant state to a highly metabolic state, accompanied by membrane system reconstruction, enzyme activation, and the initiation of DNA replication. This series of processes makes the endogenous antioxidant defense system extremely vulnerable, and cell membrane lipids, functional proteins, and DNA are easily damaged by ROS.

[0003] In current technology, glycine betaine, as an endogenous compatible solute, is an important biochemical protectant. Under salt and alkali stress, glycine betaine molecules stabilize the native conformation of proteins and the phospholipid bilayer structure of biological membranes through a "preferential repulsion" effect, thereby buffering the physicochemical damage directly caused by this stress. However, the molecular structure of glycine betaine lacks active functional groups that can provide electrons or hydrogen atoms, and therefore cannot directly participate in redox reactions that quench free radicals. Thus, in the early stages of seed germination, the glycine betaine oxidative damage defense line is weak. Summary of the Invention

[0004] (1) Technical problems to be solved

[0005] The purpose of this invention is to provide a method for germinating chili seeds, which addresses the problem of weak oxidative damage defenses of glycine betaine under soil salinization stress caused by drought.

[0006] (2) Technical solution

[0007] A method for germinating chili seeds includes the following steps:

[0008] S1. Preparation of soaking solution: Add component A to deionized water and stir continuously until completely dissolved to obtain component A soaking solution; component A is a composite soaking agent, comprising the following components in parts by weight: 55–65 parts polyethylene glycol, 5–7 parts potassium nitrate, and 0.2–0.4 parts zinc sulfate heptahydrate;

[0009] S2. Pretreatment: Place the dried chili seeds in a soaking container, pour in the soaking solution of component A, and let them soak at room temperature and under light conditions to obtain soaked chili seeds; drain the soaked chili seeds to obtain pretreated chili seeds;

[0010] S3. Preparation of coating agent: Component B is added to deionized water while stirring. After stirring evenly, a gel-like slurry is obtained. After standing, a gel-like coating agent is obtained. Component B is a composite coating dry powder, comprising the following components in parts by weight: 20-25 parts modified sodium polyacrylate, 3-5 parts modified glycine betaine, 3-5 parts sodium alginate, and 2-4 parts potassium humate. The modified sodium polyacrylate is sulfonic acid-modified sodium polyacrylate, and the modified glycine betaine is polyhydroxy modified glycine betaine.

[0011] S4. Coating treatment: Mix the pretreated chili seeds with the gel-like coating agent and mix them from bottom to top, turning them over. After mixing, dry the seeds. When the coating on the seed surface is completely dry, non-sticky, and in a dispersed granular state, the coated chili seeds are obtained, and the germination treatment of the chili seeds is completed.

[0012] Furthermore, in step S1, the volume (mL) of deionized water is in a ratio of 3:1 to the mass (g) of component A.

[0013] Furthermore, in step S3, the ratio of the volume (mL) of deionized water to the mass (g) of component B is 0.6:1.

[0014] Furthermore, the modified sodium polyacrylate is prepared by the following method:

[0015] S11. Add deionized water to the reactor, turn on the stirrer and introduce high-purity nitrogen gas; add sodium hydroxide solution dropwise to acrylic acid while cooling and stirring in an ice-water bath to obtain mixed solution A;

[0016] S12. Transfer mixed solution A to the reactor described above, then add 2-acrylamide-2-methylpropanesulfonic acid monomer and N,N'-methylenebisacrylamide in sequence, and stir until completely dissolved to obtain mixed solution B;

[0017] S13. Heat the mixed solution B, add ammonium persulfate solution dropwise to obtain mixed solution C, stir, and when mixed solution C can be stretched into continuous gel filaments without breaking, a gel is initially formed. Then heat the solution to react and obtain a hydrated gel.

[0018] S14. Cut the hydrated gel into pieces and soak them in anhydrous ethanol. Replace the ethanol with anhydrous ethanol for soaking. When the conductivity of the soaking solution is measured twice consecutively and is less than 5 μS / cm, and the pH value is stable between 6.5 and 7.5, an alcohol gel is obtained. Dry the alcohol gel to obtain a dry hard solid. Grind the dry hard solid and sieve it to obtain modified sodium polyacrylate.

[0019] Furthermore, the mass-to-volume ratio of ammonium persulfate to water in the ammonium persulfate solution is 5 g / 100 mL.

