Application of apigenin and preparation thereof in improving drought tolerance of wheat plants

Abstract

The invention discloses application of apigenin and a preparation thereof in improving drought tolerance of wheat plants. The technical problem to be solved is how to improve the drought tolerance of wheat plants. The invention specifically discloses application of apigenin or salt thereof or solvate thereof in any one of the following aspects: A1) application in improving the drought tolerance of gramineous plants; a2) application in preparation of a product for improving the drought tolerance of gramineous plants. The drought tolerance of the wheat seedlings can be improved by applying the apigenin or the salt thereof or the solvate of the apigenin and the salt to the wheat seedlings, and the method can be used for industrial production.

Description

Technical Field

[0001] The invention specifically relates to application of apigenin and preparations thereof in improving drought resistance of wheat plants. Background Art

[0002] Wheat is an important cereal crop in the world and plays an important role in my country's agriculture and economy. However, many abiotic stresses during the growth process, such as drought, salt, floods, low temperature, etc., seriously affect the growth and yield of wheat. In recent years, due to global warming, drought stress has become the main factor affecting wheat growth.

[0003] Flavonoids are important secondary metabolites and a class of low molecular weight phenolic compounds, mainly including flavonoids and flavonols, isoflavones and anthocyanins. In plants, flavonoids have many functions besides antioxidants, such as regulating cell growth, antibacterial, and resisting biotic and abiotic stresses. Apigenin is a type of flavonoid. Summary of the invention

[0004] The technical problem solved by the invention is how to improve the drought resistance of wheat.

[0005] In order to solve the above technical problems, the present invention provides the following applications.

[0006] Use of apigenin or its salt or solvate thereof in any of the following:

[0007] A1) Application in improving drought tolerance of grass plants;

[0008] A2) Application in the preparation of products for improving drought tolerance of Gramineae plants.

[0009] In the above, the drought resistance indicator may be at least one of survival rate, aboveground fresh weight, relative water content, chlorophyll a content, chlorophyll b content, chlorophyll content, carotenoid content, POD activity, soluble protein content, ABA content, salicylic acid content, ethylene content, brassinolide content, and superoxide anion content.

[0010] In the above, the apigenin can be used to improve the survival rate, fresh weight of aboveground parts, relative water content, chlorophyll a content, chlorophyll b content, chlorophyll content, carotenoid content, POD activity, soluble protein content, ABA content, salicylic acid content and / or ethylene content.

[0011] In the above, the apigenin can reduce the brassinolide content and / or the superoxide anion content.

[0012] In the above, the above-ground part may be leaves.

[0013] In the above application, the product for improving drought resistance of Gramineae plants is a drought-resistant preparation.

[0014] In the above application, the drought-resistant preparation is a liquid preparation.

[0015] In the above, apigenin or its salt, or their solvate is used in the preparation of drought-resistant preparations, and the drought-resistant preparations can improve the tolerance of Gramineae plants to drought stress.

[0016] In the above, the effective concentration of apigenin may be 20 μM-100 μM.

[0017] The drought-resistant preparation may contain, in addition to the active ingredient, a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient. The pharmaceutically acceptable carrier may be a solid carrier or a liquid carrier.

[0018] The drought-resistant preparations mentioned above may be in various dosage forms, including but not limited to liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule, etc.

[0019] In the above application, the effective concentration of apigenin in the liquid is 20 μM-100 μM.

[0020] In order to solve the above technical problems, the present invention also provides a method for improving the drought resistance of Gramineae plants.

[0021] The method comprises applying apigenin or its salt, or their solvate to Gramineae plants to improve the drought tolerance of the plants.

[0022] In the above, the method of application may be root application.

[0023] In the above method, the application is achieved by root application.

[0024] In the above, the root application may be root irrigation.

[0025] In the above method, the dosage form of the apigenin or its salt, or their solvate is a liquid.

