Method for improving cold stress resistance of rice in seedling stage and application

Abstract

The invention belongs to the field of crop stress resistance regulation and control, and discloses a method for improving cold stress resistance of rice in the seedling stage and application of the method, particularly a method for improving cold stress resistance of rice seedlings through exogenous application of 5-methoxyl-tryptamine and application of the 5-methoxyl-tryptamine in agricultural production, the use concentration of 5-methoxyl-tryptamine is 0 [mu] mol / L, 1 [mu] mol / L, 10 [mu] mol / L or 50 [mu] mol / L, and the use concentration of 5-methoxyl-tryptamine is 0 [mu] mol / L, 1 [mu] mol / L, 10 [mu] mol / L or 50 [mu] mol / L; the plant height, the root length, the fresh weight, the dry weight, the dry matter content, the chlorophyll content and the hydrogen peroxide content of rice to which 1 mu mol / L 5-methoxytryptamine is applied are all remarkably improved compared with rice to which treatment is not applied, the effect is particularly the best under the cold stress condition, damage of cold stress to rice in the seedling stage can be relieved, and therefore the rice yield is increased. Therefore, the invention provides a theoretical basis for researching the mechanism of improving the cold stress resistance of the rice in the seedling stage by the 5-methoxytryptamine, and provides practical guidance for researching the cold tolerance of plants.

Description

Technical Field

[0001] This invention belongs to, but is not limited to, the field of crop stress resistance regulation technology, and particularly relates to a method and application for improving the resistance of rice seedlings to cold stress. Background Technology

[0002] Rice is one of my country's important food crops, and its growth and development are quite sensitive to temperature, especially during the seedling stage. If rice seedlings are subjected to cold stress, they will exhibit stunted growth, yellowing leaves, and hindered root development, which can lead to death in severe cases and significantly reduce rice yield. Currently, there is a lack of efficient and low-cost chemical intervention methods.

[0003] Currently, methods to improve the cold resistance of rice mainly include traditional breeding, genetic engineering breeding, and the application of chemical inducers. Traditional breeding achieves cold resistance by screening for cold-resistant varieties, but the breeding cycle is long and genetic background is limited, making it difficult to quickly meet production needs. Although genetic engineering breeding can directionally improve cold resistance traits, there are controversies surrounding the safety of gene editing, limiting its widespread application. Existing chemical regulatory reagents are mostly chemically synthesized substances, which can easily cause residual pollution, and the effects of some reagents are unstable, making it difficult to meet the needs of green agriculture development. Therefore, finding an efficient, safe, and easy-to-operate method to improve the cold stress resistance of rice seedlings has become an important need in current agricultural production.

[0004] 5-Methoxytryptamine is a naturally occurring indole compound with no residual pollution risk, meeting the requirements of green agriculture development. As a melatonin precursor, 5-Methoxytryptamine has advantages such as lower cost and easier absorption by plants.

[0005] The closest existing technology: In research, exogenous melatonin (chemical name N-acetyl-5-methoxytryptamine) has been used to improve the tolerance of rice seedlings (seedling stage) to low-temperature stress. For example, Kang et al. enhanced melatonin levels in rice through transgenic methods and found that transgenic seedlings had higher chlorophyll content under cold stress conditions, exhibiting stronger cold resistance. This is remarkably similar to your method—treating seedlings with a solution containing 5-methoxytryptamine to enhance cold resistance—in terms of both the type of substance and the purpose of the treatment.

[0006] However, this existing technology has the following shortcomings:

[0007] 1. Opposite direction: Existing technologies focus on increasing melatonin levels rather than directly using precursors or intermediates such as 5-methoxytryptamine. Therefore, their mechanisms of action differ from those of the methods described above in terms of the substances themselves and their metabolic pathways.

[0008] 2. The treatment method is unclear: The transgenic method increases melatonin through gene expression rather than by soaking the roots in an exogenous solution, which does not conform to the specific operation process of "root soaking + different concentrations + timed treatment". Summary of the Invention

[0009] To address the problems existing in the prior art, this invention provides a method and application for improving the resistance of rice seedlings to cold stress.

[0010] The present invention is implemented as follows: a method for improving the cold stress resistance of rice seedlings, characterized in that the method includes the following steps: treating rice with a 5-methoxytryptamine solution during the rice seedling stage.

[0011] Furthermore, the concentration of 5-methoxytryptamine used is 0-50 μmol / L.

