Application of exogenous choline chloride in alleviating CdCl2 stress on okra seedlings

Abstract

The invention discloses the application of exogenous choline chloride in alleviating the stress effect of CdCl2 on okra seedlings, and belongs to the research field of technology for alleviating plant heavy metal stress. The invention protects the application of choline chloride in alleviating the damage of okra caused by CdCl2 stress. The invention uses the okra variety Taiwan Wufu as the test material, studies the relationship between choline chloride and the leaf damage, photosynthesis, antioxidant enzyme system, and cell membrane system of okra under CdCl2 stress, explores the mechanism of action of choline chloride in plants resisting heavy metal stress, and provides a theoretical basis for its safe production in okra; the invention will have broad application space and market prospects in the agricultural field.

Description

Technical Field

[0001] The invention belongs to the research field of technology for alleviating heavy metal stress in plants, and specifically relates to a method for alleviating the stress of okra seedlings with CdCl 2 Application in stress injury. Background Art

[0002] Cadmium is a non-essential nutrient element for plants. Low concentrations of cadmium promote plant growth to a certain extent, but high concentrations of cadmium are highly toxic to plants. With the development of industry, industrial wastewater and pollutants enter the soil and pollute the environment. The problem of cadmium pollution in my country's soil is serious. Studies have shown that excessive cadmium can also cause stress in plants, inhibit the synthesis of chlorophyll and photosynthesis. Under cadmium stress, plants will also show symptoms such as slow growth, short plants, chlorosis, and reduced yields. High concentrations of cadmium will destroy the metabolic balance of active oxygen in plants. The increased accumulation of active oxygen leads to membrane lipid peroxidation and reduced activity of antioxidant enzymes in plants.

[0003] Okra is rich in protein, free amino acids, carotenoids, various vitamins, mineral elements such as phosphorus, iron, potassium, calcium, and sticky substances composed of pectin and polysaccharides. It has multiple health functions and is very popular among 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 fruits will also affect human health. Therefore, it is crucial to alleviate the damage of cadmium stress to okra.

[0004] Choline chloride (CC), as a new type of plant growth regulator, has a wide range of applications in agricultural development. Its low toxicity and non-polluting properties have increased its application range. The present invention's research shows that exogenous choline chloride can enhance the tolerance of plants to abiotic stress and alleviate the damage caused by adversity to plants. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of CdCl 2 The problem of high Cd content in okra seedlings grown under stress was solved by providing choline chloride in alleviating the CdCl 2 Application in stress injury.

[0006] Choline chloride in alleviating plant CdCl 2 Stress damage or increase plant CdCl 2 Application in stress resistance.

[0007] Use of choline chloride in at least one of the following (a1)-(a29):

[0008] (a1) Increase CdCl 2Chlorophyll a content of okra under stress;

[0009] (a2) Preparation of CdCl 2 Products of chlorophyll a content of okra under stress;

[0010] (a3) Increase CdCl 2 Chlorophyll b content of okra under stress;

[0011] (a4) Preparation of CdCl 2 Products of chlorophyll b content of okra under stress;

[0012] (a5) Increase CdCl 2 Total chlorophyll content of okra under stress;

[0013] (a6) Preparation of CdCl 2 Products of total chlorophyll content of okra under stress;

[0014] (a7) Increase CdCl 2 Total carotenoid content of okra under stress;

[0015] (a8) Preparation of CdCl 2 Products of total carotenoid content of okra under stress;

[0016] (a9) Increase CdCl 2 Relative water content of okra under stress;

[0017] (a10) Preparation of CdCl 2 products of relative water content of okra under stress;

[0018] (a11) Increase CdCl 2 SOD activity of okra under stress;

[0019] (a12) Preparation of CdCl 2 Products of SOD activity of okra under stress;

[0020] (a13) Increase CdCl 2 GPX activity of okra under stress;

[0021] (a14) Preparation of CdCl 2 Products of GPX activity of okra under stress;

[0022] (a15) Increase CdCl 2 CAT activity of okra under stress;

[0023] (a16) Preparation of CdCl 2 Products of CAT activity of okra under stress;

[0024] (a17) Increase CdCl 2 GR activity of okra under stress;

[0025] (a18) Preparation of CdCl 2 Products of GR activity of okra under stress;

[0026] (a19) Increase CdCl 2 APX activity of okra under stress;

