Method for improving cadmium resistance of cotton by exogenously applying citric acid

Abstract

The invention relates to the field of cadmium-polluted soil treatment, aims to solve the technical problem of how to promote growth of cotton in cadmium-polluted soil, and provides a method for improving cadmium resistance of cotton by applying citric acid from an external source. The method for enhancing the cadmium resistance of the cotton is preliminarily established, reference is provided for promoting the growth of the cotton in the cadmium-polluted soil, and the application of the cotton in repairing the cadmium-polluted soil is further promoted.

Description

Technical Field

[0001] This invention relates to the field of cadmium-contaminated soil remediation, and specifically discloses a method for improving the cadmium tolerance of cotton by applying citric acid exogenously. Background Technology

[0002] Cadmium (Cd), a transitional element in Group IIB of the periodic table, is commonly found in soils and zinc (Zn) minerals (Brzóska and Moniuszko-Jakoniuk 2001). It ranks seventh among the top 20 most toxic metals globally and is classified as a Group 1 carcinogen (Hussain, Ashraf et al. 2021). Since the Industrial Revolution, human activities (such as phosphate fertilizer application, tailings disposal, metal smelting, mining operations, and fossil fuel combustion) have led to a significant and widespread increase in Cd levels in global soils, far exceeding the original environmental background value (0.097 mg / kg). For example, the average Cd concentration in European soils is 0.33 mg / kg, and the average concentration in Chinese farmland is 0.19 mg / kg (Zhang, Yang et al. 2024).

[0003] Against this backdrop of severe Cd pollution, phytoremediation technology has become an important soil remediation strategy due to its environmental and economic advantages (Singh, Kumar et al. 2022). However, Cd, due to its high solubility and mobility, is easily absorbed by plants and accumulates in different organs, severely inhibiting plant growth, damaging morphological structures, leading to physiological metabolic disorders, stunted growth, reduced yields, and even death, posing a significant challenge to the efficiency of phytoremediation (Goncharuk and Zagoskina 2023). At this juncture, the crucial role of low molecular weight organic acids (LMWOAs) becomes apparent. As natural products of plant root exudates, microbial secretions, and soil organic matter decomposition, LMWOAs play an indispensable role in enhancing plant Cd absorption efficiency and improving plant tolerance under Cd stress through their natural chelating ability, regulatory effects on plant physiology, and improvement of the soil environment (Song, Cheng et al. 2025).

[0004] Citric acid (CA) has been widely recognized as a key organic acid for reducing Cd toxicity in plants, with significantly higher efficacy than other common LMWOAs. As a common organic acid secreted by plant roots, CA can effectively chelate Cd in the soil. 2+ This enhances its bioavailability (Wang, Duan et al. 2023). More importantly, CA can accumulate in large quantities in plants, serving as intracellular Cd. 2+The key chelating agent binds free toxic Cd. 2+ By combining to form low-toxicity or non-toxic complexes, separation can be achieved, thereby directly reducing Cd. 2+ The cytotoxicity of Cd (Chen, Yu et al. 2024). CA plays a central role in combating Cd-induced oxidative stress, and exogenous addition of CA can effectively alleviate Cd oxidative stress. 2+ The mechanism of induced reactive oxygen species bursts includes restoring intracellular CA levels and protecting the structure and activity of antioxidant enzymes, enabling plants to more effectively scavenge reactive oxygen species and maintain redox balance (Xue, Zhang et al. 2023).

[0005] Cotton, as an important global fiber crop, not only has significant economic value but also shows potential in phytoremediation of Cd-contaminated soils due to its relatively strong cadmium absorption capacity and tolerance. However, high concentrations of Cd stress can adversely affect the inherent phenotypic plasticity of cotton (i.e., its ability to adapt to environmental changes by adjusting its growth and physiological characteristics) (An, Hong et al. 2022).

[0006] Given that the early growth stage of seedlings (hypocotyl and radicle development period) is a critical period for plants to establish resistance, this invention uses germinating cotton seeds for experiments and develops a method to enhance their cadmium tolerance in cadmium-polluted environments by adding citric acid.

