Application of exogenous spermine in relieving stress effect of Cd on plants

Abstract

The invention belongs to the technical field of plant cultivation, and particularly discloses an application of exogenous spermine (Spm) in relieving stress of Cd on plants, an okra variety, Taiwan Wufu, China, is taken as a test material, and the relationship between the Spm and osmotic regulation, leaf damage, photosynthesis, an antioxidant enzyme system and a cell membrane system of the okra under stress of CdCl2 is researched. The action mechanism of Spm in resisting heavy metal stress by plants is discussed, and a theoretical basis is provided for safe production of okra; the invention has wide application space and market prospect in the field of agriculture.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant cultivation, and relates to the research on plant growth under heavy metal stress, in particular to the application of exogenous spermine in relieving the stress of Cd on plants. BACKGROUND

[0002] Cadmium is a non-essential nutrient element for plants. Low concentration of cadmium can promote the growth of plants to a certain extent, but high concentration of cadmium has a great toxic effect on plants. With the development of industry, industrial wastewater and pollutants enter the soil to pollute the environment, and the problem of cadmium pollution in soil in China is serious. Studies have shown that excessive cadmium can also cause plant stress, inhibit the synthesis of chlorophyll and photosynthesis, and under cadmium stress, plants will also show symptoms such as growth retardation, dwarf, chlorosis and yield reduction. High concentration of cadmium can destroy the active oxygen metabolism balance in the plant body, and the accumulation of active oxygen increases to cause membrane lipid peroxidation, and the activity of antioxidant enzymes in the plant body decreases.

[0003] Okra contains rich protein, free amino acids, carotenoids, various vitamins and mineral elements such as phosphorus, iron, potassium and calcium, and sticky substances composed of pectin and polysaccharides, and has many health care functions, so it is deeply welcomed by consumers. Studies have shown that the growth of okra leaves and the activity of antioxidant enzymes are inhibited under cadmium stress, the quality of okra fruits decreases under cadmium stress, and the accumulation of cadmium in the fruits also affects human health. Therefore, it is very important to alleviate the damage of cadmium stress to okra.

[0004] Heavy metal pollution has a great impact on the growth and development of plants, and how to improve the tolerance of plants to heavy metal pollution has become an important problem in the current research field. Polyamines are small molecule aliphatic nitrogen bases produced in the metabolic process of organisms, which are the general term of putrescine (Put), spermidine (SPD), spermine (Spm) and cadaverine (CAD), have biological activity, and widely exist in biological organisms. They not only participate in the basic metabolism of cells such as cell proliferation, differentiation and programmed death, but also participate in the response to environmental stress.

[0005] Among them, the application of Spm in plant breeding has been widely reported, such as the prior art (Wang W, Shi S, Kang W, He L, Nan P. Effects of Spermine Seed Soaking on Seed Germination and Seedling Growth of Medicago polymorpha under Drought Stress [J]. Grassland and Turf, 2025, (No. 4).) explored the effects of spermine (Spm) seed soaking on seed germination and seedling growth of Medicago polymorpha under drought stress. However, the research on the effect of Spm on plant heavy metal stress resistance is still blank. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide application of exogenous spermine in relieving stress of CdCl2 on okra, the present application takes okra variety China Taiwan Wufu as test material, studies the relationship between spermine and CdCl2 stress of okra in osmotic regulation, leaf damage, photosynthesis, antioxidant enzyme system and the like, and explores the mechanism of spermine in resisting heavy metal stress of plants, so as to provide a theoretical basis for safe production of okra.

[0007] The present application is realized by the following technical solutions: Application of Spm in relieving stress damage of CdCl2 on plants or improving CdCl2 stress resistance of plants.

[0008] Further, the plant is a dicotyledon, a malvaceae plant or okra.

[0009] Further, the application process specifically is that the plant is sprayed with Spm with a concentration of 0.1-10.0 mmol / L.