[0027] (a20) Preparation of CdCl 2 Products of APX activity of okra under stress;

[0028] (a21) Increase CdCl 2 POD activity of okra under stress;

[0029] (a22) Preparation of CdCl 2 Products of POD activity of okra under stress;

[0030] (a23) Reduction of CdCl 2 MDA content of okra under stress;

[0031] (a24) Preparation of reduced CdCl 2 Products of MDA content of okra under stress;

[0032] (a25) Reduce CdCl 2 Electrolyte osmosis of okra under stress;

[0033] (a26) Preparation of reduced CdCl 2 Products of electrolyte osmotic pressure of okra under stress;

[0034] (a27) Reduction of CdCl 2 H of okra under stress 2 O 2 content;

[0035] (a28) Preparation of reduced CdCl 2 H of okra under stress 2 O 2 Products with high content;

[0036] (a29) Increase the CdCl content of okra 2 Stress resistance.

[0037] The above application is to spray the plants with choline chloride at a concentration of 0.1-6.0 mmol / L.

[0038] A method for cultivating okra, comprising spraying okra with choline chloride at a concentration of 0.1-6.0 mmol / L. Preferably, the okra is sprayed with choline chloride at a concentration of 1.0-4.0 mmol / L. Further preferably, the okra is sprayed with choline chloride at a concentration of 1.5-3.0 mmol / L. Most preferably, the okra is sprayed with choline chloride at a concentration of 3.0 mmol / L.

[0039] A method to alleviate okra CdCl 2 The method of stress damage is to spray okra with choline chloride at a concentration of 0.1-6.0 mmol / L. Preferably, the okra is sprayed with choline chloride at a concentration of 1.0-4.0 mmol / L. Further preferably, the okra is sprayed with choline chloride at a concentration of 1.5-3.0 mmol / L. Most preferably, the okra is sprayed with choline chloride at a concentration of 3.0 mmol / L.

[0040] The present invention also protects choline chloride against CdCl 2 Application of stress in okra breeding.

[0041] The present invention also protects the above method in the case of resistance to CdCl 2 Application of stress in okra breeding.

[0042] The above-mentioned plant is any one of the following (c1) to (c3):

[0043] (c1) dicotyledons;

[0044] (c2) Malvaceae;

[0045] (c3) Okra.

[0046] In any of the above applications or methods, the okra is specifically okra in the seedling stage.

[0047] Any of the above-mentioned okra can be Taiwan Wufu okra.

[0048] The exogenous choline chloride provided by the present invention can alleviate the 2 Application in stress of okra seedlings: Exogenous spraying of choline chloride can increase the CdCl 2 Stress resistance. 0.1 mmol / L CdCl 2The results of stress germination rate test showed that the germination rate and fresh weight of okra seeds treated with 1.5 and 3.0 mmol / L choline chloride were significantly higher than those treated with 0.0 and 6.0 mmol / L choline chloride, and increased by 55%, 112% and 45%, 106% respectively compared with 0.0 mmol / L choline chloride plants. Among them, the okra seedlings treated with 3.0 mmol / L choline chloride had the best effect. 2 The results of the stress pot experiment showed that okra seedlings treated with exogenous choline chloride grew well and had improved resistance. The fresh weight and dry weight of potted okra seedlings treated with 1.5, 3.0 and 6.0 mmol / L choline chloride were significantly higher than those treated with 0.0 mmol / L choline chloride, and increased by 68%, 135%, 20% and 180%, 253%, 163% respectively compared with 0.0 mmol / L choline chloride plants. Among them, the okra seedlings treated with 3.0 mmol / L choline chloride had the best effect and expressed strong CdCl 2 Stress resistance: Specifically, the okra seedlings treated with choline chloride had increased chlorophyll a content, increased chlorophyll b content, increased total chlorophyll content, increased total carotenoid content, increased relative water content, increased SOD activity, increased GPX activity, increased CAT activity, increased GR activity, increased APX activity, increased POD activity, decreased electrolyte permeability, and decreased H 2 O 2 content and reduced the content of malondialdehyde.