[0026] In the above method, the concentration of apigenin in the liquid used is 20μM-100μM.

[0027] In the above method, the concentration of apigenin in the seed coating is 20 or 100 μM.

[0028] In any of the above uses or methods, the plant may be any of the following:

[0029] D1) Triticum plants;

[0030] D2) Wheat.

[0031] In the above, the wheat may be the wheat variety Chang 6878.

[0032] In the above, the wheat may be wheat seedlings. The wheat seedlings may be seedlings in the seedling stage. The seedling stage may be 7 days of growth.

[0033] Beneficial Effects

[0034] The present invention discloses the application of apigenin and its preparation in improving the drought resistance of wheat plants. The technical problem solved is how to improve the drought resistance of wheat plants. Specifically disclosed is the application of apigenin or its salt or their solvate in any of the following: A1) application in improving the drought resistance of gramineous plants; A2) application in preparing products for improving the drought resistance of gramineous plants. Applying apigenin or its salt or their solvate to wheat seedlings can improve the drought resistance of wheat seedlings and can be used in industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is the effect of exogenous apigenin on the phenotype of wheat seedlings under drought stress in Example 1.

[0036] Figure 2 The effect of apigenin on wheat seedling growth indexes under drought stress in Example 1; (A) survival rate, (B) aboveground fresh weight, (C) aboveground dry weight, (D) relative water content. The control group was subjected to drought stress, and 20, 50, and 100 μM were subjected to drought stress and 20, 50, and 200 μM apigenin were added, respectively. The bars represent the mean ± standard deviation of 3 repetitions; different letters indicate statistically significant differences (P<0.05) after Duncan's multiple range test.

[0037] Figure 3 The effect of apigenin on the physiological indexes of wheat seedlings under drought stress in Example 2; (A) chlorophyll a content, (B) chlorophyll b content, (C) chlorophyll content, (D) carotenoid content. The control group was subjected to drought stress, and 20, 50, and 100 μM were subjected to drought stress and 20, 50, and 200 μM apigenin were added. The bars represent the mean ± standard deviation of 3 repetitions; different letters indicate statistically significant differences (P<0.05) after Duncan's multiple range test.

[0038] Figure 4The effect of apigenin on the physiological indexes of wheat seedlings under drought stress in Example 2; (A) superoxide anion content, (B) POD activity, (C) soluble protein content. The control group was subjected to drought stress, and 20, 50, and 100 μM were subjected to drought stress and 20, 50, and 200 μM apigenin were added. The bars represent the mean ± standard deviation of 3 repetitions; different letters indicate statistically significant differences (P<0.05) after Duncan's multiple range test.

[0039] Figure 5 The effect of apigenin on the physiological indexes of wheat seedlings under drought stress in Example 2; the effect of apigenin on (A) ABA content, (B) salicylic acid (SA) content, (C) brassinolide (BR) content, and (D) ethylene content of wheat seedlings under drought stress. The control group was subjected to drought stress, and 20, 50, and 100 μM were respectively subjected to drought stress and 20, 50, and 200 μM apigenin were added. The bars represent the mean ± standard deviation of 3 repetitions; different letters indicate statistically significant differences (P<0.05) after Duncan's multiple range test. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0041] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0042] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology and laboratory operation procedures used herein are terms and routine procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0043] The term "kit" refers to any delivery system used to deliver substances. In reaction assays, such delivery systems include systems that store, transport or deliver reaction reagents (e.g., oligonucleotides, enzymes, etc. in appropriate containers) and / or support materials (e.g., buffers, instructions for performing the assay, etc.) from one location to another. For example, a kit comprises one or more housings (e.g., boxes) containing the relevant reaction reagents and / or support materials.

[0044] The terms "include", "including", "have", "contain" and the like are all open terms, meaning including but not limited to.