[0010] Further improvement schemes of the present application are: The application of Spm is at least one of the following (a1)-(a28): (a1) increasing proline content of okra under CdCl2 stress; (a2) preparing a product for increasing proline content of okra under CdCl2 stress; (a3) increasing soluble protein content of okra under CdCl2 stress; (a4) preparing a product for increasing soluble protein content of okra under CdCl2 stress; (a5) increasing chlorophyll a content of okra under CdCl2 stress; (a6) preparing a product for increasing chlorophyll a content of okra under CdCl2 stress; (a7) increasing chlorophyll b content of okra under CdCl2 stress; (a8) preparing a product for increasing chlorophyll b content of okra under CdCl2 stress; (a9) increasing total chlorophyll content of okra under CdCl2 stress; (a10) preparing a product for increasing total chlorophyll content of okra under CdCl2 stress; (a11) increasing total carotenoid content of okra under CdCl2 stress; (a12) preparing a product for increasing total carotenoid content of okra under CdCl2 stress; (a13) increasing SOD activity of okra under CdCl2 stress; (a14) preparing a product for improving SOD activity of okra under CdCl2 stress; (a15) improving CAT activity of okra under CdCl2 stress; (a16) preparing a product for improving CAT activity of okra under CdCl2 stress; (a17) improving APX activity of okra under CdCl2 stress; (a18) preparing a product for improving APX activity of okra under CdCl2 stress; (a19) improving POD activity of okra under CdCl2 stress; (a20) preparing a product for improving POD activity of okra under CdCl2 stress; (a21) reducing MDA content of okra under CdCl2 stress; (a22) preparing a product for reducing MDA content of okra under CdCl2 stress; (a23) reducing electrolyte permeability of okra under CdCl2 stress; (a24) preparing a product for reducing electrolyte permeability of okra under CdCl2 stress; (a25) reducing H2O2 content of okra under CdCl2 stress; (a26) preparing a product for reducing H2O2 content of okra under CdCl2 stress; (a27) reducing O 2- content of okra under CdCl2 stress; (a28) preparing a product for reducing O 2- content of okra under CdCl2 stress.

[0011] Further, the application process is: using Spm with a concentration of 0.1 - 10.0 mmol / L to carry out foliar spraying on okra seedlings.

[0012] Further improvement of the application is: An okra cultivation method, comprising the following steps: using Spm with a concentration of 0.1 - 10.0 mmol / L to carry out foliar spraying on the okra.

[0013] Preferably, the concentration of the Spm is 0.1 - 1.0 mmol / L; More preferably, the concentration of the Spm is 1.0 mmol / L.

[0014] A method for relieving CdCl2 stress damage of okra, comprising the following steps: using Spm with a concentration of 0.1 - 10.0 mmol / L to carry out foliar spraying on okra seedlings.

[0015] Preferably, the concentration of the Spm is 0.1-1.0 mmol / L. More preferably, the concentration of the Spm is 1.0 mmol / L.

[0016] The application also protects the application of Spm in the breeding of CdCl2 stress-resistant okra.

[0017] Compared with the prior art, the application has the following beneficial effects: The application provides an application of exogenous Spm in relieving the stress of CdCl2 on okra seedlings: spraying exogenous Spm can improve the CdCl2 stress resistance of okra plants. The results of the 0.1 mmol / L CdCl2 stress germination rate test show that the germination rate and fresh weight of okra seeds treated with 0.1 mmol / L, 1.0 mmol / L and 10.0 mmol / L Spm are significantly higher than those of the 0.0 mmol / L Spm treatment, and are increased by 66%, 140%, 43% and 91%, 151%, 65% respectively compared with the 0.0 mmol / L Spm plants, and the effect of the okra seedlings treated with 1.0 mmol / L Spm is the best. The results of the 0.1 mmol / L CdCl2 stress potting test show that the okra seedlings treated with exogenous Spm grow well and have improved resistance, and the fresh weight and dry weight of the potting okra seedlings treated with 0.1 mmol / L, 1.0 mmol / L and 10.0 mmol / L Spm are significantly higher than those of the 0.0 mmol / L Spm treatment, and are increased by 103%, 173%, 32% and 231%, 383%, 47% respectively compared with the 0.0 mmol / L Spm plants, and the effect of the okra seedlings treated with 1.0 mmol / L Spm is the best, and the okra seedlings express very strong CdCl2 stress resistance; specifically, the proline content, soluble protein content, chlorophyll a content, chlorophyll b content, total chlorophyll content, total carotenoid content, SOD activity, CAT activity, APX activity, POD activity, malondialdehyde content, electrolyte permeability, H2O2 content and O 2- content of the okra seedlings treated with Spm are increased, and the O

[0018] The results show that the Spm has important application in relieving the stress of CdCl2 on the okra seedlings, the Spm can promote the growth of the okra seedlings under the stress of CdCl2 and improve the tolerance of the okra seedlings to the stress of CdCl2 by using the Spm with a determined concentration as a crop relieving agent, the relationship between the Spm and the osmosis regulation, leaf damage, photosynthesis, antioxidant enzyme system and cell membrane system of the okra under the stress of CdCl2 is researched, the mechanism of the Spm in resisting the stress of heavy metals is discussed, and a theoretical basis is provided for safe production of the okra, and the application space and market prospect of the application in the agricultural field are wide. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 The effects of different concentrations (0.0, 0.1, 1.0 and 10.0 mmol / L) of Spm on the germination rate and fresh weight of okra seedlings under normal conditions and the stress of 0.1 mmol / L CdCl2 are determined.

