Method for improving drought tolerance of cotton by exogenous application of melatonin

Abstract

The invention relates to the field of plant planting, aims to solve the technical problem of how to improve the drought tolerance of cotton, and provides a method for improving the drought tolerance of cotton by applying melatonin externally. According to the technical scheme, the application of the melatonin to improvement of the drought tolerance of the cotton is provided. The method for enhancing the drought tolerance of the cotton is preliminarily established, and reference is provided for promoting the growth of the cotton in a drought environment.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation, and specifically discloses a method for improving the drought resistance of cotton by exogenously applying melatonin. Background Technology

[0002] As one of the most common meteorological disasters globally, drought has a wide-ranging and profound impact on human life, affecting multiple fields such as agriculture, ecology, economy, and health, and often exhibiting persistence and cascading effects. Agricultural impacts: Drought reduces groundwater and soil moisture, severely impacting crop growth and potentially causing famine and livelihood risks. Ecosystem risks: Drought exposes ecosystems to significant risks, potentially leading to biodiversity loss and ecological imbalance. Socioeconomic problems: Drought triggers social conflicts, with forced migrations due to resource scarcity affecting the lives of millions. These impacts demonstrate that drought is not merely a natural phenomenon, but a major threat to human society and the ecological environment.

[0003] Cotton is an important strategic resource and a vital economic crop, playing a crucial role in agricultural economic development. Existing research has found that drought stress severely restricts cotton growth and development, affecting factors such as plant height, leaf dry weight, stem dry weight, leaf area index, number of fruit nodes, fiber quality, canopy development, and root system development. Therefore, against the backdrop of global warming, studying the impact of meteorological drought on cotton yield will contribute to a deeper understanding of the cotton industry's development prospects and promote sustainable economic development.

[0004] Seed germination is a crucial process in a plant's life cycle, transitioning from dormancy to vegetative growth. It is a core link in the plant's life cycle and directly affects the survival and continuation of the species. The cotton seedling stage refers to the growth phase from seed germination to bud formation (usually 30-40 days after sowing). This period is critical for cotton root development, seedling morphology formation, and the development of stress resistance. Drought during the seedling stage not only restricts seedling growth but can also lead to a decline in yield and quality later on. However, currently, there are few measures to improve cotton germination rate and seedling drought tolerance under drought conditions. Summary of the Invention

[0005] This invention improves the drought resistance of cotton during the germination and seedling stages by applying melatonin exogenously, thus promoting cotton growth under drought stress.

[0006] 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 melatonin (CA, CAS No. 73-31-4) in improving the drought resistance of cotton.

[0007] The application is any one or more of the following:

[0008] Y1. Application in improving drought resistance during cotton germination; Y2. Application in improving drought resistance in cotton seedlings.

[0009] In the above applications, the melatonin is prepared as a solution when used.

[0010] In the above applications, the solvent of the solution can be water.

[0011] In the above application, the melatonin described in Y1 is used during cotton seed germination, and the concentration of melatonin in the solution is 500 µM.

[0012] In the above application, the melatonin described in Y2 is used when the cotton has grown two to three true leaves, and the concentration of melatonin in the solution is 50 µM.

[0013] The present invention also provides a method for improving the drought resistance of cotton during the germination period, including the step of adding melatonin during cotton seed germination.

[0014] In the above method, the melatonin is prepared as a solution when used, and the concentration of melatonin in the solution can be 500µM.

[0015] In the above method, the solvent of the solution can be water.

[0016] In the above method, the addition of melatonin involves soaking the seeds in a melatonin solution.

[0017] The present invention also provides a method for improving the drought resistance of cotton seedlings, including the step of adding melatonin when the cotton has grown two to three true leaves.

[0018] In the above method, the melatonin is prepared as a solution when used, and the concentration of melatonin in the solution can be 50µM.

[0019] In the above method, the solvent of the solution can be water.

[0020] In the above method, the addition of melatonin involves foliar spraying of a melatonin solution for 5-7 days.

[0021] This invention develops a method to enhance the drought resistance of cotton, providing an effective strategy for promoting its growth in arid environments. Attached Figure Description

[0022] Figure 1 This illustrates the effect of different treatments on seed germination in Example 1 of the present invention. Figure 1 Photo A shows the phenotypic images of H177 germination on day 3 in cotton plants treated with different concentrations of melatonin. Figure 1 B represents the relative germination potential. Figure 1 C represents the length of the radicle. Figure 1 D represents fresh weight. The significance analysis result was P < 0.05. The significance analysis result was P<0.01.

[0023] Figure 2 This describes the effect of applying different concentrations of melatonin externally on cotton seedlings in Example 1 of the present invention. Figure 2 A represents the phenotype of H177 seedlings at different concentrations of melatonin applied topically; Figure 2 B represents the leaf water content; Figure 2 C represents the chlorophyll content of the leaves; Figure 2 D represents the expression level of drought-resistant transcription factors. The significance analysis result was P < 0.05. The significance analysis result was P<0.01. Detailed Implementation

[0024] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative and not intended to limit the scope of the invention. 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.