[0049] The results show that the choline chloride of the present invention can alleviate the 2 It has important applications in the stress of okra seedlings. Choline chloride can promote the growth of okra seedlings in CdCl 2 Stress and improve the resistance of okra seedlings to CdCl 2 Tolerance to stress, choline chloride and CdCl 2 The relationship between leaf damage, photosynthesis, antioxidant enzyme system and cell membrane system of okra under stress is explored, and the mechanism of action of choline chloride in plants resisting heavy metal stress is explored, providing a theoretical basis for its safe production in okra; the present invention will have broad application space and market prospects in the agricultural field. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 The present invention has different concentrations (0.000, 0.025, 0.050, 0.100, 0.200, 0.400 and 0.800 mmol / L) of CdCl 2 Effect on the germination rate of okra variety Taiwan Wufu seeds.

[0051] Figure 2 The normal conditions of the present invention and 0.1 mmol / LCdCl 2 Effects of different concentrations (0.0, 1.5, 3.0 and 6.0 mmol / L) of choline chloride on the germination rate and fresh weight of okra seeds under stress.

[0052] Figure 3 The normal conditions of the present invention and 0.1 mmol / LCdCl 2 Identification of resistance of okra seedlings to the alleviating effect of different concentrations (0.0, 1.5, 3.0 and 6.0 mmol / L) of choline chloride under stress.

[0053] Figure 4 The normal conditions of the present invention and 0.1 mmol / LCdCl 2 Effects of 3.0 mmol / L choline chloride on the contents of photosynthetic pigments (chlorophyll a, chlorophyll a, total chlorophyll, and total carotenoids) in okra seedlings under stress.

[0054] Figure 5 The normal conditions of the present invention and 0.1 mmol / LCdCl 2 Effect of 3.0 mmol / L choline chloride on malondialdehyde content, electrolyte osmotic rate, relative water content and H in okra seedlings under stress 2 O 2 The influence of content.

[0055] Figure 6 The normal conditions of the present invention and 0.1 mmol / LCdCl 2 Effects of 3.0 mmol / L choline chloride on the activities of key enzymes (SOD, GPX, CAT, GR, APX, POD) in the ROS scavenging system of okra seedlings under stress. Implementation

[0056] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagents unless otherwise specified. The quantitative tests in the following examples were repeated three times, and the results were averaged.

[0057] In the embodiment, okra ( Abelmoschus sculentus) variety Taiwan Wufu was used as the experimental material. Okra variety Taiwan Wufu, reference [Feibing Wang, Gaolei Ren, Fengsheng Li, Sitong Qi, YanXu, Bowen Wang, Yulin Yang, Yuxiu Ye, Qing Zhou, Xinhong Chen. A chalconesynthase gene AeCHS from Abelmoschus sculentus regulates flavonoidaccumulation and abiotic stress tolerance in transgenic Arabidopsis . 2018,40:97], preserved by the laboratory of Jiangsu Plant Production and Processing Practice Education Center, School of Life Sciences and Food Engineering, Huaiyin Institute of Technology.

[0058] Choline chloride (CC) was purchased from Sigma.

[0059] The Cd donor is cadmium chloride (CdCl 2 ·2H 2 O) was purchased from Sigma.

[0060] Example 1 Okra seed germination rate suitable for CdCl 2 Analysis of stress concentration

[0061] Referring to the method of Wang Yonghui et al. (2014) [Wang Yonghui, Chen Jianping, Zhang Peitong, Cai Liwang, Shi Qinghua, Wang Haiyang. Screening of salt-tolerant materials of okra and correlation analysis of salt tolerance during germination period. Journal of Southwest Agriculture, 2014, 27(2): 788-792], 50 plump and uniformly sized okra seeds were selected, disinfected with 5% hypochlorous acid solution for 5 min, then rinsed with distilled water for 3 times, and placed in a transparent plastic fresh-keeping box with two layers of filter paper and an inner diameter of 12 cm. 8 CdCl concentrations of 0.000, 0.025, 0.050, 0.100, 0.200, 0.400 and 0.800 mmol / L were set. 2 concentration, where 0.000 mmol / L CdCl 2 The concentration is the control, CdCl 2 Place 10 mL of CdCl in each fresh-keeping box to be treated. 2Solution, repeated six times. The fresh-keeping box was placed in an artificial climate box for germination, with a day / night temperature of 28℃ / 25℃, a day / night temperature of 12 h each, and a humidity of 60%±1%. The radicle length of 0.2 mm was used as the seed germination mark, and the number of seeds that germinated was counted on the 5th day. Seed germination rate = (number of germinated seeds on the 5th day / total number of seeds tested) × 100%.