[0020] Figure 2 The effects of different concentrations (0.0, 0.1, 1.0 and 10.0 mmol / L) of Spm on the relieving effect resistance of okra seedlings under normal conditions and the stress of 0.1 mmol / L CdCl2 are determined.

[0021] Figure 3 The effects of different concentrations (0.0, 0.1, 1.0 and 10.0 mmol / L) of Spm on the proline and soluble protein contents of okra seedlings under normal conditions and the stress of 0.1 mmol / L CdCl2 are determined.

[0022] Figure 4 The effects of different concentrations (0.0, 0.1, 1.0 and 10.0 mmol / L) of Spm on the photosynthetic pigment contents (chlorophyll a, chlorophyll a, total chlorophyll and total carotenoids) of okra seedlings under normal conditions and the stress of 0.1 mmol / L CdCl2 are determined.

[0023] Figure 5 The effects of different concentrations (0.0, 0.1, 1.0 and 10.0 mmol / L) of Spm on the malondialdehyde content, electrolyte permeability, H2O2 content and O 2- content of okra seedlings under normal conditions and the stress of 0.1 mmol / L CdCl2 are determined.

[0024] Figure 6 The effects of different concentrations (0.0, 0.1, 1.0 and 10.0 mmol / L) of Spm on the activities of key enzymes (SOD, CAT, APX and POD) of ROS scavenging system of okra seedlings under normal conditions and the stress of 0.1 mmol / L CdCl2 are determined. Detailed Implementation

[0025] The present invention will now be described in detail with reference to specific embodiments.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples are all commercially available biochemical reagents. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0027] Okra ( Abelmoschus esculentus The okra variety Wufu from Taiwan, China, was used as the experimental material. Reference: [Feibing Wang, Gaolei Ren, Fengsheng Li, SitongQi, Yan Xu, Bowen Wang, Yulin Yang, Yuxiu Ye, Qing Zhou, Xinhong Chen. Achalcone synthase gene] AeCHS from Abelmoschus esculentus regulates flavonoidaccumulation and abiotic stress tolerance in transgenic Arabidopsis [2018, 40:97], preserved by the Jiangsu Provincial Plant Production and Processing Practice Education Center Laboratory, College of Life Sciences and Food Engineering, Huaiyin Institute of Technology.

[0028] Spm (Spermine, abbreviated Spm) was purchased from Sigma.

[0029] The Cd donor was cadmium chloride (CdCl2·2H2O), purchased from Sigma-Aldrich.

[0030] Example 1: Analysis of CdCl2 stress resistance in okra seedlings regulated by exogenous Spm 1. Analysis of germination rate of okra seeds under CdCl2 stress after treatment with different concentrations of Spm (1) Test methods The method of Wang Yonghui et al. (2014) was used to select 50 seeds of the same size from okra seed material. The seeds were disinfected with 5% hypochlorous acid solution for 5 min, then washed with distilled water for 3 times. The seeds were soaked in distilled water and 0.1 mmol / L CdCl2 solution at room temperature for 24 hours, respectively. Then the seeds were placed in a transparent plastic preservation box with two layers of filter paper with an inner diameter of 12 cm. Different concentrations (0, 0.1, 1.0, 10.0 mmol / L) of Spm were used for treatment, including: (i) distilled water treatment under normal conditions; (ii) 0.1 mmol / L CdCl2 with the addition of 0.0, 0.1, 1.0 and 10.0 mmol / L Spm, respectively. Each treatment was repeated six times. The preservation box was placed in an artificial climate chamber for germination, with white / day temperature of 28℃ / 25℃, 12 hours of white / day and black / day, and humidity of 60% ± 1%. The 0.2 mm of radicle length was used as the seed germination marker, and the number of seeds germinated on the 5th day was counted. The seed germination rate = (the number of seeds germinated on the 5th day / total number of seeds tested) x 100%.