[0045] The term "pharmaceutically acceptable" means that the substance or composition must be compatible with the other ingredients of the formulation and / or the plant to be treated with it chemically and / or toxicologically. Preferably, the "pharmaceutically acceptable" of the present invention refers to that approved by the national government or listed in the Chinese Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, particularly in humans.

[0046] The term "pharmaceutically acceptable excipient" is a pharmaceutically acceptable carrier, solvent, suspending agent or excipient used to deliver the active ingredient (e.g., RGD peptide or TGF-β1 signaling pathway inhibitor) in the drug of the present invention to the plant. Exemplary excipients can be liquid or solid, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes / colorants, flavor enhancers, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents, emulsifiers, sprays, compressed air or other suitable gases, or other suitable inactive ingredients used in combination with the pharmacological compound. Examples of excipients include various lactose, mannitol, oils such as corn oil, buffers such as PBS, saline, polyethylene glycol, glycerol, polypropylene glycol, dimethyl sulfoxide, amides such as dimethylacetamide, proteins such as albumin, monosaccharides and oligosaccharides such as glucose, lactose, cyclodextrin and starch.

[0047] The terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. Further, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0048] The term "effective" refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity upon administration to a subject in need thereof. Note that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that would be effective if administered alone. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the specific drug or drugs employed, the mode of administration, and the like.

[0049] Example 1 Effect of exogenous apigenin on wheat seedling phenotype under drought stress

[0050] 1.1 Materials and methods

[0051] The molecular formula of apigenin is C 15 H 10 O5, CAS number is 520-36-5.

[0052] 25% PEG culture medium: Add the following reagents to one liter of water to prepare Holland culture medium: 0.23615 g of calcium nitrate tetrahydrate, 0.27218 g of potassium phosphate, 0.24647 g of magnesium sulfate heptahydrate, 0.11825 g of potassium sulfide, 0.22058 g of calcium chloride dihydrate, 0.00006683 g of boric acid, 0.00003629 g of sodium molybdate, 0.00012484 g of copper sulfate pentahydrate, 0.00028756 g of zinc sulfate heptahydrate, 0.000169 g of manganese sulfate, 0.27802 g of ferrous sulfate heptahydrate, and 0.3722 g of disodium ethylenediaminetetraacetic acid. Add 250 g of PEG to the above one liter of water to prepare 25% PEG culture medium.

[0053] 25% PEG + 20μM apigenin culture solution: Add the following reagents to one liter of water to prepare Holland culture solution: 0.23615 g of calcium nitrate tetrahydrate, 0.27218 g of potassium phosphate, 0.24647 g of magnesium sulfate heptahydrate, 0.11825 g of potassium sulfide, 0.22058 g of calcium chloride dihydrate, 0.00006683 g of boric acid, 0.00003629 g of sodium molybdate, 0.00012484 g of copper sulfate pentahydrate, 0.00028756 g of zinc sulfate heptahydrate, 0.000169 g of manganese sulfate, 0.27802 g of ferrous sulfate heptahydrate, and 0.3722 g of disodium ethylenediaminetetraacetic acid. Add 250 g of PEG and 5.4048 mg of apigenin to the above one liter of water to prepare 25% PEG + 20μM apigenin culture solution.

[0054] 25% PEG + 50μM apigenin culture solution: Add the following reagents to one liter of water to prepare Holland culture solution: 0.23615 g of calcium nitrate tetrahydrate, 0.27218 g of potassium phosphate, 0.24647 g of magnesium sulfate heptahydrate, 0.11825 g of potassium sulfide, 0.22058 g of calcium chloride dihydrate, 0.00006683 g of boric acid, 0.00003629 g of sodium molybdate, 0.00012484 g of copper sulfate pentahydrate, 0.00028756 g of zinc sulfate heptahydrate, 0.000169 g of manganese sulfate, 0.27802 g of ferrous sulfate heptahydrate, and 0.3722 g of disodium ethylenediaminetetraacetic acid. Add 250 g of PEG and 13.512 mg of apigenin to the above one liter of water to prepare 25% PEG + 50μM apigenin culture solution.