[0062] The results showed that under normal conditions (0.000 mmol / L CdCl 2 ) under the condition of germination of okra seeds, the results are shown in Figure 1 . At 0.025, 0.050, 0.100, 0.200, 0.400 and 0.800 mmol / L CdCl 2 After the concentration treatment, okra seed germination was inhibited. 2 The seed germination rates were 92.67%, 82.00%, 49.33%, 22.67%, 13.33% and 1.33% respectively. Figure 1 Based on the germination rate test results, 0.1mmol / L CdCl 2 as heavy metal stress concentration.

[0063] Example 2 Exogenous choline chloride regulates CdCl in okra seedlings 2 Stress resistance identification and analysis

[0064] 1. Resistance of okra seeds to CdCl after treatment with different concentrations of choline chloride 2 Analysis of germination rate under stress

[0065] Referring to the method of Wang Yonghui et al. (2014) [Wang Yonghui, Chen Jianping, Zhang Peitong, Cai Liwang, Shi Qinghua, Wang Haiyang. Screening of salt-tolerant materials of okra and correlation analysis of salt tolerance during germination period. Journal of Southwest Agriculture, 2014, 27(2): 788-792], 50 plump and uniform seeds of okra were selected and disinfected with 5% hypochlorous acid solution for 5 min, then rinsed with distilled water for 3 times, and then hydrated in clean water and 0.1 mmol / L CdCl at room temperature. 2 The samples were immersed in the solution for 24 h and then placed in a transparent plastic fresh-keeping box with an inner diameter of 12 cm and covered with two layers of filter paper. They were then treated with different concentrations of choline chloride (0.0, 1.5, 3.0 and 6.0 mmol / L). The treatments included: (i) adding 0.0, 1.5, 3.0 and 6.0 mmol / L choline chloride to distilled water under normal conditions; (ii) adding 0.1 mmol / L CdCl 2Under the same conditions, 0.0, 1.5, 3.0 and 6.0 mmol / L choline chloride were added respectively. Each treatment was repeated six times. The fresh-keeping boxes were placed in an artificial climate box for germination, with a day / night temperature of 28℃ / 25℃, a day / night of 12 h each, and a humidity of 60%±1%. The radicle length of 0.2 mm was used as the seed germination mark, and the number of seeds that germinated was counted on the 5th day. Seed germination rate = (number of germinated seeds on the 5th day / total number of seeds tested) × 100%.

[0066] The results showed that 0.1 mmol / L CdCl 2 The results of the stress germination rate test showed that Figure 2 , 1.5 and 3.0mmol / L choline chloride treatment okra seed germination rate ( Figure 2 A) and fresh weight ( Figure 2 B) was significantly higher than that of 0.0 and 6.0 mmol / L choline chloride treatments, and increased by 55%, 112% and 45%, 106% respectively compared with 0.0 mmol / L choline chloride plants. Among them, the okra seedlings treated with 3.0 mmol / L choline chloride had the best effect.

[0067] 2. Resistance of okra seedlings to CdCl after treatment with different concentrations of choline chloride 2 Stress resistance analysis

[0068] Referring to the method 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 anddrought tolerance in okra plants based on applied attapulgite clay. Advances in Biochemistry, 2022, 10:1-10], Taiwan okra seedlings were transplanted into 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 well watered with half-Hoagland solution for 4 weeks until the seedlings grew new leaves. Subsequently, CdCl 2Stress and choline chloride treatment, the treatment includes: (i) under normal conditions, each pot of okra seedlings was irrigated with clean water once every 2 days for 4 weeks; at the same time, it was sprayed with 0.0, 1.5, 3.0 and 6.0 mmol / L choline chloride aqueous solution 3 times a day. (ii) CdCl 2 Stress treatment: each pot of okra seedlings was treated with 100 mL of 0.1 mmol / L CdCl 2 Solution irrigation was carried out every 2 days for 4 weeks, and at the same time, aqueous solutions containing 0.0, 1.5, 3.0 and 6.0 mmol / L choline chloride were sprayed 3 times a day.