[0031] (2) Test results The results showed that the 0.1 mmol / L CdCl2 stress germination rate test results showed that the germination rate of okra seeds treated with 0.1, 1.0, 10.0 mmol / L Spm was significantly higher than that of 0.0 mmol / L Spm treatment. Figure 1 A) and fresh weight ( Figure 1 B) were significantly higher than 0.0 mmol / L Spm treatment, which increased by 66%, 140%, 43% and 91%, 151%, 65%, respectively, compared with 0.0 mmol / L Spm plants, and the effect of 1.0 mmol / L Spm treatment on okra seedlings was the best.

[0032] 2. Analysis of okra seedling resistance to CdCl2 stress after treatment with different concentrations of Spm (1) Test method Methods: The methods of Li et al. (2022) [Hengpeng Li, Shasha Yang, Wenya Wu, Chunyan Wang, Yanyang Li, Chenzhong Wan, Yuxiu Ye, Xinhong Chen, Zunxin Wang, Laibao Hu, Feibing Wang. Physiological and biochemical mechanisms of improving salt and drought tolerance in okra plants based on applied attapulgite clay. Advances in Biochemistry, 2022, 10:1-10] were used to transplant the seedlings of Abelmoschus manihot L. var. Taiwan Wu-Fu to plastic pots (19 cm in diameter) containing a mixture of turf, humus, and vermiculite (1:1:1, v / v / v) in a greenhouse. All seedlings were watered with half-Hogland solution for 4 weeks until new leaves grew. Subsequently, CdCl2 stress and Spm treatment were performed, including: (i) normal conditions, each pot of okra seedling plants was irrigated with water every 2 days for 4 weeks, and sprayed with 0.0, 0.1, 1.0, and 10.0 mmol / L Spm solution every day for 3 times.

[0033] (2) Test results The results showed that the pot experiment results of 0.1 mmol / L CdCl2 stress indicated that the growth of okra seedling plants treated with exogenous Spm was good and the resistance was improved Figure 2 A), the fresh weight (B) and dry weight (C) of pot-grown okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm were significantly higher than those treated with 0.0 mmol / L Spm, which increased by 103%, 173%, 32%, and 231%, 383%, 47%, respectively, compared with 0.0 mmol / L Spm plants, among which the effect of 1.0 mmol / L Spm treatment on okra seedlings was the best, showing very strong CdCl2 stress resistance. Figure 2 Figure 2 Therefore, the results of phenotypic identification showed that the okra plants treated with exogenous 1.0 mmol / L Spm had the best CdCl2 stress resistance phenotype, which was used to analyze the physiological and biochemical mechanisms of inducing CdCl2 stress resistance.

[0034] Therefore, the results of phenotypic identification showed that the okra plants treated with exogenous 1.0 mmol / L Spm had the best CdCl2 stress resistance phenotype, which was used to analyze the physiological and biochemical mechanisms of inducing CdCl2 stress resistance. ​

[0035] Example 2: Determination of physiological and biochemical indicators of resistance of okra seedlings to CdCl2 stress 1. Proline content determination (1) Test method Under normal conditions, the free proline content of plants is very low, but when subjected to salt, drought and other stresses, free amino acids will accumulate in large quantities, and the accumulation index is related to the stress resistance of plants. Therefore, proline can be used as a biochemical indicator of plant stress resistance.

[0036] The determination method is described in the reference

Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao. AtMYB12 regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana . Molecular Genetics and Genomics, 2016, 291:1545-1559

[0037] (2) Test results The experimental results are shown in Figure 3 A. The results showed that after 0.1 mmol / L CdCl2 stress treatment, the proline content of the pot-grown okra seedlings treated with 0.1, 1.0 and 10.0 mmol / L Spm was significantly higher than that treated with 0.0 mmol / L Spm, which increased by 57%, 75% and 39% respectively compared with 0.0 mmol / L Spm plants, and the proline content of the okra seedlings treated with 1.0 mmol / L Spm was significantly higher than that of the okra plants without Spm treatment.

[0038] 2. Determination of soluble protein content (1) Test method Plant soluble proteins have a protective effect on cell membranes and are also the material basis for maintaining normal life activities of plants, and play an important protective role in the process of plants suffering from high temperature stress.