[0055] 25% PEG + 100μM apigenin culture solution: Add the following reagents to one liter of water to prepare Holland culture solution: 0.23615g of calcium nitrate tetrahydrate, 0.27218g of potassium phosphate, 0.24647g of magnesium sulfate heptahydrate, 0.11825g of potassium sulfide, 0.22058g of calcium chloride dihydrate, 0.00006683g of boric acid, 0.00003629g of sodium molybdate, 0.00012484g of copper sulfate pentahydrate, 0.00028756g of zinc sulfate heptahydrate, 0.000169g of manganese sulfate, 0.27802g of ferrous sulfate heptahydrate, and 0.3722g of disodium ethylenediaminetetraacetic acid. Add 250g of PEG and 27.024mg of PEG to the above one liter of water to prepare 25% PEG + 100μM apigenin culture solution.

[0056] Select the plump wheat variety Chang 6878 for drought tolerance identification. Pick an appropriate amount of seeds in a large round dish, disinfect with 2.5% NaClO, soak for 30 minutes, and wash with clean water 3-6 times. Place the seeds at room temperature 20°C for germination for 3 days, pick the germinated seeds with consistent growth status, and move them to a black light-proof plant hydroponic box 96-well plate with pointed tweezers. There is one hole between every two seeds, and they are planted in a cross-type, that is, 48 ​​rice seeds of the same strain are planted on each plate, and three biological replicates are set for each strain. The transplanted wheat plants are moved to the greenhouse for culture. Start with tap water for 3 days, change to 1× Hogland nutrient solution for 4 days, change the nutrient solution every three days, and after 7 days of culture, take the wheat seedlings for drought treatment (25% PEG) and different concentrations of apigenin treatment (25% PEG + 20, 50, 100 μM apigenin) respectively. The specific method is as follows:

[0057] 25% PEG culture medium group: The wheat seedlings cultured in 1× Hoagland nutrient solution for 4 days were transferred to 25% PEG culture medium. The phenotype was observed and photographed on the 0th day. After culturing in 25% PEG culture medium for 16 and 22 days, the seedlings were transferred to 1× Hoagland nutrient solution. After 3 days of rehydration under normal conditions, the phenotype was observed and photographed. The survival rate, aboveground fresh weight, aboveground dry weight, and relative water content were calculated. Three samples were used each time and the experiment was repeated 3 times. SPSS11.5 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation. Duncan multiple range test was used. Different letters indicate statistically significant differences (P < 0.05).

[0058] The difference between the 25% PEG+20 μM apigenin culture medium group and the 25% PEG culture medium group is that the 25% PEG culture medium is replaced by 25% PEG+20 μM apigenin culture medium.

[0059] The difference between the 25% PEG+50 μM apigenin culture medium group and the 25% PEG culture medium group is that the 25% PEG culture medium is replaced by 25% PEG+50 μM apigenin culture medium.

[0060] The difference between the 25% PEG+100 μM apigenin culture medium group and the 25% PEG culture medium group is that the 25% PEG culture medium is replaced by 25% PEG+100 μM apigenin culture medium.

[0061] Survival rate: The survival rate of each treatment group after rehydration was calculated, and the standard was that the roots regained elasticity and the leaves turned green. Survival rate = surviving seedlings / total number of seedlings.

[0062] Fresh weight of aboveground parts: The fresh weight of aboveground parts of each treatment group after rehydration was counted. The aboveground parts of the seedlings were quickly placed on a scale for weighing and the numbers were recorded. The aboveground parts were leaves.

[0063] Dry weight of aboveground part: The dry weight of underground part was counted for each treatment group after rehydration. The underground part of the seedlings was packaged and placed in an oven. After 12 hours, the weight was weighed until there was no change in weight. This was the dry weight, and the aboveground part was the leaves.