[0007] References

[0008] An, M., D. Hong, D. Chang, C. Zhang, H. Fan and K. Wang (2022). "Polymer amendment regulates cadmium migration in cadmium contaminated cottonfield: Insights from genetic adaptation and CAnotypic plasticity." Sci TotalEnviron 807(Pt 3): 151075. Brzóska, MM and J. Moniuszko-Jakoniuk (2001). "Interactions between cadmium and zinc in the organism." Food Chem Toxicol 39(10): 967-980. Chen, K., B. Yu, W. Xue, Y. Sun, C. Zhang, X. Gao, X. Zhou, Y. Deng,J. Yang and B. Zhang (2024). "Citric acid inhibits Cd absorption andtransportation by improving the antagonism of essential elements in riceorgans." Toxics 12(6). Goncharuk, E. A. and N. V. Zagoskina (2023). "Heavy Metals, TheirPhytotoxicity, and the Role of CAnolic Antioxidants in plant stress responseswith focus on cadmium: Review." Molecules 28(9). Hussain, B., M. N. Ashraf, R. Shafeeq Ur, A. Abbas, J. Li and M.Farooq (2021). "Cadmium stress in paddy fields: Effects of soil conditionsand remediation strategies." Sci Total Environ 754: 142188. Singh, S., V. Kumar, D. S. Dhanjal, P. Parihar, P. C. Ramamurthy andJ. Singh (2022). 12 - Phytoremediation of heavy metals, metalloids, andradionuclides: Prospects and challenges. Phytoremediation Technology for theRemoval of Heavy Metals and Other Contaminants from Soil and Water. V. Kumar,M. P. Shah and S. K. Shahi, Elsevier: 253-276. Song, Y., Q. Cheng and B. Zhao (2025). "Exogenous organic acids promoted phytoremediation by Hydrangea macrophylla in cadmium contaminated soil." Ecotoxicol Environ Saf 290: 117551. Wang, Y., W. Duan, C. Lv, Z. Wei, Y. Zhu, Q. Yang, Y. Liu, Z. Shen, Y. Xia, K. Duan and L. Quan (2023). "Citric acid and poly-glutamic acid promote the phytoextraction of cadmium and lead in Solanum nigrum L. grown in compound Cd-Pb contaminated soils." Bull Environ Contam Toxicol 110(1): 37. Xue, W., X. Zhang, C. Zhang, C. Wang, Y. Huang and Z. Liu (2023). "Mitigating the toxicity of reactive oxygen species induced by cadmium via restoring citrate valve and improving the stability of enzyme structure in rice." ChemosCAre 327: 138511. Zhang, X., M. Yang, H. Yang, R. Pian, J. Wang and A. M. Wu (2024). "The uptake, transfer, and detoxification of cadmium in plants and its exogenous effects." Cells 13(11).。 Summary of the Invention

[0009] This invention promotes the elongation of the radicle after germination of cotton seeds under cadmium stress by applying citric acid exogenously, thereby improving the cadmium tolerance of cotton, promoting the growth of cotton in cadmium-stressed environments, and further promoting the application of cotton in the remediation of cadmium-contaminated soil.

[0010] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of the present invention is to provide the application of citric acid (CA, CAS No. 77-92-9).

[0011] The application of citric acid is any one or more of the following: Y1. Application in promoting the elongation of cotton radicles in cadmium-contaminated soil; Y2. Application in improving the cadmium resistance of cotton; Y3. Application of cotton in the remediation of cadmium-contaminated soil.

[0012] In the above applications, the citric acid is prepared as a solution, and the content of the citric acid in the solution can be 1 mM.

[0013] In the above applications, the solvent of the solution can be water.

[0014] In the above applications, the citric acid is used during cotton seed germination.

[0015] The present invention also provides a method for improving the cadmium resistance of cotton, including the step of adding citric acid during cotton seed germination.

[0016] In the above method, the citric acid is prepared as a solution when used, and the content of the citric acid in the solution can be 1 mM.

[0017] In the above method, the solvent of the solution can be water.

[0018] The present invention also provides a method for remediating cadmium-contaminated soil, comprising the steps of planting cotton on cadmium-contaminated soil and adding citric acid when the cotton seeds germinate.

[0019] In the above method, the citric acid is prepared as a solution when used, and the content of the citric acid in the solution can be 1 mM.

[0020] In the above method, the solvent of the solution can be water.

[0021] This invention develops a method to enhance the cadmium tolerance of cotton, providing an effective strategy for promoting its growth in cadmium-contaminated soil and advancing the application of cotton in phytoremediation technologies for heavy metal-contaminated soil. Attached Figure Description

[0022] Figure 1 This illustrates the effect of different treatments on seed germination in Example 1 of the present invention. Figure 1In the images of each variety, the three on the left are the embryo phenotypes of the ddH2O group, the three in the middle are the embryo phenotypes of the Cd group, and the three on the right are the embryo phenotypes of the Cd+CA group. Detailed Implementation

[0023] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0025] The citric acid (CA, CAS number 77-92-9) used in the following examples is a product of Solarbio, catalog number 77-92-9.

[0026] Example 1 Eight upland cotton materials were randomly selected (see Table 1), all of which are described in the non-patent literature "Han Mingge. Cotton germplasm resistant to Cd". 2+ Identification and GhHMP1 Cloning [D]. Urumqi: Xinjiang Agricultural University, 2018.”, which is available to the public from the applicant for replication of this experiment. Seeds of all upland cotton materials were identified and preserved for stress resistance.

[0027] Table 1

[0028] Cotton seeds were delinted with concentrated sulfuric acid and dried. Then, plump, uniformly sized, smooth seeds were selected and sterilized with a 75% ethanol solution for 5-10 minutes. The seeds were then rinsed repeatedly with distilled water 5-8 times and soaked in sterile water for 24 hours. Seeds showing signs of germination were selected for subsequent experiments.

[0029] 1. Citric acid treatment concentration screening Select H242 and TM-1 from 8 materials for citric acid treatment concentration screening. Germinate the white seeds of these two materials using the vertical double-layer filter paper method with different liquids X, and screen the citric acid treatment concentration.