[0039] The determination method is referenced in the following literature: [Hong Zhai, Feibing Wang, Zengzhi Si, Jinxi Huo, LeiXing, Yanyan An, Shaozhen He, Qingchang Liu. A] myo -inositol-1-phosphatesynthase gene, IbMIPS1 [This study, published in Plant Biotechnology Journal, 2016, 14:592-602, investigated the soluble protein content of okra seedlings. The okra plants were leaves from okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm aqueous solution for two weeks under both the unstressed and pot-grown conditions described above. The experiment was repeated three times, and the average value was taken.]

[0040] (2) Test results The experimental results are shown in Figure 3 The results showed that after treatment with 0.1 mmol / L CdCl2, the soluble protein content of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm was significantly higher than that treated with 0.0 mmol / L Spm, increasing by 3%, 38%, and 8%, respectively, compared with plants treated with 0.0 mmol / L Spm. Among them, the soluble protein content of okra seedlings treated with 1.0 mmol / L Spm was significantly higher than that of okra plants not treated with Spm.

[0041] 3. Determination of photosynthetic pigment content (1) Test methods Chloroplasts are important organelles in plants, responsible for physiological processes such as photosynthesis and responses to abiotic stress. Photosynthetic pigments in chloroplasts include chlorophyll and carotenoids, with chlorophyll further divided into chlorophyll a and chlorophyll b. Abiotic stress affects plant photosynthesis, increasing the number of free radicals in chloroplasts, which destroy chlorophyll and thus impair photosynthesis. The content of photosynthetic pigments reflects a plant's ability to perform photosynthesis.

[0042] The determination method was based on the reference [Lichtenthaler Hartmut K, Buschmann Claus. Chlorophylls and carotenoids: measurement and characterization by UV-VISspectroscop. Current Protocols in Food Analytical Chemistry, 2001, 1], and the photosynthetic pigment content of okra seedlings was detected. The okra plants were leaves from okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm aqueous solution for two weeks under both the unstressed and 0.1 mmol / L CdCl2 treatments in the potted plant identification process. The experiment was repeated three times, and the average value was taken.

[0043] (2) Test results The experimental results are shown in Figure 4 The results showed that after treatment with 0.1 mmol / L CdCl2, the chlorophyll a content of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm was significantly reduced. Figure 4 A) Chlorophyll b ( Figure 4 B) Total chlorophyll ( Figure 4 C) and total carotenoid content ( Figure 4 D) The contents were significantly higher than those treated with 0.0 mmol / L Spm, increasing by 40%, 54%, 13% and 59%, 72%, 56% and 43%, 57%, 20% and 72%, 107%, and 48% respectively compared with plants treated with 0.0 mmol / L Spm. Among them, the contents of chlorophyll a, chlorophyll b, total chlorophyll and total carotenoids in okra seedlings treated with 1.0 mmol / L Spm were significantly higher than those in okra plants not treated with Spm.

[0044] 4. MDA content determination (1) Test methods When plant organs age or are damaged under stress, membrane lipid peroxidation often occurs. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of plant stress damage. That is, the higher the MDA content, the greater the degree of plant stress damage.

[0045] The determination method is referenced in the following literature: [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao]. AtMYB12regulates flavonoids accumulation andabiotic stress tolerance in transgenic Arabidopsis thaliana [Molecular Genetics and Genomics, 2016, 291:1545-1559], to detect the MDA content of okra seedlings. Okra plants were leaves from okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm aqueous solution for two weeks under both the unstressed and pot-grown conditions described above. The experiment was repeated three times, and the average value was taken.

[0046] (2) Test results The experimental results are shown in Figure 5 The results showed that after treatment with 0.1 mmol / L CdCl2 stress, the MDA content of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm was significantly lower than that treated with 0.0 mmol / L Spm, decreasing by 47%, 61%, and 39% respectively compared with plants treated with 0.0 mmol / L Spm. Among them, the MDA content of okra seedlings treated with 1.0 mmol / L Spm was significantly lower than that of okra plants not treated with Spm.

[0047] 5. Electrolyte permeability measurement (1) Test methods Electrolyte permeability is an indicator of the permeability of plant cell membranes. The lower the value, the more intact the cell membrane and the better its function.

[0048] The determination method is referenced in the following literature: [Yujia Liu, Xiaoyu Ji, Xianguang Nie, Min Qu, LeiZheng, Zilong Tan, Huimin Zhao, Lin Huo, Shengnan Liu, Bing Zhang, YuchengWang]. ArabidopsisAtbHLH112 regulates the expression of genes involved in inabiotic stress tolerance by binding to their E-box and GCG-box motifs. [NewPhytologist, 2015, 207:692-709] Electrolyte osmotic rate of okra seedlings was detected. Okra plants were leaves from okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm aqueous solution for two weeks under the above-mentioned potted plant identification without stress and under the above-mentioned potted plant identification with 0.1 mmol / L CdCl2 treatment. The experiment was repeated three times, and the results were averaged.