[0020] Furthermore, the modified glycine betaine is prepared by the following method:

[0021] S21. Tris(hydroxymethyl)aminomethane, 1-bromododecane, sodium iodide and anhydrous potassium carbonate are added to a reactor, and then N,N-dimethylformamide is added to the reactor as a solvent; stirring is started, and the reaction is heated under nitrogen protection. After the reaction is completed, the mixture is cooled to room temperature and filtered to obtain filtrate A.

[0022] S22. Pour filtrate A into ice water, extract the aqueous phase with ethyl acetate, wash the organic phase with saturated brine, dry the organic phase with anhydrous sodium sulfate, filter, and distill under reduced pressure to obtain the crude product; purify the crude product with an eluent to obtain oily substance B;

[0023] S23. Add the obtained oily substance B to anhydrous ethanol, and under ice-water bath cooling and stirring, obtain mixed solution D. Add the first mixed solvent dropwise to mixed solution D. After the addition is complete, obtain mixed solution E.

[0024] S24. Remove the ice bath, heat the mixed solution E to 70-75℃ and stir to react. After the reaction is complete, cool the mixed solution E to room temperature, distill under reduced pressure to obtain a viscous residue. Add anhydrous ethanol to the residue, stir to dissolve, filter while hot to obtain filtrate B. Cool filtrate B, let it stand, and filter under suction to obtain crystal A.

[0025] S25. Wash crystal A with anhydrous ethanol, then wash with the second mixed solvent to obtain crystal B. Dry crystal B to obtain modified glycine betaine.

[0026] Furthermore, the eluent in step S22 is composed of ethyl acetate and methanol, with a volume ratio of ethyl acetate to methanol of 10:1.

[0027] Furthermore, the first mixed solvent is obtained by dissolving sodium chloroacetate and sodium hydroxide in deionized water.

[0028] Furthermore, the second mixed solvent is composed of ethyl acetate and n-hexane in a volume ratio of 1:1.

[0029] (3) Beneficial effects

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] 1. The modified glycine betaine in this invention introduces a hydroxyl group into its molecule. The hydroxyl group acts as a hydrogen atom donor, reducing hydroxyl radicals to stable products through a hydrogen atom transfer reaction, thus eliminating their ability to attack biomolecules and inhibiting the oxidative damage of reactive oxygen species to cell membrane lipids, functional proteins, and DNA. The oxygen atom in the hydroxyl group contains a lone pair of electrons, which can chelate free transition metal ions in the cell, reducing the activity of these ions in catalyzing the Fenton reaction and reducing the generation of hydroxyl radicals from the source. At the same time, its fully retained "preferential repulsion" effect continues to maintain the stability of protein and membrane structures.

[0032] 2. The modified sodium polyacrylate in this invention overcomes the failure problem of sodium polyacrylate under salt and alkali stress by introducing strongly ionized sulfonate groups. The sulfonate and carboxylate groups together form a high-capacity ion exchange network, which preferentially adsorbs and fixes harmful ions such as sodium ions, forming a chemical buffer zone around the seed, thereby reducing the initial ion signal that triggers the outbreak of reactive oxygen species from the physicochemical source. Attached Figure Description

[0033] Figure 1 This is a flowchart illustrating the preparation process of a method for germinating chili seeds.

[0034] Figure 2 This is a graph showing the germination potential of chili seeds in Example 1 and Comparative Example 9 on day 4.

[0035] Figure 3 The image shows the growth of chili seedlings in Examples 1-3 and Comparative Examples 1-2 on day 30. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention; that is, the described embodiments are only a part of the embodiments of the invention, not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0037] It should be noted that relational terms such as "first" and "second" are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0038] Example 1: As Figure 1 As shown in the figure, a preferred embodiment of the present invention provides a method for germinating chili seeds, comprising the following steps:

[0039] S1. Preparation of soaking solution: Add 100g of component A to 300mL of deionized water at 28℃ and stir continuously until completely dissolved to obtain component A soaking solution; Component A is a composite soaking agent, comprising the following components in parts by weight: 60 parts polyethylene glycol, 6 parts potassium nitrate, and 0.3 parts zinc sulfate heptahydrate;

[0040] S2. Pretreatment: Place 100g of dried chili seeds into a soaking container, pour in 300mL of component A soaking solution, and let it stand and soak for 8 hours at room temperature and under light conditions to obtain soaked chili seeds; spread the soaked chili seeds on gauze and drain for 10 minutes until there is no obvious water film on the surface but it is still moist to obtain pretreated chili seeds.