[0064] Relative water content: The relative water content of each treatment group after rehydration was statistically analyzed. The plant tissue was cut and quickly placed in an aluminum box of known weight, and the fresh weight (Wf) was weighed. After weighing the fresh weight, the sample was immersed in distilled water or wrapped in wet gauze that was saturated with water for 6-8 hours. After taking it out, the surface water of the sample was wiped dry with absorbent paper and weighed; the sample was immersed in distilled water for 1 hour, taken out, wiped dry, and weighed until the saturated weight of the sample was approximately the same, and the saturated fresh weight of the sample (Wt) was obtained; if the time taken to reach water saturation is known in advance, the saturated fresh weight can be measured by weighing it once, and then drying it and weighing the dry weight (Wd). Calculate according to the following formula: Relative water content = (Wf-Wd) / (Wt-Wd)×100%.

[0065] 1.2 Results and Analysis

[0066] The Chang 6878 wheat seedlings were subjected to drought treatment and apigenin was applied to the seedlings. The results showed that the drought tolerance of wheat was improved under the treatment of apigenin at various concentrations. Under the conditions of 20μM and 100μM apigenin treatment, the drought tolerance was significantly higher than that of the drought group. When 20μM and 100μM apigenin were applied at the same time as drought treatment, it can be seen that the survival rate, growth potential and other traits of the recipients were slightly better than those of the wheat seedlings under drought treatment ( Figure 1 We also measured the aboveground fresh weight, aboveground dry weight and relative water content. After applying 20μM and 100μM apigenin, the aboveground fresh weight, aboveground dry weight and relative water content increased significantly ( Figure 1 , Figure 2 ; Figure 1 In the data, control is the 25% PEG culture medium group, 20μM is the 25% PEG+20μM apigenin culture medium group, 50μM is the 25% PEG+50μM apigenin culture medium group, and 1000μM is the 25% PEG+1000μM apigenin culture medium group; before drought is the phenotype before drought treatment, after 16 days of drought is the phenotype 16 days after drought treatment, after 22 days of drought is the phenotype 22 days after drought treatment, and after 3 days of rehydration is the phenotype 3 days after rehydration), indicating that the flavonoid compound apigenin may play an important role in improving wheat drought tolerance.

[0067] Example 2 Effects of exogenous apigenin on physiological parameters of wheat seedlings under drought stress

[0068] 1.1 Materials and methods

[0069] The drought-treated wheat seedlings in the above-mentioned 25% PEG culture medium group, 25% PEG + 20 μM apigenin culture medium group, 25% PEG + 60 μM apigenin culture medium group and 25% PEG + 100 μM apigenin culture medium group were treated with drought for one week (7 days). 0.1 g of sample was required for each index, placed in a centrifuge tube and immediately transferred to liquid nitrogen for storage. According to the requirements of the kit, the chlorophyll content, antioxidant enzyme (POD), oxide (superoxide anion), osmotic substance (soluble protein), and plant hormone (ABA, ET, SA, BR) were measured respectively. Three samples were collected each time, and each experiment was repeated 3 times. SPSS11.5 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation. Duncan multiple range test was used. Different letters indicate statistically significant differences (P<0.05).

[0070] Chlorophyll a content: Cut the leaves of different treatment groups into small pieces, place them in a 10 ml acetone-ethanol (1:1) mixture, and keep them away from light for 24 hours until all the leaves turn white. Filter the solution and measure the absorbance at 645 nm, 663 nm and 440 nm. Ca is chlorophyll a; Cb is chlorophyll b; V is the total volume; W is the fresh weight of the leaves. The calculation formula is as follows.