[0049] (2) Test results The experimental results are shown in Figure 5 The results showed that after treatment with 0.1 mmol / L CdCl2 stress, the electrolyte osmotic rates of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm were significantly lower than those treated with 0.0 mmol / L Spm, decreasing by 50%, 59%, and 33%, respectively, compared with plants treated with 0.0 mmol / L Spm. Among them, the electrolyte osmotic rate of okra seedlings treated with 1.0 mmol / L Spm was significantly lower than that of okra plants not treated with Spm.

[0050] 6. Determination of H2O2 content (1) Test methods When plants are under stress or aging, the increased metabolism of reactive oxygen species (ROS) leads to the accumulation of H2O2. H2O2 can directly or indirectly oxidize intracellular macromolecules such as nucleic acids and proteins, and damage cell membranes, thereby accelerating cell aging and disintegration. Therefore, the higher the H2O2 content, the greater the degree of damage the plant suffers from environmental stress.

[0051] The determination method is referenced in the following literature: [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao]. AtMYB12 regulates flavonoids accumulation andabiotic stress tolerance in transgenic Arabidopsis thaliana[Molecular Genetics and Genomics, 2016, 291:1545-1559], to detect the H2O2 content in okra seedlings. Okra plants were leaves from okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm solution for two weeks under both the unstressed and pot-grown conditions described above. The experiment was repeated three times, and the average result was taken.

[0052] (2) Test results The experimental results are shown in Figure 5 The results showed that after treatment with 0.1 mmol / L CdCl2 stress, the H2O2 content of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm was significantly lower than that of the 0.0 mmol / L Spm treatment, decreasing by 46%, 67%, and 29% respectively compared with the 0.0 mmol / L Spm plants. Among them, the H2O2 content of okra seedlings treated with 1.0 mmol / L Spm was significantly lower than that of okra plants not treated with Spm.

[0053] 7. O 2- Content determination (1) Test methods Superoxide anion (O 2- Free radicals are reactive oxygen species produced by plants under stress conditions. They can directly act on biomolecules such as proteins and nucleic acids, or they can be derived into other reactive oxygen species, causing damage to cell structure and function. Therefore, measuring the production and scavenging rate of superoxide anion free radicals in plant tissues under stress conditions can indirectly reveal the extent of tissue and cell damage and the strength of resistance. 2- Its content is closely related to plant stress resistance, and its content can be used as an indicator to assess the strength of plant resistance.

[0054] Determination method reference [Feibing Wang, Chenzhong Wan, Wenya Wu, Yanning Zhang, Yuanxin Pan, Xiaomeng Chen, Chun Li, Jiali Pi, Zunxin Wang, Yuxiu Ye, Xinhong Chen. Methyl jasmonate (MeJA) enhances salt tolerance of okra ( Abelmoschus esculentusL.) plants by regulating ABA signaling, osmotic adjustment substances, photosynthesis and ROS metabolism. Scientia Horticulturae, 2023, 319:112145, Detection of O in okra seedlings 2- Content. Okra plants were leaves of okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm aqueous solution for two weeks under the above-mentioned unstressed and potted plant identification conditions, respectively. The experiment was repeated three times, and the average value was taken.

[0055] (2) Test results The experimental results are shown in Figure 5 The results showed that after treatment with 0.1 mmol / L CdCl2, the levels of O2 in potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm were significantly reduced. 2- The O content was significantly lower than that of the 0.0 mmol / L Spm treatment, decreasing by 38%, 58%, and 11% respectively compared to plants treated with 0.0 mmol / L Spm. Specifically, the O content in okra seedlings treated with 1.0 mmol / L Spm was significantly lower. 2- The content was significantly lower than that of okra plants that were not treated with Spm.

[0056] 8. SOD activity assay (1) Test methods Superoxide dismutase (SOD) activity can serve as a physiological and biochemical indicator of plant stress resistance. The lower the SOD activity, the greater the degree of stress damage suffered by the plant.