[0041] S3. Preparation of coating agent: Add 6 mL of deionized water to a dry container, then add 10 g of component B to the deionized water while stirring. Stir at 800 rpm for 3 minutes until uniform, and obtain a gel-like slurry. Let stand for 5 minutes to obtain a gel-like coating agent. Component B is a composite coating dry powder, comprising the following components in parts by weight: 22.5 parts modified sodium polyacrylate, 4 parts modified glycine betaine, 4 parts sodium alginate, and 3 parts potassium humate. The modified sodium polyacrylate is sulfonic acid-modified sodium polyacrylate, and the modified glycine betaine is polyhydroxy modified glycine betaine.

[0042] S4. Coating treatment: Pour the pretreated chili seeds mixed with the gel coating agent into a container, and gently stir from bottom to top and turn over for 2 minutes. After mixing, use a low-temperature drying device at 35℃ to dry for 25 minutes. When the seed coating is completely dry, non-sticky, and in a dispersed granular state, the coated chili seeds are obtained, and the germination treatment of chili seeds is completed.

[0043] The modified sodium polyacrylate is prepared by the following method:

[0044] S11. In a 250mL four-necked flask equipped with a mechanical stirrer, thermometer, constant pressure dropping funnel and nitrogen inlet tube, add 30mL of deionized water, turn on the stirrer and introduce high-purity nitrogen gas, bubble for 25 minutes to create an inert reaction environment; place 6.5g of acrylic acid in a 50mL single-necked round-bottom flask, and slowly add 6.0mL of 10mol / L sodium hydroxide solution dropwise while cooling in an ice-water bath and stirring to obtain mixed solution A;

[0045] S12. Transfer mixed solution A to the above four-necked flask, add 3.5g of 2-acrylamide-2-methylpropanesulfonic acid monomer and 0.008g of N,N'-methylenebisacrylamide to the four-necked flask in sequence, and stir until completely dissolved to obtain mixed solution B;

[0046] S13. Heat the mixed solution B to 60°C and slowly add ammonium persulfate solution dropwise using a constant pressure dropping funnel to obtain mixed solution C. Mixed solution C is stirred continuously at 60°C for 2 hours. When mixed solution C can be stretched into continuous gel filaments without breaking, a gel is initially formed. Then, the reaction temperature is slowly raised to 75°C and the reaction continues at this temperature to obtain a hydrated gel.

[0047] S14. Cut the hydrated gel into pieces and soak them in 200 mL of anhydrous ethanol to obtain a soaking solution. Replace the anhydrous ethanol every 6 hours for 24 hours. When the conductivity of the soaking solution is less than 5 μS / cm for two consecutive measurements and the pH value is stable at 7.0, an alcohol gel is obtained. Place the alcohol gel in a forced-air drying oven and dry it at 75°C for 36 hours until constant weight to obtain a dry hard solid. Grind the dry hard solid with a pulverizer and sieve it through an 80-mesh sieve to obtain modified sodium polyacrylate.

[0048] The ammonium persulfate solution is obtained by dissolving 0.05 g of ammonium persulfate in 1 mL of deionized water.

[0049] The modified glycine betaine was prepared by the following method:

[0050] S21. On a dry 250mL three-necked round-bottom flask, install a mechanical stirrer, a reflux condenser, and a thermometer. Add 3.0g of tris(hydroxymethyl)aminomethane, 6.2g of 1-bromododecane, 0.38g of sodium iodide, and 3.45g of anhydrous potassium carbonate to the flask. Add 20mL of N,N-dimethylformamide as a solvent. Start stirring and, under nitrogen protection, raise the temperature to 90℃. Reflux the reaction at this temperature for 30 hours. After the reaction is complete, cool to room temperature and filter to obtain filtrate A.

[0051] S22. Pour filtrate A into 100 mL of ice water. Extract the aqueous phase with ethyl acetate (3 × 30 mL), wash the organic phase with saturated brine (2 × 30 mL), dry the organic phase with anhydrous sodium sulfate overnight, filter, and distill under reduced pressure at 40 °C using a rotary evaporator to obtain the crude product. Purify the crude product by silica gel column chromatography with 400 mL of eluent to obtain oily substance B.