[0069] The results showed that 0.1 mmol / L CdCl 2 The results of the pot experiment showed that Figure 3 The okra seedlings treated with exogenous choline chloride grew well and had improved resistance. The fresh weight and dry weight of potted okra seedlings treated with 1.5, 3.0 and 6.0 mmol / L choline chloride were significantly higher than those treated with 0.0 mmol / L choline chloride, and increased by 68%, 135%, 20% and 180%, 253%, 163% respectively compared with those treated with 0.0 mmol / L choline chloride. Among them, the okra seedlings treated with 3.0 mmol / L choline chloride had the best effect, expressing strong CdCl 2 Stress resistance.

[0070] Therefore, the phenotypic identification results showed that okra plants treated with exogenous 3.0 mmol / L choline chloride had the best CdCl 2 Stress resistance phenotype for analysis of induced CdCl 2 Physiological and biochemical mechanisms of stress resistance.

[0071] Example 3 Okra seedlings resistance to CdCl 2 Determination of physiological and biochemical indicators of stress resistance

[0072] Chloroplasts are important organelles for plants to carry out photosynthesis and respond to stress. The photosynthetic pigments in chloroplasts include chlorophyll and carotenoids, of which chlorophyll is divided into chlorophyll a and chlorophyll b. Stress affects plant photosynthesis, increases the number of free radicals in chloroplasts, and thus destroys chlorophyll, affecting plant photosynthesis. The content of photosynthetic pigments reflects the ability of plants to carry out photosynthesis.

[0073] The determination method was based on the literature [LichtenthalerHartmut 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 the leaves of okra seedlings without stress in the above potted plant identification, the leaves of okra seedlings sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted plant identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0074] The experimental results are shown in Figure 4 (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1mmol / L CdCl 2 After stress treatment, chlorophyll a ( Figure 4 A) Chlorophyll b ( Figure 4 B) Total chlorophyll ( Figure 4 C) and total carotenoid content ( Figure 4 D) was significantly higher than that of okra plants not treated with choline chloride.

[0075] When plant organs age or suffer damage under adverse conditions, membrane lipid peroxidation often occurs. Malondialdehyde (MDA) is the final decomposition product of membrane lipid peroxidation, and its content can reflect the degree of damage suffered by the plant under adverse conditions. That is, the higher the MDA content, the greater the degree of damage suffered by the plant under adverse conditions.

[0076] Determination method references [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], the MDA content of okra seedlings was detected. The okra plants were the leaves of okra seedlings without stress in the above potted identification, sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0077] The experimental results are shown in Figure 5 A (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the MDA content in okra plants treated with exogenous choline chloride was significantly lower than that in okra plants not treated with choline chloride.

[0078] Electrolyte permeability is an indicator to measure the permeability of plant cell membranes. The lower the value, the more intact the cell membrane and the better its functionality.

[0079] Determination method references [Yujia Liu, Xiaoyu Ji, Xianguang Nie, Min Qu, Lei Zheng, Zilong Tan, Huimin Zhao, Lin Huo, Shengnan Liu, Bing Zhang, Yucheng Wang. Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs. New Phytologist, 2015, 207:692-709], the electrolyte permeability of okra seedlings was detected. Okra plants were the leaves of okra seedlings without stress in the above potted identification, sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and 0.1 mmol / L CdCl in the above potted identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0080] The experimental results are shown in Figure 5 B (Normal is blank control, CdCl 2stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the electrolyte permeability in okra plants treated with exogenous choline chloride was significantly lower than that in okra plants not treated with choline chloride.

[0081] Relative water content is an important indicator reflecting the water status of plants. The relative water content of plant tissues is positively correlated with plant stress resistance, and the relative water content of cells reflects the degree of stress on cells.

[0082] Determination method reference [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 the relative water content of okra seedlings. The okra plants were the leaves of the okra seedlings without stress in the above potted identification, the leaves of the okra seedlings sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of the okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0083] The experimental results are shown in Figure 5 C (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the relative water content in okra plants treated with exogenous choline chloride was significantly higher than that in okra plants not treated with choline chloride.

[0084] 5. H 2 O 2 Content determination

[0085] When plants are in adverse conditions or aging, the metabolism of active oxygen in the body is enhanced, causing H 2 O 2 Accumulation occurs. 2 O 2It can directly or indirectly oxidize intracellular nucleic acids, proteins and other biological macromolecules and damage cell membranes, thereby accelerating cell aging and disintegration. 2 O 2 The higher the content, the greater the degree of damage the plant suffers from adversity.