[0057] The determination method is referenced in the following literature: [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao]. AtMYB12 regulates flavonoids accumulation andabiotic stress tolerance in transgenic Arabidopsis thaliana[Molecular Genetics and Genomics, 2016, 291:1545-1559], to detect SOD activity in okra seedlings. Okra plants were leaves from okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm solution for two weeks under both the unstressed and 0.1 mmol / L CdCl2 treatments in the pot experiment. The experiment was repeated three times, and the average value was taken.

[0058] (2) Test results The experimental results are shown in Figure 6 The results showed that after treatment with 0.1 mmol / L CdCl2 stress, the SOD activity of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm was significantly higher than that treated with 0.0 mmol / L Spm, increasing by 7%, 30%, and 6% respectively compared with plants treated with 0.0 mmol / L Spm. Among them, the SOD activity of okra seedlings treated with 1.0 mmol / L Spm was significantly higher than that of okra plants not treated with Spm.

[0059] 9. CAT Activity Assay (1) Test methods Catalase (CAT) is widely present in plant tissues and is one of the important protective enzymes. Its function is to remove H2O2 produced during metabolism to avoid the oxidative damage to cells caused by the accumulation of H2O2. Therefore, its activity level is related to the plant's stress resistance.

[0060] The determination method reference is: [Yufeng Yang, Shikai Guan, Hong Zhai, Shaozhen He, Qingchang Liu. Development and evaluation of a storage root-bearing sweet potato somatic hybrid between] Ipomoea batatas (L.) Lam. and I. triloba [L. Plant Cell, Tissue and Organ Culture, 2009, 99:83-89], to detect CAT activity in okra seedlings. Okra plants were leaves from okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm aqueous solution for two weeks under both the unstressed and pot-grown conditions described above. The experiment was repeated three times, and the average value was taken.

[0061] (2) Test results The experimental results are shown in Figure 6 The results showed that after treatment with 0.1 mmol / L CdCl2 stress, the CAT activity of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm was significantly higher than that treated with 0.0 mmol / L Spm, increasing by 77%, 142%, and 29% respectively compared with plants treated with 0.0 mmol / L Spm. Among them, the CAT activity of okra seedlings treated with 1.0 mmol / L Spm was significantly higher than that of okra plants not treated with Spm.

[0062] 10. APX Activity Assay (1) Test methods Ascorbate peroxidase (APX) is one of the important antioxidant enzymes in plant reactive oxygen species metabolism, especially a key enzyme in chloroplasts for scavenging H2O2, and also a major enzyme in vitamin C metabolism. APX activity can serve as a physiological and biochemical indicator of plant stress resistance, and its level is related to plant stress resistance.

[0063] The determination method is referenced in the following literature: [Hong Zhai, Feibing Wang, Zengzhi Si, Jinxi Huo, LeiXing, Yanyan An, Shaozhen He, Qingchang Liu. A] myo -inositol-1-phosphatesynthase gene, IbMIPS1 [This study investigated the APX activity in okra seedlings. The okra seedlings were selected from leaves of okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm aqueous solution for two weeks under both the no-stress and 0.1 mmol / L CdCl2 treatments in the pot experiments. The experiment was repeated three times, and the average value was taken.]

[0064] (2) Test results The experimental results are shown in Figure 6The results showed that after treatment with 0.1 mmol / L CdCl2 stress, the APX activity of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm was significantly higher than that treated with 0.0 mmol / L Spm, increasing by 41%, 51%, and 16% respectively compared with plants treated with 0.0 mmol / L Spm. Among them, the APX activity of okra seedlings treated with 1.0 mmol / L Spm was significantly higher than that of okra plants not treated with Spm.

[0065] 11. POD activity assay (1) Test methods Peroxidase (POD) activity can serve as a physiological and biochemical indicator of plant stress resistance. The lower the POD activity, the greater the degree of stress damage suffered by the plant.

[0066] The determination method is referenced in the following literature: [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao]. AtMYB12 regulates flavonoids accumulation andabiotic stress tolerance in transgenic Arabidopsis thaliana [Molecular Genetics and Genomics, 2016, 291:1545-1559], to detect POD activity in okra seedlings. Okra plants were leaves from okra seedlings treated with different concentrations (0.0, 0.1, 1.0, and 10.0 mmol / L) of Spm aqueous solution for two weeks under both the unstressed and pot-grown conditions described above. The experiment was repeated three times, and the average result was taken.