[0012] Furthermore, the concentration of 5-methoxytryptamine used is 0 μmol / L, 1 μmol / L, 10 μmol / L, or 50 μmol / L.

[0013] Furthermore, the treatment method is root soaking.

[0014] Furthermore, the method for improving the cold stress resistance of rice seedlings involves soaking the roots in a solution containing 5-methoxytryptamine for 2 hours, followed by cold stress treatment.

[0015] Furthermore, the cold stress temperature is 4°C.

[0016] Another objective of this invention is to provide an application for improving the resistance of rice seedlings to cold stress, wherein the rice varieties are Minghui 63 and Minghui 86.

[0017] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0018] First, this invention significantly improves the physiological adaptability of rice plants under low-temperature stress by applying 5-methoxytryptamine during the seedling stage. Experimental results show that the treated rice exhibits significantly increased root length and plant height under cold stress, indicating that this substance can regulate physiological and biochemical processes related to cell division and elongation, mitigating the inhibitory effect of low temperature on root and aboveground growth, thereby expanding the area for nutrient absorption and light energy utilization. Simultaneously, both fresh and dry weight of the plants are increased, with increased dry matter content, reflecting improved biomass accumulation and material storage capacity while enhancing cold resistance. The increased chlorophyll content in leaves stabilizes the thylakoid membrane structure of chloroplasts, reduces damage to photosynthetic structures caused by low temperature, and maintains high photosynthetic efficiency and energy supply. The increased hydrogen peroxide content in leaves may trigger cold-resistance-related signaling pathways, enhancing active defense responses.

[0019] In terms of safety, 5-methoxytryptamine is a naturally occurring indole compound with no residual pollution risk, aligning with the development direction of green agriculture and sustainable planting. Its metabolic products in rice are non-toxic and have no adverse effects on human health, and will not cause harmful accumulation in grain products, making it suitable for widespread use in edible crops. This characteristic lays a solid ecological and food safety foundation for the agricultural application of this method.

[0020] In application, the method of this invention is characterized by its simplicity and low equipment requirements. It can be implemented without relying on complex agricultural machinery or precision devices, making it easy to promote in large-scale field planting. Farmers can complete the treatment during the seedling stage through conventional spraying, root soaking, or root irrigation. The technology is easy to master, has low input costs, and yields quick results, ensuring both cold resistance and production efficiency and economic benefits for rice.

[0021] Secondly, 5-methoxytryptamine, as a precursor to melatonin, has a structure similar to melatonin, but research on its application in rice under low-temperature stress is still in its early stages, and systematic application protocols have not yet been established domestically or internationally. 5-methoxytryptamine not only has the advantage of being cheaper than melatonin, but may also possess similar functions, such as enhancing cold resistance by regulating ROS scavenging, thus filling a gap in research on the functions of tryptamines in plants.

[0022] As a major rice-producing country, China frequently experiences low-temperature damage (such as early spring low temperatures in Northeast China and late spring frosts in the south), leading to yield reductions of 10%-30%. Statistics show that China loses approximately 3-5 million tons of rice annually due to low temperatures, and globally, over 15 million hectares of rice fields are threatened by cold weather. Traditional breeding and agronomic measures (such as mulching and spraying antifreeze agents) have limited effectiveness, necessitating the use of novel bioactive substances to enhance cold resistance. Firstly, using 5-methoxytryptamine to improve rice's resistance to cold stress is a biochemical method to enhance crop cold resistance, mitigating the impact of extreme weather on food systems. Secondly, this method overcomes the problems of long cycles and chemical residues associated with transgenic breeding, providing a rapid and safe solution. Finally, this approach reduces reliance on chemical fertilizers and pesticides, responding to the national call for "emission reduction and carbon sequestration" and "high and stable yields."

[0023] Tryptamines primarily function as neurotransmitters or hallucinogens in animals, and early studies neglected their potential physiological roles in plants. In previous research, tryptamines were almost never classified as plant signaling molecules. This study further confirms the independent function of tryptamines in plants. Attached Figure Description

[0024] Figure 1 The phenotypic diagrams of Minghui 63 and Minghui 86 under different concentrations of 5-methoxytryptamine treatment at 28°C and 4°C are provided in the embodiments of the present invention.