[0052] S23. In a 50 mL single-necked round-bottom flask, the obtained oily substance B is dissolved in 15 mL of anhydrous ethanol. Under the cooling and stirring of an ice-water bath, a mixed solution D is obtained. The first mixed solvent is slowly added dropwise to the mixed solution D using a constant pressure dropping funnel, keeping the temperature below 30 °C. After the addition is completed, a mixed solution E is obtained.

[0053] S24. Remove the ice bath, heat the mixed solution E to 75°C, and stir the reaction at this temperature for 15 hours. After the reaction is complete, cool the mixed solution E to room temperature and distill it under reduced pressure at 40°C on a rotary evaporator to obtain a viscous residue. Add 30 mL of anhydrous ethanol at 65°C to the residue, stir to dissolve, filter while hot to obtain filtrate B, cool filtrate B to 0°C, let it stand, and filter under suction to obtain crystal A.

[0054] S25. Wash crystal A twice with 10 mL of anhydrous ethanol at 0 °C, and then wash it once with 10 mL of a second mixed solvent at 0 °C to obtain crystal B. Place crystal B in a vacuum drying oven at 40 °C and below -0.095 MPa and dry for 24 hours to obtain modified glycine betaine.

[0055] The eluent in step S22 is composed of ethyl acetate and methanol, with a volume ratio of ethyl acetate to methanol of 10:1.

[0056] The first mixed solvent was obtained by dissolving 11.1 g of sodium chloroacetate and 4.8 g of sodium hydroxide in 20 ml of deionized water.

[0057] The second mixed solvent is composed of ethyl acetate and n-hexane in a volume ratio of 1:1.

[0058] Example 2: This example is based on Example 1, but differs from Example 1 in that component A in this example includes the following components in parts by weight: 55 parts polyethylene glycol, 5 parts potassium nitrate, and 0.2 parts zinc sulfate heptahydrate.

[0059] Component B comprises the following components in parts by weight: 20 parts modified sodium polyacrylate, 3 parts modified glycine betaine, 3 parts sodium alginate, and 2 parts potassium humate.

[0060] The rest is the same as in Example 1.

[0061] Example 3: This example is based on Example 1, but differs from Example 1 in that component A in this example includes the following components in parts by weight: 65 parts polyethylene glycol, 7 parts potassium nitrate, and 0.4 parts zinc sulfate heptahydrate.

[0062] Component B comprises the following components in parts by weight: 25 parts modified sodium polyacrylate, 5 parts modified glycine betaine, 5 parts sodium alginate, and 4 parts potassium humate.

[0063] The rest is the same as in Example 1.

[0064] Comparative Example 1: This comparative example is based on Example 1, except that the modified sodium polyacrylate described in this comparative example does not introduce sulfonate groups. The rest is the same.

[0065] The modified sodium polyacrylate is prepared by the following method:

[0066] A1. In a 250 mL four-necked flask equipped with a mechanical stirrer, thermometer, constant pressure dropping funnel and nitrogen inlet tube, add 30 mL of deionized water, turn on the stirrer and introduce high-purity nitrogen gas, bubble for 25 minutes to remove dissolved oxygen and create an inert reaction environment; place 6.5 g of acrylic acid in a 50 mL single-necked round-bottom flask, and slowly add 6.0 mL of 10 mol / L sodium hydroxide solution while cooling in an ice-water bath and stirring to obtain mixed solution A;

[0067] A2. Under continuous nitrogen purging and stirring, mixed solution A is slowly added dropwise to the above four-necked flask through a constant pressure dropping funnel. After the addition is complete, 0.013g of N,N'-methylenebisacrylamide is added, and stirring is continued for 12 minutes until the mixture is homogeneous, thus obtaining the first mixed solution. The temperature is raised to stabilize at 65℃, and ammonium persulfate solution is quickly added to the first mixed solution. The temperature is maintained at 65℃, and nitrogen purging is continued. Within 10 minutes, a non-flowing elastic gel block is formed. The gel is then cured at 70℃ for 1.5 hours to obtain sodium polyacrylate hydrogel.

[0068] A3. Cut the sodium polyacrylate hydrogel into blocks and place them in a forced-air drying oven. Dry them at 75°C for 8 hours until constant weight is achieved. After drying, blocky material is obtained. The dried blocky material is crushed by a pulverizer and passed through a 40-mesh sieve to obtain modified sodium polyacrylate.