[0067] (2) Test results The experimental results are shown in Figure 6 The results showed that after treatment with 0.1 mmol / L CdCl2 stress, the POD activity of potted okra seedlings treated with 0.1, 1.0, and 10.0 mmol / L Spm was significantly higher than that treated with 0.0 mmol / L Spm, increasing by 35%, 82%, and 41% respectively compared with plants treated with 0.0 mmol / L Spm. Among them, the POD activity of okra seedlings treated with 1.0 mmol / L Spm was significantly higher than that of okra plants not treated with Spm.

[0068] The results of physiological and biochemical index measurements showed that exogenous Spm treatment significantly improved the CdCl2 stress resistance of okra plants. This study investigated the relationship between Spm and CdCl2 stress on okra's osmotic regulation, leaf damage, photosynthesis, antioxidant enzyme system, and cell membrane system, exploring the mechanism of Spm in plant resistance to heavy metal stress and providing a theoretical basis for its safe production in okra.

[0069] The above description of the embodiments is only for illustrating the technical concept and features of the present invention. Its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. Those skilled in the art can obviously easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the above embodiments should not be used to limit the scope of protection of the present invention. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be covered within the scope of protection of the present invention.

Claims

1. Application of Spm in alleviating CdCl2 stress damage in plants or improving CdCl2 stress resistance in plants.

2. The application according to claim 1, characterized in that: The plant in question is a dicotyledonous plant, a plant of the Malvaceae family, or okra.

3. The application according to claim 1 or 2, characterized in that: Spraying was performed on the plants using Spm at a concentration of 0.1-10.0 mmol / L.

4. The application of Spm is characterized by, It is at least one of the following (a1)-(a28): (a1) Increase the proline content of okra under CdCl2 stress; (a2) Prepare a product that increases the proline content of okra under CdCl2 stress; (a3) Increase the soluble protein content of okra under CdCl2 stress; (a4) Prepare a product that increases the soluble protein content of okra under CdCl2 stress; (a5) Increase the chlorophyll a content of okra under CdCl2 stress; (a6) Prepare a product that increases the chlorophyll a content of okra under CdCl2 stress; (a7) Increase the chlorophyll b content of okra under CdCl2 stress; (a8) Prepare a product that increases the chlorophyll b content of okra under CdCl2 stress; (a9) Increase the total chlorophyll content of okra under CdCl2 stress; (a10) Prepare a product that increases the total chlorophyll content of okra under CdCl2 stress; (a11) Increase the total carotenoid content of okra under CdCl2 stress; (a12) Prepare a product that increases the total carotenoid content of okra under CdCl2 stress; (a13) Increases the SOD activity of okra under CdCl2 stress; (a14) Prepare a product that enhances the SOD activity of okra under CdCl2 stress; (a15) Increases CAT activity in okra under CdCl2 stress; (a16) Prepare a product that enhances the CAT activity of okra under CdCl2 stress; (a17) Increases APX activity in okra under CdCl2 stress; (a18) Prepare a product that enhances the APX activity of okra under CdCl2 stress; (a19) Increases the POD activity of okra under CdCl2 stress; (a20) Prepare a product that improves the POD activity of okra under CdCl2 stress; (a21) Reduces the MDA content of okra under CdCl2 stress; (a22) Prepare a product that reduces the MDA content of okra under CdCl2 stress; (a23) Reduce the electrolyte permeability of okra under CdCl2 stress; (a24) Prepare a product that reduces the electrolyte permeability of okra under CdCl2 stress; (a25) Reduce the H2O2 content of okra under CdCl2 stress; (a26) Prepare a product that reduces the H2O2 content of okra under CdCl2 stress; (a27) Reduces O3 in okra under CdCl2 stress 2- content; (a28) Preparation of O2-reducing agent for okra under CdCl2 stress 2- Products with high content.

5. The application according to claim 4, characterized in that: Spraying okra seedlings with Spm at a concentration of 0.1-10.0 mmol / L was used.

6. A method for cultivating okra, characterized in that, The okra was foliar sprayed with Spm at a concentration of 0.1-10.0 mmol / L.

7. A method for alleviating CdCl2 stress injury in okra, characterized in that: Foliar spraying of okra seedlings was carried out using Spm at a concentration of 0.1-10.0 mmol / L.

8. The method according to claim 6 or 7, characterized in that: The okra seedlings were foliar sprayed with Spm at a concentration of 0.1-1.0 mmol / L.

9. The method according to claim 8, characterized in that: The okra seedlings were foliar sprayed with Spm at a concentration of 1.0 mmol / L.

10. Application of Spm in the breeding of okra resistant to CdCl2 stress.

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