[0025] 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.

[0026] H177 (preserved in this laboratory) in the upland cotton drought-resistant material in the following examples is described in the non-patent literature "Melatonin regulated through GhTDC5 "Enhances tryptophan decarboxylase against drought stress in cotton," which is available to the public from the applicant for replication of the experiment.

[0027] The melatonin (CA, CAS number 73-31-4) used in the following examples is a product of Solarbio, catalog number Cat#M8600.

[0028] Example 1 H177, a drought-resistant upland cotton variety, was selected as the experimental material. The seeds of H177 were delinted with concentrated sulfuric acid and dried. Then, plump and uniformly sized smooth seeds were selected and sterilized with 75% ethanol solution for 5-10 minutes. The seeds were then washed with distilled water 5 times (1 minute each time) and then dried in a warm and dry environment for later use as sterilized seeds.

[0029] 1. Melatonin increases the germination rate of cotton seeds under drought stress. Seeds disinfected with H177 were divided into 6 groups for soaking treatment: 4 groups of seeds were soaked in melatonin solutions of different concentrations (250 µM, 500 µM, 750 µM and 1000 µM), and the other two groups of seeds were soaked in ddH2O. All 6 groups were soaked in the dark for 12 h, and then subjected to drought stress treatment for 3 days using the vertical double-layer filter paper method. The growth of cotton hypocotyls after 3 days of drought treatment with different concentrations of melatonin was observed.

[0030] The specific steps for drought stress treatment using the vertical double-layer filter paper method are as follows: First, lay the filter paper flat on a plastic plate and moisten it with distilled water. Arrange the seeds with their growth points facing the same direction on the filter paper. Roll up the filter paper horizontally along the seed position, enclosing the seed in the middle of the filter paper, so that the filter paper forms a straight cylinder. Place the cylindrical filter paper vertically in a beaker and incubate it in the dark at 28°C for 3 days, keeping the filter paper moist.

[0031] The specific settings for the 6 groups are as follows: Control group (CK): seeds were soaked in ddH2O and then germinated using the vertical double-layer filter paper method with ddH2O.

[0032] 12% PEG6000 (i.e. 12% PEG) group: after soaking in ddH2O, the seeds were germinated using the vertical double-layer filter paper method with 12% PEG6000 solution.

[0033] 12% PEG6000+250 µM MT (i.e. 12% PEG+250 µM MT) group: after soaking in 250 µM melatonin solution, the seeds were germinated using the vertical double-layer filter paper method with 12% PEG6000 solution.

[0034] 12% PEG6000+500 µM MT (i.e., 12% PEG+500 µM MT) group: after soaking in 500 µM melatonin solution, the seeds were germinated using the vertical double-layer filter paper method with 12% PEG6000 solution.

[0035] 12% PEG6000+750 µM MT (i.e. 12% PEG+750 µM MT) group: after soaking in 750 µM melatonin solution, the seeds were germinated using the vertical double-layer filter paper method with 12% PEG6000 solution.

[0036] 12% PEG6000+1000 µM MT (i.e. 12% PEG+1000 µM MT) group: after soaking in 1000 µM melatonin solution, the seeds were germinated using the vertical double-layer filter paper method with 12% PEG6000 solution.

[0037] Set up 3 replicates, with 10 seeds per replicate group.

[0038] The growth of cotton hypocotyls was observed after 3 days of drought treatment with different concentrations of melatonin. Seed germination rate was recorded, hypocotyl length was measured using ImageJ, and seed fresh weight was weighed.

[0039] See results Figure 1 The results showed that in all six cotton samples, treatment with 12% PEG6000 inhibited hypocotyl elongation, while the addition of melatonin significantly alleviated this inhibition. Soaking seeds in 500 µM melatonin showed the best effect, with the highest relative germination potential, and the most significant increases in root length and fresh weight. This indicates that exogenous melatonin can alleviate the inhibition of hypocotyl elongation in drought-stressed cotton and improve the germination rate of cotton under drought stress.

[0040] 2. Melatonin enhances drought resistance in seedlings. Using upland cotton variety H177 as the experimental material, seeds were sown and cultured until two to three true leaves emerged before the experiment began. A total of six groups were set up. In a dark environment, four groups were sprayed with different concentrations of melatonin solution (20 µM, 50 µM, 100 µM, and 200 µM), while the other two groups were sprayed with water. After 5-7 days of continuous foliar spraying, the seedlings were washed, and their roots were placed in a 12% PEG6000 solution (the roots of one group sprayed with water were placed in water as a control group, CK). Plant phenotypes were observed, and samples were taken, flash-frozen in liquid nitrogen, and stored at -80℃. Leaf water content, chlorophyll content, and the expression level of drought-resistant transcription factors were measured. The specific setup of the six groups is as follows: Control group (CK): After spraying with clean water for 5-7 days, wash the seedlings and put the roots into clean water.