[0069] Comparative Example 2: This comparative example is based on Example 1, except that the modified glycine betaine described in this comparative example does not introduce three hydroxyl groups. The rest is the same.

[0070] The modified glycine betaine was prepared by the following method:

[0071] B1. Weigh 9.4g of sodium chloroacetate and add it to a 250mL three-necked round-bottom flask equipped with a magnetic stirrer, reflux condenser and thermometer. Add 50mL of deionized water and stir to dissolve to obtain a second mixed solution. Add 3.2g of sodium hydroxide solid to the second mixed solution and stir to dissolve to obtain a third mixed solution.

[0072] B2. Under ice-water bath cooling and stirring, 18g of 30% trimethylamine aqueous solution was slowly added dropwise to the third mixed solution, keeping the temperature below 30℃. After the addition was completed, the ice bath was removed, and the mixture was heated to 65℃. The mixture was then refluxed and stirred at this temperature for 12 hours. After the reaction was completed, the mixture was cooled to room temperature and connected to a rotary evaporator. The mixture was then subjected to vacuum distillation at a water bath temperature of 40℃ to obtain residue B.

[0073] B3. Add concentrated hydrochloric acid dropwise to residue B while stirring and monitoring with precision pH paper. Adjust the pH of the solution to 1.5 to obtain an acidified solution. Transfer the acidified solution to a 250 mL beaker, add 100 mL of anhydrous ethanol, stir vigorously to form a large amount of white precipitate, filter, and obtain filtrate C. Transfer the obtained filtrate C to a round-bottom flask again and distill under reduced pressure at 40 °C using a rotary evaporator to obtain a syrup-like substance.

[0074] B4. Add 20 mL of anhydrous ethanol at 65 °C to the syrup, stir to dissolve, and then slowly add 30 mL of ethyl acetate dropwise while stirring to obtain a turbid substance;

[0075] B5. After the turbid substance was placed in an ice-water bath at 0℃ and left to stand overnight, it was filtered to obtain crystal C. Crystal C was washed three times with anhydrous ethanol and ethyl acetate at 0℃ in a volume ratio of 1:1. The washed crystal C was transferred to a petri dish and dried in a vacuum drying oven at 45℃ and below -0.095MPa for 36 hours until constant weight was obtained to obtain modified glycine betaine.

[0076] Comparative Example 3: This comparative example is based on Example 1, except that it does not include modified sodium polyacrylate. The rest are the same.

[0077] Comparative Example 4: This comparative example is based on Example 1, except that it does not include modified glycine betaine. All other parts are the same.

[0078] Comparative Example 5: This comparative example is based on Example 1, except that it does not include modified sodium polyacrylate and modified glycine betaine. The rest are the same.

[0079] Comparative Example 6: This comparative example is based on Example 1, except that it does not include sodium alginate. The rest are the same.

[0080] Comparative Example 7: This comparative example is based on Example 1, except that it does not include potassium humate. The rest are the same.

[0081] Comparative Example 8: This comparative example is based on Example 1, except that it does not include sodium alginate and potassium humate. The rest are the same.

[0082] Comparative Example 9: This comparative example differs from Example 1 in that the chili seeds are not coated. The steps include:

[0083] S1. Preparation of soaking solution: Add component A to 300 mL of deionized water at 28 °C and stir continuously until completely dissolved to obtain component A soaking solution; component A is a composite soaking agent, comprising the following components in parts by weight: 60 parts polyethylene glycol, 6 parts potassium nitrate, and 0.3 parts zinc sulfate heptahydrate;

[0084] S2. Pretreatment: Place 100g of dried chili seeds into a soaking container, pour in 300mL of component A soaking solution, and let it stand and soak for 8 hours at room temperature and under light conditions to obtain soaked chili seeds; spread the soaked chili seeds on gauze and drain for 10 minutes until there is no obvious water film on the surface but it is still moist to obtain pretreated chili seeds.

[0085] Example 1: "Zhongjiao 107" chili seeds of uniform size from the same batch were selected. A mixed aqueous solution prepared with 150 mmol / L sodium chloride and 20 mmol / L sodium bicarbonate was used to simulate the extract of typical arid saline-alkali soil and as the stress medium during the germination stage. This mixed aqueous solution effectively simulated the stress characteristics of saline-alkali soil. The 150 mmol / L sodium chloride mainly simulated osmotic stress and ion poisoning, while the 20 mmol / L sodium bicarbonate introduced an alkaline environment, simulating the high pH stress caused by the accumulation of bicarbonate ions in saline-alkali soil. This combination can well reflect the typical ionic composition and chemical properties of the extract of saline-alkali soil in arid regions.