[0071] Chlorophyll a = (12.7 663 - 2.69 645) × / (1000);

[0072] Chlorophyll b = (22.9 645-4.68 663) × / (1000);

[0073] Total chlorophyll = (20 645 + 8.02 663) × / (1000);

[0074] Carotenoids = (1000 470-3.27 -104) / 229× / (1000);

[0075] Antioxidant enzyme (POD) activity: The kit was purchased from Beijing Solebow Technology Co., Ltd., with the item number BC0090. The tissue mass (g): extract volume (mL) was adjusted to a ratio of 1:5-10 (it is recommended to weigh about 0.1g of tissue and add 1mL of extract). Ice bath homogenization was performed. Centrifuge at 8000g for 10min at 4℃, take the supernatant, and place on ice for testing. Add the reagents in the kit in sequence, mix immediately and time, record the absorbance value A1 at 470nm at 30s and the absorbance value A2 after 1min30s. Calculate ΔA=A2-A1. The calculation formula is POD (U / g mass)=ΔA×V total ÷ (W×V sample ÷V total sample) ÷ 0.01 ÷ T = 7133×ΔA ÷ W

[0076] Oxide (superoxide anion) content: The kit was purchased from Suzhou Keming Biotechnology Co., Ltd. According to the ratio of tissue mass (g): extract volume (mL) of 1:5-10 (it is recommended to weigh about 0.1g tissue and add 1mL extract), ice bath homogenize, then centrifuge at 10000g, 4℃ for 20min, take the supernatant and place it on ice for testing. Preheat the spectrophotometer / enzyme reader for 30min and adjust the wavelength to 530nm. Mix the sample and reagent as required, centrifuge at 8000g, 25℃ for 5min, carefully draw 200mL of the upper aqueous phase into a micro quartz cuvette or 96-well plate, and measure A530. ΔA=A determination-A blank.

[0077] Superoxide anion content (nmol / g fresh weight) = 148.76x (ΔA+0.0027) ÷ W Soluble protein content: The kit was purchased from Suzhou Keming Biotechnology Co., Ltd., ADS-F-SP002-96. Weigh about 0.1g of tissue, add imL of extract (the extract can be enzyme extraction buffer, distilled water, or physiological saline), homogenize in an ice bath, centrifuge at 12000rpm, 4℃ for 10min, take the supernatant, which is the test solution. Mix well, keep warm at 60℃ for 30min, transfer all to a 1mL glass cuvette, and measure the absorbance value A at 562nm, ΔA = A determination - A blank. .

[0078] ABA content: The kit was purchased from Shanghai Abermate Co., Ltd. The required strips were taken out from the aluminum foil bag after equilibration at room temperature for 60 minutes. The remaining strips were sealed with a ziplock bag and returned to 4°C. Standard wells and sample wells were set. 50uL of standard with different concentrations was added to each standard well. 10μL of the sample to be tested was first added to the sample well to be tested, and then 40uL of the sample diluent was added. Then 100uL of horseradish peroxidase (HRP)-labeled detection antibody was added to each well of the standard well and sample well. μL, seal the reaction wells with a sealing film, incubate in a 37℃ water bath or incubator for 60min, discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1min, discard the washing solution, pat dry on absorbent paper, repeat the washing process 5 times (you can also use a plate washer), add 50uL of substrate A and B to each well, incubate at 37℃ in the dark for 15min, add 50μL of stop solution to each well, and measure the OD value of each well at a wavelength of 450nm within 15min. Bring in the standard curve.

[0079] Salicylic acid (SA) content: obtained from Jiangsu Yutong Biotechnology Co., Ltd. Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips with a ziplock bag and return them to 4°C. Set up standard wells and sample wells, add 50μL of different concentrations of standard wells; add 10μL of the sample to be tested to the sample well, and then add 40μL of sample diluent; do not add to the blank wells. Except for the blank wells, add 100μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard well and sample well, seal the reaction wells with a sealing film, and incubate at 37°C in a water bath or incubator for 60min. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times (you can also use a plate washer to wash the plate). Add 50μL of substrate A and B to each well and incubate at 37°C in the dark for 15min. Add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 min, and bring in the standard curve.