[0086] Determination method references [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], detecting H in okra seedlings 2 O 2 The okra plants were the leaves of okra seedlings without stress in the above potted plants, the leaves of okra seedlings sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted plants. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0087] The experimental results are shown in Figure 5 D (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, H 2 O 2 The content was significantly lower than that in okra plants not treated with choline chloride.

[0088] Superoxide dismutase (SOD) activity can be used as a physiological and biochemical indicator of plant stress resistance. The lower the SOD activity, the greater the degree of damage to the plant from adversity.

[0089] Determination method references [Feibing Wang, Weili Kong, Gary Wong, Lifeng Fu, Rihe Peng, Zhenjun Li, Quanhong Yao. AtMYB12regulates flavonoids accumulation and abiotic stress tolerance in transgenic Arabidopsis thaliana . Molecular Genetics and Genomics, 2016, 291:1545-1559], detect the SOD activity of okra seedlings. Okra plants were the leaves of okra seedlings without stress in the above potted identification, sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0090] The experimental results are shown in Figure 6 A (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the SOD activity in okra plants treated with exogenous choline chloride was significantly higher than that in okra plants not treated with choline chloride.

[0091] Glutathione peroxidase (GPX) is an important peroxide-degrading enzyme widely present in the body. It is a key enzyme for removing reactive oxygen species in the body and plays an important role in plant stress resistance.

[0092] Determination method references: [Hong Zhai, Feibing Wang, Zengzhi Si, Jinxi Huo, Lei Xing, Yanyan An, Shaozhen He, Qingchang Liu. myo -inositol-1-phosphatesynthase gene, IbMIPS1 , enhances salt and drought tolerance and stem nematoderesistance in transgenic sweetpotato. Plant Biotechnology Journal, 2016, 14:592-602], detect GPX activity in okra seedlings. Okra plants were the leaves of okra seedlings without stress in the above potted identification, sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted identification. 2Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0093] The experimental results are shown in Figure 6 B (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the GPX activity in okra plants treated with exogenous choline chloride was significantly higher than that in okra plants not treated with choline chloride.

[0094] Catalase (CAT) is widely present in plant tissues and is one of the important protective enzymes. Its function is to remove H produced in metabolism. 2 O 2 , to avoid H 2 O 2 The accumulation of oxidative damage to cells, so its activity is related to the plant's stress resistance.

[0095] Determination method reference [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 the leaves of okra seedlings without stress in the above potted identification, sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0096] The experimental results are shown in Figure 6 C (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the CAT activity in okra plants treated with exogenous choline chloride was significantly higher than that in okra plants not treated with choline chloride.

[0097] Glutathione reductase (GR) is a ubiquitous flavin enzyme that participates in the defense system of cellular stress, and its activity is related to the stress resistance of plants.

[0098] Determination method reference [Wenjin Zhang, ZhicaiXie, Lianhong Wang, Ming Li, Duoyong Lang, Xinhui Zhang. Silicon alleviates salt and drought stress of Glycyrrhizauralensis Seeding by altering antioxidant metabolism and osmoticadjustment. Journal of Plant Research, 2017, 130:611-624], and the GR activity of okra seedlings was detected. The okra plants were the leaves of okra seedlings without stress in the above potted identification, the leaves of okra seedlings sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0099] The experimental results are shown in Figure 6 D (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the GR activity in okra plants treated with exogenous choline chloride was significantly higher than that in okra plants not treated with choline chloride.

[0100] Ascorbate peroxidase (APX) is one of the important antioxidant enzymes in plant active oxygen metabolism, especially in scavenging H 2 O 2 APX is a key enzyme in the metabolism of vitamin C. APX activity can be used as a physiological and biochemical indicator of plant stress resistance, and its activity is related to the stress resistance of plants.

[0101] Determination method references: [Hong Zhai, Feibing Wang, Zengzhi Si, Jinxi Huo, Lei Xing, Yanyan An, Shaozhen He, Qingchang Liu. myo -inositol-1-phosphatesynthase gene, IbMIPS1, enhances salt and drought tolerance and stem nematoderesistance in transgenic sweetpotato. Plant Biotechnology Journal, 2016, 14:592-602], detect APX activity in okra seedlings. Okra plants were the leaves of okra seedlings without stress in the above potted identification, sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0102] The experimental results are shown in Figure 6 E (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the APX activity in okra plants treated with exogenous choline chloride was significantly higher than that in okra plants not treated with choline chloride.