[0086] Preparation of the blank control group for chili seeds: 100g of dried chili seeds were placed in a soaking container, and 300mL of deionized water at 28℃ was poured in. The mixture was soaked for 8 hours at room temperature and under dark conditions. After soaking, the seeds were spread on gauze and drained for 10 minutes until there was no obvious water film on the surface but the seeds remained moist. This was the chili seeds for the blank control group. The experimental group consisted of chili seeds treated according to the methods of Examples 1-3 and Comparative Examples 1-9. The chili seeds of the blank control group and the experimental group were evenly placed in a petri dish (containing 10mL of the above-mentioned extract) lined with two layers of moist filter paper and germinated in an artificial climate chamber under the following conditions: day / night temperature 28℃ / 20℃, relative humidity 70%, and darkness. A total of 13 treatments were prepared, including the blank control group, the groups of Examples 1-3, and the groups of Comparative Examples 1-9. Each treatment was replicated 4 times, with 50 seeds per replicate, forming the test seeds.

[0087] The test metrics and methods are as follows:

[0088] 1. Germination Indicators: The number of germinated seedlings was recorded daily (defined as the length of the radicle breaking through the seed coat being no less than 2 mm). The germination potential on day 4 and the final germination rate on day 8 were calculated. On day 8 of germination, 10 seedlings were randomly selected from each replicate, and the length of their radicles was measured using calipers, and their fresh weight was weighed using an analytical balance with a density of 0.01%. The results are shown in Table 1.

[0089] The formula for calculating germination potential is:

[0090] Germination potential (%) = (Cumulative number of normally germinated seeds on day 4 / Total number of seeds tested) × 100%;

[0091] The formula for calculating the final germination rate is:

[0092] Final germination rate (%) = (Cumulative number of normally germinated seeds on day 8 / Total number of seeds tested) × 100%;

[0093] Table 1. Results of chili seed germination indicators:

[0094]

[0095] As shown in Table 1, Examples 1-3 exhibited the best germination potential, final germination rate, and radicle growth indicators for chili seeds under simulated drought and saline-alkali stress, demonstrating the effectiveness of the present invention. The lower indicators in Comparative Examples 1 and 2 indicate that both modified sodium polyacrylate and modified glycine betaine are indispensable. The indicators in Comparative Examples 5 and 9 showed no substantial difference from the blank control group, further confirming that a complete coating system containing modified sodium polyacrylate and modified glycine betaine is crucial for protecting seed germination.

[0096] Based on the above experiments (more seeds) Figure 2 This is a graph showing the germination potential of chili seeds in Example 1 and Comparative Example 9 on day 4. Figure 3 The graph shows the growth of chili seedlings from Examples 1-3 and Comparative Examples 1-2 on day 30; as shown. Figure 2 and Figure 3 As shown, the germination and seedling growth of the chili seeds after germination treatment according to the embodiment are better than those of the comparative example.

[0097] 2. Physiological and biochemical indicators: On the third day of germination, embryonic tissue from chili seeds was taken for testing. The results are shown in Table 2.

[0098] (1) Determination of superoxide anion generation rate: Weigh 0.2g of chili seed embryo tissue and immerse it in 3mL of 50mmol / L phosphate buffer (pH 7.8) containing 0.1% nitroblue tetrazolium. Incubate at 25℃ in the dark for 1 hour. Remove the embryo tissue and rinse with distilled water to obtain stained embryo tissue. Place the stained embryo tissue in a test tube, add 5mL of N,N-dimethylformamide, and extract in an 80℃ water bath for 30 minutes until the embryo tissue is colorless. After cooling, centrifuge and measure the absorbance of the supernatant at a wavelength of 595nm.

[0099] The formula for calculating the superoxide anion generation rate is:

[0100] Superoxide anion generation rate (ΔA) 595 ·g -1 ·min -1 )=(A 595试验组 -A 595空白对照组 ) / [fresh weight of tissue (g) × reaction time (min)];

[0101] A in the formula 595 The absorbance value of the embryonic tissue at a wavelength of 595 nm is given.