[0041] 12% PEG6000 (i.e. 12% PEG) group: After spraying with water for 5-7 days, wash the seedlings and put the roots into a 12% PEG6000 solution.

[0042] 12% PEG6000+20 µM MT (i.e. 12% PEG+20 µM MT) group: After spraying with 20 µM melatonin solution for 5-7 days, wash the seedlings and put the roots into 12% PEG6000 solution.

[0043] 12% PEG6000+50 µM MT (i.e. 12% PEG+50 µM MT) group: After spraying with 50 µM melatonin solution for 5-7 days, wash the seedlings and put the roots into 12% PEG6000 solution.

[0044] 12% PEG6000+100 µM MT (i.e. 12% PEG+100 µM MT) group: After spraying with 100 µM melatonin solution for 5-7 days, wash the seedlings and put the roots into 12% PEG6000 solution.

[0045] 12% PEG6000+200 µM MT (i.e. 12% PEG+200 µM MT) group: After spraying with 200 µM melatonin solution for 5-7 days, wash the seedlings and put the roots into 12% PEG6000 solution.

[0046] Set up 3 replicates, with 10 plants per replicate group.

[0047] Leaf moisture content determination method: Sampling: Select leaves from the same growth position of each plant (avoid diseased or insect-damaged leaves), quickly rinse off surface dust with clean water, and absorb surface moisture with absorbent paper (to avoid residual moisture affecting the results). Weigh immediately and record as fresh weight (FW). Place in an oven, first at 105℃ for 30 minutes to blanch (to stop enzyme activity and prevent organic matter decomposition), then adjust to 80-85℃ to dry to constant weight and record as dry weight (DW).

[0048] Leaf moisture content (%) = (fresh weight - dry weight) / fresh weight × 100%.

[0049] Method for determining chlorophyll content: Take 0.1 g of fresh cotton leaves, cut them into small pieces, and place them in a 2 mL centrifuge tube. Quickly freeze the tube in liquid nitrogen and then shake and grind it. Add 1 mL of 80% acetone to the EP tube and place it in a refrigerator at 4℃ for 12 h (the EP tube can be inverted every 4 hours). Centrifuge and take 100 μL of the supernatant, then dilute it with 900 μL of 80% acetone. Measure the absorbance of the extract at 663 nm, 652 nm, and 645 nm, using 80% acetone as a blank control. Substitute the values ​​into the formula to calculate the chlorophyll content.

[0050] Chlorophyll a content (mg / g) = (12.72 × A663 - 2.59 × A645) × Vs / 1000w Chlorophyll b content (mg / g) = (22.88 × A654 - 4.67 × A663) × Vs / 1000w Vs: Total volume of extract (mL) w: Sample mass (g) The gene expression levels of several drought-related transcription factors, including GhNAC3, GhbZIP15, GhNAC2, and GhAGL16, were determined using quantitative real-time immunoassay, with UBQ7 as an internal reference gene. The primer sequences used are shown in Table 1. Table 1 Primer sequences used in quantitative fluorescence assay

[0051] See results Figure 2 In the six cotton samples, treatment with 12% PEG6000 resulted in stress, leaf water loss, and decreased chlorophyll content. Melatonin addition significantly alleviated drought stress in cotton, with foliar spraying of 50 µM melatonin showing the best phenotype, highest leaf water content, and highest chlorophyll content. This indicates that exogenous melatonin can alleviate the inhibition of drought stress in cotton and enhance its drought resistance.

[0052] The above results indicate that external application of melatonin can effectively alleviate drought stress and improve the tolerance of cotton to drought stress.

[0053] 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 melatonin in improving the drought resistance of cotton.

2. The application according to claim 1, characterized in that: The application described is the use of melatonin to improve the drought resistance of cotton during the germination stage.

3. The application according to claim 2, characterized in that: The melatonin is used during cotton seed germination, and is prepared as a solution with a concentration of 500 µM.

4. The application according to claim 1, characterized in that: The application described is the use of melatonin to improve the drought resistance of cotton seedlings.

5. The application according to claim 4, characterized in that: The melatonin is used when the cotton has grown two to three true leaves. The melatonin is prepared as a solution and the concentration of melatonin in the solution is 50 µM.

6. A method for improving drought resistance during the germination period of cotton, characterized in that: The method includes the step of adding melatonin during cotton seed germination.

7. The method according to claim 6, characterized in that: The melatonin is prepared as a solution when added: the melatonin content in the solution is 500 µM, and the addition of melatonin is achieved by soaking seeds in the melatonin solution.

8. A method for improving drought resistance in cotton seedlings, characterized in that: This includes the step of adding melatonin when the cotton plant has grown two to three true leaves.

9. The method according to claim 8, characterized in that: The melatonin is prepared as a solution when added: the melatonin content in the solution is 50 µM, and the melatonin is added by foliar spraying the melatonin solution for 5-7 days.

Images