[0103] Peroxidase (POD) activity can be used as a physiological and biochemical indicator of plant stress resistance. The lower the POD activity, the greater the degree of damage to the plant caused by adversity.

[0104] Determination method references [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], detect the POD activity of okra seedlings. Okra plants were the leaves of okra seedlings without stress in the above potted identification, sprayed with 3.0 mmol / L choline chloride aqueous solution for 2 weeks, and the leaves of okra seedlings sprayed with 0.1 mmol / L CdCl in the above potted identification. 2 Treatment: Spray the leaves of okra seedlings with 3.0 mmol / L choline chloride aqueous solution for 2 weeks. The experiment was repeated three times and the results were averaged.

[0105] The experimental results are shown in Figure 6F (Normal is blank control, CdCl 2 stress refers to heavy metal stress). The results showed that 0.1 mmol / L CdCl 2 After stress treatment, the POD activity in okra plants treated with exogenous choline chloride was significantly higher than that in okra plants not treated with choline chloride.

[0106] The results of physiological and biochemical indexes showed that exogenous choline chloride treatment increased the CdCl 2 Stress resistance. Studies on choline chloride and CdCl 2 The relationship between okra's leaf damage, photosynthesis, antioxidant enzyme system, and cell membrane system under stress was explored to explore the mechanism of choline chloride in plants' resistance to heavy metal stress, providing a theoretical basis for its safe production in okra.

Claims

1. Application of choline chloride in alleviating CdCl2 stress damage of okra or improving CdCl2 stress resistance of okra.

2. Use of choline chloride in at least one of the following (a1) to (a28): (a1) Increase the chlorophyll a content of okra under CdCl2 stress; (a2) preparing a product for increasing the chlorophyll a content of okra under CdCl2 stress; (a3) Increase the chlorophyll b content of okra under CdCl2 stress; (a4) preparing a product for increasing the chlorophyll b content of okra under CdCl2 stress; (a5) Increase the total chlorophyll content of okra under CdCl2 stress; (a6) preparing a product for increasing the total chlorophyll content of okra under CdCl2 stress; (a7) Increase the total carotenoid content of okra under CdCl2 stress; (a8) preparing a product for increasing the total carotenoid content of okra under CdCl2 stress; (a9) Increase the relative water content of okra under CdCl2 stress; (a10) preparing a product for increasing the relative water content of okra under CdCl2 stress; (a11) Improve the SOD activity of okra under CdCl2 stress; (a12) preparing a product for improving the SOD activity of okra under CdCl2 stress; (a13) Improve the GPX activity of okra under CdCl2 stress; (a14) preparing a product for improving the GPX activity of okra under CdCl2 stress; (a15) Improve the CAT activity of okra under CdCl2 stress; (a16) preparing a product for improving CAT activity of okra under CdCl2 stress; (a17) Improved GR activity of okra under CdCl2 stress; (a18) preparing a product for improving GR activity of okra under CdCl2 stress; (a19) Improved APX activity of okra under CdCl2 stress; (a20) preparing a product for improving APX activity of okra under CdCl2 stress; (a21) Improve the POD activity of okra under CdCl2 stress; (a22) preparing a product for improving the POD activity of okra under CdCl2 stress; (a23) Reduce the MDA content of okra under CdCl2 stress; (a24) preparing a product for reducing the MDA content of okra under CdCl2 stress; (a25) Reduce the electrolyte permeability of okra under CdCl2 stress; (a26) preparing a product for reducing electrolyte permeability of okra under CdCl2 stress; (a27) Reduce the H2O2 content of okra under CdCl2 stress; (a28) Preparation of products for reducing the H2O2 content of okra under CdCl2 stress.

3. The use according to claim 1 or 2, characterized in that: Choline chloride was sprayed on the plants with a concentration of 0.1-6.0 mmol / L.

4. A method for alleviating CdCl2 stress damage in okra, characterized in that: Choline chloride with a concentration of 0.1-6.0 mmol / L was sprayed on the leaves of okra.

5. The method according to claim 4, characterized in that: Choline chloride with a concentration of 1.0-4.0 mmol / L is used to spray the okra on its leaves.

6. The method according to claim 5, characterized in that: Choline chloride with a concentration of 1.5-3.0 mmol / L is used to spray the okra on its leaves.

7. Application of choline chloride in breeding of okra resistant to CdCl2 stress.

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