[0102] (2) Determination of relative conductivity (cell membrane damage rate): Weigh 0.5g of embryonic tissue into a test tube, add 10mL of deionized water, soak at room temperature for 2 hours, and measure the conductivity of the extract (C1). Then boil the test tube in a boiling water bath for 15 minutes, cool to room temperature, add water to the original volume, and measure the total conductivity (C2).

[0103] Formula for calculating relative conductivity (cell membrane damage rate):

[0104] Relative conductivity (%) = (C1 / C2) × 100%;

[0105] (3) Superoxide dismutase activity assay (nitroblue tetrazolium photochemical reduction method): 0.1 mL of enzyme extraction buffer was added to 1.5 mL of 50 μmol / L phosphate buffer (pH 7.8) containing 130 mmol / L methionine, 750 μmol / L nitroblue tetrazolium, 100 μmol / L disodium EDTA, and 20 μmol / L riboflavin to obtain sample tubes. The tube containing only buffer without enzyme extraction buffer was considered the maximum reduction tube. The reaction was carried out under 4000 lux light for 20 minutes.

[0106] Formula for calculating superoxide dismutase activity:

[0107] Superoxide dismutase activity (g) -1 )=[(A 560最大还原管 -A 560样品管 ) / A 560最大还原管 ]×100%×(V 总1 / V 样1 )×(1 / (W1×t1));

[0108] A in the formula 560 The absorbance value at a wavelength of 560 nanometers; V 总1 V is the total volume of the reaction system (mL); 样1 W1 is the volume of enzyme extract added (mL); W1 is the fresh weight of the tissue corresponding to the enzyme extract (g); t1 is the light irradiation time (min); "(A 560最大还原管 -A 560样品管 ) / A 560最大还原管 "" represents the inhibition rate; "×100%" converts the inhibition rate into a percentage, used to calculate the enzyme activity units required to achieve 50% inhibition.

[0109] (4) Catalase activity assay (UV absorption method): Add 0.1 mL of enzyme extraction solution to 3 mL of 50 mmol / L phosphate buffer (pH 7.0) containing 15 mmol / L hydrogen peroxide and mix quickly. Immediately start timing at 240 nm and record the initial absorbance value and the absorbance value after 1 minute of reaction.

[0110] Formula for calculating catalase activity:

[0111] Catalase activity (μmol·g) -1 ·min -1 )=(ΔA 240 ×V 总2 ) / (ε×d×V 样2 ×W2×t2);

[0112] ΔA in the formula 240 V represents the decrease in absorbance within the first minute of the reaction. 总2The total volume of the reaction system is 3.1 mL; ε is the molar extinction coefficient of hydrogen peroxide at 240 nm, which is 0.0396 L·μmol. -1 ·cm -1 ;d is the optical path length of the cuvette (1 cm); V 样2 t2 is the volume of enzyme extract added (0.1 mL), W2 is the fresh weight of the tissue corresponding to the enzyme extract (g), and t2 is the reaction time (1 min).

[0113] Table 2. Results of physiological and biochemical indicators of chili seeds:

[0114]

[0115] As shown in Table 2, the physiological and biochemical data directly confirm the effectiveness of this invention in addressing the weakness of oxidative damage defenses at the mechanistic level. Examples 1-3 exhibited the lowest superoxide anion generation rate and relative conductivity, while possessing the highest superoxide dismutase and catalase activities. This indicates that the reactive oxygen species (ROS) burst in chili seeds treated by the method of this invention is suppressed, cell membrane integrity is better protected, and the endogenous antioxidant enzyme system is effectively maintained. The relative conductivity of Comparative Example 1 is higher than that of Examples 1-3, indicating that its external barrier failure led to severe membrane damage; the superoxide anion generation rate of Comparative Example 2 is higher than that of Examples 1-3, confirming the key role of the hydroxyl structure in the modified glycine betaine in scavenging ROS. The data of Comparative Examples 6-8 are better than those of Comparative Examples 1-4, indicating that the two core modified substances play a dominant role, and the auxiliary components have an optimizing effect. The physiological and biochemical data clearly reveal from the mechanism of action that the two key modified substances in the coating are indispensable, and the absence of any single component will lead to vulnerability in the corresponding defense links.