[0030] The specific steps of the vertical double-layer filter paper method are as follows: Cut the filter paper to A4 size and lay it flat on a plastic tray. Place the seeds on the filter paper, which has been pre-moistened with distilled water, with the growth points facing the same direction. Spacing the seeds 5-7 cm apart, roll the filter paper horizontally along the seed position, ensuring the seed is rolled in the middle of the filter paper, making the filter paper into a straight cylinder. Remember to keep the seed growth point downwards. Place the cylindrical filter paper vertically in a beaker and put the beaker in an artificial incubator at 28℃ for 24 hours in the dark for germination. After 24 hours of natural growth, add different liquids (such as liquid X) to the bottom of the beaker to keep the filter paper moist throughout the germination period. Observe the seed germination status after 3 days of treatment.

[0031] The treatment group consists of six groups, and the liquid X used in each group is as follows: ddH2O: ddH2O is used.

[0032] 4 mM CdCl2 group: An aqueous solution of CdCl2 was used, with a concentration of 4 mM.

[0033] 4 mM CdCl2 + 0.5 mM CA group: An aqueous solution of CdCl2 and citric acid was used, with the concentration of CdCl2 being 4 mM and the concentration of citric acid being 0.5 mM.

[0034] 4 mM CdCl2 + 1 mM CA group: An aqueous solution of CdCl2 and citric acid was used, with the concentration of CdCl2 being 4 mM and the concentration of citric acid being 1 mM.

[0035] 4 mM CdCl2 + 2 mM CA group: An aqueous solution of CdCl2 and citric acid was used, with the concentration of CdCl2 being 4 mM and the concentration of citric acid being 2 mM.

[0036] 4 mM CdCl2 + 3 mM CA group: An aqueous solution of CdCl2 and citric acid was used, with the concentration of CdCl2 being 4 mM and the concentration of citric acid being 3 mM.

[0037] Each group of each variety was treated with 30 seeds.

[0038] The results showed that, compared with the ddH2O group, 4 mM CdCl2 stress significantly inhibited radicle growth, and compared with the 4 mM CdCl2 group, the radicle length of the 4 mM CdCl2 + 1 mM CA group was significantly increased. Therefore, 1 mM CA treatment for 3 days was selected as the treatment condition.

[0039] 2. Effects of external application of citric acid on physiological parameters of cotton under cadmium stress Using the budding seeds of eight upland cotton varieties listed in Table 1 as experimental materials, ddH2O treatment was used as a normal growth control, and 4 mM CdCl2 aqueous solution was selected as the cadmium treatment during seed germination. Under the premise of cadmium treatment, an additional 1 mM concentration of CA was added as an external application of citric acid under cadmium treatment. The specific treatment groups are as follows: ddH2O group: using ddH2O.

[0040] Cd group: An aqueous solution of CdCl2 as the solute was used, with a concentration of 4 mM.

[0041] Cd+CA group: An aqueous solution of CdCl2 and citric acid was used, with the concentration of CdCl2 being 4 mM and the concentration of citric acid being 1 mM.

[0042] Set up 3 repetitions, and process 10 seeds per repetition per group.

[0043] After incubation at 25℃ in the dark for 3 days, the lengths of hypocotyls and radicles in each group were investigated. The results are shown in […]. Figure 1 And Table 2.

[0044] Table 2

[0045] Note: The values ​​in the table are mean ± standard deviation. Different letters after the data indicate that the difference is significant at the p < 0.05 level.

[0046] In eight cotton samples, 4 mM cadmium treatment inhibited hypocotyl and radicle elongation, while the addition of citric acid significantly alleviated the inhibition of hypocotyl and radicle elongation during germination in all eight samples. This indicates that exogenous citric acid can alleviate the inhibition of hypocotyl and radicle elongation in cotton under cadmium stress and enhance the cotton's cadmium tolerance.

[0047] The above results indicate that external application of citric acid can effectively alleviate cadmium stress and improve the tolerance of cotton to cadmium stress.

[0048] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Application of citric acid in promoting the elongation of cotton radicles in cadmium-contaminated soil.

2. Application of citric acid in improving the cadmium resistance of cotton.

3. Application of citric acid in the remediation of cadmium-contaminated soil using cotton.

4. The application according to any one of claims 1-3, characterized in that: The citric acid is used during cotton seed germination.

5. The application according to claim 4, characterized in that: The citric acid is prepared as a solution when used, and the content of the citric acid in the solution is 1 mM.

6. A method for improving the cadmium resistance of cotton, characterized in that: The method includes the step of adding citric acid during cotton seed germination.

7. The method according to claim 6, characterized in that: The citric acid is added in a solution: the content of the citric acid in the solution is 1 mM.

8. A method for remediating cadmium-contaminated soil, characterized in that: The method includes the steps of planting cotton on cadmium-contaminated soil and adding citric acid when the cotton seeds germinate.

9. The method according to claim 8, characterized in that: The citric acid is added in a solution: the content of the citric acid in the solution is 1 mM.

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