[0025] Figure 2 This is a statistical chart of the plant height of Minghui 63 and Minghui 86 under different concentrations of 5-methoxytryptamine treatment at 28℃ and 4℃, provided in the embodiments of the present invention.

[0026] Figure 3 This is a statistical chart of the root length of Minghui 63 and Minghui 86 under different concentrations of 5-methoxytryptamine treatment at 28℃ and 4℃, provided in the embodiments of the present invention.

[0027] Figure 4 This is a statistical chart of the fresh weight of Minghui 86 under different concentrations of 5-methoxytryptamine treatment at 28°C and 4°C, provided in the embodiments of the present invention.

[0028] Figure 5 This is a statistical chart of the dry weight of Minghui 86 under different concentrations of 5-methoxytryptamine treatment at 28°C and 4°C, provided in the embodiments of the present invention.

[0029] Figure 6 This is a statistical chart of the dry matter content of Minghui 86 under different concentrations of 5-methoxytryptamine treatment at 28°C and 4°C, provided in the embodiments of the present invention.

[0030] Figure 7 This is a statistical chart of chlorophyll content of Minghui 86 under different concentrations of 5-methoxytryptamine treatment at 28℃ and 4℃, provided in the embodiments of the present invention.

[0031] Figure 8 This is a statistical chart showing the hydrogen peroxide content of Minghui 86 under different concentrations of 5-methoxytryptamine treatment and at 28°C and 4°C, as provided in the embodiments of the present invention. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0033] In traditional rice production systems, low-temperature stress during the seedling stage is one of the key factors restricting stable yield. Existing technologies largely rely on variety selection or chemical regulation to improve cold resistance; however, in industrial applications, these methods often suffer from long response times, imprecise regulatory mechanisms, or insufficient environmental adaptability. Especially in major rice-producing areas prone to cold waves, seedling frost damage directly leads to poor greening and yield reduction. How to improve seedling cold resistance through exogenous regulatory means in a short period is a pressing technical challenge in agricultural production.

[0034] This application addresses the low-temperature sensitivity of rice seedlings by introducing 5-methoxytryptamine as an exogenous signaling molecule. As an indoleamine derivative, this substance can regulate redox balance at the cellular level, mitigating the accumulation of reactive oxygen species under cold stress. Unlike existing methods that rely solely on cold-resistance gene resources, this exogenous molecular intervention can regulate metabolic pathways and signal transduction in a short period, thus possessing immediate and universal industrial application value.

[0035] During the treatment process, 5-methoxytryptamine is absorbed into the rice plant through the roots, enters the vascular bundle system, and is transported to the leaves. In the cytoplasm, this molecule can reduce the excessive accumulation of hydrogen peroxide and superoxide anions by regulating the activity of antioxidant enzymes such as glutathione peroxidase and superoxide dismutase. Simultaneously, it can maintain the proportion of unsaturated fatty acids in the cell membrane, preventing membrane lipid peroxidation caused by chilling injury, thereby maintaining the integrity and functional stability of the cell membrane system.

[0036] From the perspective of signal transduction, 5-methoxytryptamine can interact with Ca... 2+ The interaction of signaling, the MAPK cascade, and the ABA-dependent pathway triggers the transcriptional expression of a series of downstream response genes. These genes include dehydration protein genes related to osmotic regulation and mitochondrial functional genes related to energy metabolism. Through this network-like regulatory mechanism, rice exhibits stronger osmotic regulation and energy supply capabilities under low-temperature conditions, providing support for physiological activities under cold stress.

[0037] At the industrial application level, this method provides a workable solution for rice seedling management. By applying 5-methoxytryptamine solution to hydroponics or seedling raising, not only can seedling loss caused by low temperatures be reduced, but seedlings also exhibit better tillering ability and uniform growth after transplanting. This means that farmers can improve seedling survival rates and final yields at a lower cost in cold years or areas with high risk of chilling injury, forming a beneficial supplement to existing fertilizer or hormone-based regulation methods.

[0038] In summary, this application, through the elucidation of the mechanism of action of 5-methoxytryptamine, establishes a complete technical pathway of "exogenous signaling molecule—antioxidant system—cold stress response." This pathway not only fundamentally solves the shortcomings of existing technologies in short-term cold resistance regulation but also provides a scalable operational model for industrial applications, demonstrating significant technological advancement and practical application value. The establishment of this mechanism provides new insights for coping with extreme climates in the rice industry and also offers a reference framework for stress regulation research in other crops.