[0116] Finally, it should be noted that the above description is only 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 germinating chili seeds, characterized in that, Includes the following steps: S1. Preparation of soaking solution: Add component A to deionized water and stir to obtain component A soaking solution; component A is a composite soaking agent, comprising the following components in parts by weight: 55–65 parts polyethylene glycol, 5–7 parts potassium nitrate, and 0.2–0.4 parts zinc sulfate heptahydrate; S2. Pretreatment: Pour component A soaking solution into dried chili seeds and let them soak in the solution at room temperature and under light conditions to obtain soaked chili seeds; drain the soaked chili seeds to obtain pretreated chili seeds; S3. Preparation of coating agent: Component B is added to deionized water while stirring. After stirring evenly, a gel-like slurry is obtained. After standing, a gel-like coating agent is obtained. Component B is a composite coating dry powder, comprising the following components in parts by weight: 20-25 parts modified sodium polyacrylate, 3-5 parts modified glycine betaine, 3-5 parts sodium alginate, and 2-4 parts potassium humate. The modified sodium polyacrylate is sulfonic acid-modified sodium polyacrylate, and the modified glycine betaine is polyhydroxy modified glycine betaine. S4. Coating treatment: The pretreated chili seeds are mixed with a gel-like coating agent and then dried to obtain coated chili seeds, thus completing the germination treatment of the chili seeds; The modified sodium polyacrylate is prepared by the following method: S11. Add deionized water to the reactor, turn on the stirrer and introduce high-purity nitrogen gas; add sodium hydroxide solution dropwise to acrylic acid while cooling and stirring in an ice-water bath to obtain mixed solution A; S12. Transfer mixed solution A to the above reactor, then add 2-acrylamide-2-methylpropanesulfonic acid monomer and N,N'-methylenebisacrylamide in sequence, stir, and obtain mixed solution B; S13. After heating the mixed solution B, add ammonium persulfate solution dropwise and stir to obtain a hydrated gel; S14. Cut the hydrated gel into pieces and soak them in anhydrous ethanol to obtain an alcohol gel; dry the alcohol gel to obtain a dry hard solid; grind and sieve the dry hard solid to obtain modified sodium polyacrylate. The modified glycine betaine was prepared by the following method: S21. Tris(hydroxymethyl)aminomethane, 1-bromododecane, sodium iodide and anhydrous potassium carbonate are added to a reactor, and then N,N-dimethylformamide is added to the reactor; the reaction is carried out under nitrogen protection and heated. After the reaction is completed, the mixture is cooled to room temperature and filtered to obtain filtrate A. S22. Pour filtrate A into ice water, extract the aqueous phase with ethyl acetate, wash the organic phase with saturated brine, and dry the organic phase with anhydrous sodium sulfate; filter and distill under reduced pressure to obtain crude product; purify the crude product with eluent to obtain oily substance B; S23. Add the obtained oily substance B to anhydrous ethanol, and under ice-water bath cooling and stirring, obtain mixed solution D. Add the first mixed solvent dropwise to mixed solution D. After the addition is complete, obtain mixed solution E. S24. Remove the ice bath, heat and stir the mixed solution E. After the reaction is complete, cool the mixed solution E to room temperature, distill under reduced pressure, add the obtained product to anhydrous ethanol, stir to dissolve, filter while hot to obtain filtrate B, cool filtrate B, let it stand, filter under suction to obtain crystal A; S25. Wash crystal A with anhydrous ethanol, then wash with the second mixed solvent to obtain crystal B. Dry crystal B to obtain modified glycine betaine.

2. The method for germinating chili seeds according to claim 1, characterized in that, In step S1, the volume ratio of deionized water to the mass ratio of component A is 3:1; the volume unit of deionized water is mL, and the mass unit of component A is g.

3. The method for germinating chili seeds according to claim 1, characterized in that, In step S3, the volume ratio of deionized water to the mass of component B is 0.6:1; the volume of deionized water is in mL, and the mass of component B is in g.

4. The method for germinating chili seeds according to claim 1, characterized in that, The mass-to-volume ratio of ammonium persulfate to water in the ammonium persulfate solution is 5 g / 100 mL.

5. The method for germinating chili seeds according to claim 1, characterized in that, The eluent in step S22 is composed of ethyl acetate and methanol, with a volume ratio of ethyl acetate to methanol of 10:

1.

6. The method for germinating chili seeds according to claim 1, characterized in that, The first mixed solvent is obtained by dissolving sodium chloroacetate and sodium hydroxide in deionized water.

7. The method for germinating chili seeds according to claim 1, characterized in that, The second mixed solvent is composed of ethyl acetate and n-hexane in a volume ratio of 1:1.

Images