[0080] Brassinolide (BR) content: obtained from Jiangsu Yutong Biotechnology Co., Ltd., item number BLL105482E. Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips with a ziplock bag and return to 4°C. Set up standard wells and sample wells, add 50μL of different concentrations of standard wells to each standard well; add 10μL of the sample to be tested to the sample well, and then add 40μL of sample diluent; do not add to the blank well. Except for the blank well, add 100μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard well and sample well, seal the reaction well with a sealing film, and incubate at 37°C in a water bath or incubator for 60min. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times (you can also use a plate washer to wash the plate). Add 50μL of substrate A and B to each well and incubate at 37°C in the dark for 15min. Add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 min, and bring in the standard curve.

[0081] Ethylene content: obtained from Jiangsu Yutong Biotechnology Co., Ltd. Take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, and seal the remaining strips with a ziplock bag and return them to 4°C. Set up standard wells and sample wells, add 50μL of different concentrations of standard wells to each standard well; add 10μL of the sample to be tested to the sample well first, and then add 40μL of sample diluent; do not add to the blank well. Except for the blank well, add 100μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard well and sample well, seal the reaction well with a sealing film, and incubate at 37°C in a water bath or incubator for 60min. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution, let stand for 1min, shake off the washing solution, pat dry on absorbent paper, and repeat the washing process 5 times (you can also use a plate washer to wash the plate). Add 50μL of substrate A and B to each well and incubate at 37°C in the dark for 15min. Add 50 μL of stop solution to each well, measure the OD value of each well at a wavelength of 450 nm within 15 min, and bring in the standard curve.

[0082] 1.2 Experimental Results

[0083] After applying apigenin, the content of oxides decreased significantly, and the content of antioxidant enzymes, chlorophyll and plant hormones increased significantly. Figure 3 , Figure 4 , Figure 5 ; Figure 3 , Figure 4 and Figure 5 Control is the 25% PEG culture medium group, 20μM is the 25% PEG + 20μM apigenin culture medium group, 50μM is the 25% PEG + 50μM apigenin culture medium group, and 1000μM is the 25% PEG + 1000μM apigenin culture medium group; Figure 3 In the figure, A is the chlorophyll a content, B is the chlorophyll b content, C is the chlorophyll content, and D is the carotenoid content; Figure 4 In the table, A is the superoxide anion content, B is the POD activity, and C is the soluble protein content; Figure 5 In the graph, A is the ABA content, B is the salicylic acid (SA) content, C is the brassinolide (BR) content, and D is the ethylene content).

[0084] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of apigenin or its salt or its solvate in any of the following: A1) Application in improving drought tolerance of grass plants; A2) Application in the preparation of products for improving drought tolerance of Gramineae plants.

2. The use according to claim 1, characterized in that The product for improving drought resistance of gramineous plants is a drought-resistant preparation.

3. The use according to claim 2, characterized in that The drought-resistant preparation is a liquid preparation.

4. The use according to claim 3, characterized in that The effective concentration of apigenin in the liquid is 20 μM-100 μM.

5. A method for improving drought tolerance of Gramineae plants, characterized in that: The method comprises applying apigenin or its salt, or their solvate to Gramineae plants to improve the drought tolerance of the plants.

6. The method according to claim 5, characterized in that The application is effected by root application.

7. The method according to claim 5, characterized in that The dosage form of the apigenin or its salt, or their solvate is a liquid.

8. The method according to claim 6 or 7, characterized in that The concentration of apigenin in the liquid used is 20 μM-100 μM.

9. The method according to claim 8, characterized in that The concentration of apigenin in the seed coating was 20 or 100 μM.

10. The use according to any one of claims 1 to 4 or the method according to any one of claims 6 to 9, characterized in that: The plant may be any of the following: D1) Triticum plants; D2) Wheat.

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