[0039] This invention provides a method for improving the cold stress resistance of rice seedlings, comprising the following steps: treating rice with 5-methoxytryptamine during the rice seedling stage.

[0040] The concentration of 5-methoxytryptamine used is 0 μmol / L, 1 μmol / L, 10 μmol / L, or 50 μmol / L. In this invention, the preferred concentration of 5-methoxytryptamine used is 1 μmol / L.

[0041] The treatment method is root soaking.

[0042] The treatment method involves root immersion for 2 hours, which allows 5-methoxytryptamine to be absorbed through the roots, providing a foundation for cold resistance in the early stages of seedling growth.

[0043] The rice varieties mentioned are Minghui 63 and Minghui 86.

[0044] Example 1: Seed selection and seedling cultivation

[0045] Select plump, disease-free seeds of 'Minghui 63' and 'Minghui 86', disinfect them with 10% sodium hypochlorite solution for 8 minutes, rinse them 5 times with distilled water, and then place them in petri dishes containing moist germination paper. Grow them in the dark at 28℃. After 3 days, transplant the germinated rice seedlings into hydroponic boxes containing Kimura nutrient solution, and place them at 28℃ with 150 μmol·m² light. -2 ·s -1 It was grown for one week under a light cycle of 16 hours of light and 8 hours of light.

[0046] Example 2: Preparation of Mother Solution and Working Solution

[0047] Mother liquor: 5-methoxytryptamine is prepared by mixing 5-methoxytryptamine with an additive; the concentration of the 5-methoxytryptamine solution is 0 mmol / L, 1 mmol / L, 10 mmol / L, or 50 mmol / L, and the additive is an ethanol solution.

[0048] Working solution: Take 80 μL of the stock solution of 5-methoxytryptamine and add it to 800 mL of rice culture medium to obtain the working concentrations of 0 μmol / L, 1 μmol / L, 10 μmol / L and 50 μmol / L.

[0049] Example 3: Cold stress treatment

[0050] 5-Methoxytryptamine and cold stress treatment: Rice was divided into two groups. Each group was cultured in Kimura culture medium containing 0 μmol / L, 1 μmol / L, 10 μmol / L, and 50 μmol / L 5-methoxytryptamine for 2 hours. Then, one group was subjected to cold stress treatment (temperature 4℃, light 16h / dark 8h, treatment 36h), and the other group served as a room temperature control (temperature 28℃, light 16h / dark 8h).

[0051] Example 4: Index Measurement

[0052] After cold stress treatment, seedlings recovered for 6 days (temperature 28℃, 16h light / 8h dark). Phenotypes of each group of seedlings were recorded, and plant height, root length, fresh weight, dry weight, chlorophyll content, and hydrogen peroxide content were statistically analyzed. Plant height: Measured using a ruler from the root-stem junction to the tip of the tallest leaf when naturally extended. Root length: Measured from the root-stem junction to the tip of the longest root when naturally extended. Fresh weight: After removing whole rice plants from the hydroponic container, the surface moisture was absorbed with filter paper, and the fresh weight of a single plant was measured using a precision balance. Dry weight: The rice samples with measured fresh weights were placed in kraft paper bags, dried in a 45℃ oven for 3 days, and the dry weight of a single plant was measured using a precision balance. Chlorophyll content: The fully expanded leaves of the rice were clamped with a SPAD instrument, and values ​​were read from 3-5 sites, and the average value was taken.

[0053] Hydrogen peroxide content: H2O2 was determined using a titanium sulfate method assay kit (A06421, Nanjing Jiancheng, China).

[0054] like Figure 1 , Figure 2 , Figure 3 As shown, treatment with 1 μmol / L 5-methoxytryptamine increased the length of both the aboveground and underground parts of rice, especially under cold stress. With increasing 5-methoxytryptamine concentrations (10 μmol / L, 50 μmol / L), the growth of both aboveground and underground parts of rice was inhibited. This demonstrates the dual effect of 5-methoxytryptamine on rice growth; it only promotes growth within a suitable concentration range, while excessively high concentrations have an inhibitory effect.

[0055] like Figure 4 , Figure 5 , Figure 6 As shown, for Minghui 86, 1 μmol / L of 5-methoxytryptamine increased the dry matter accumulation of rice (dry matter accumulation rate (%) = (dry weight ÷ wet weight) × 100%), especially under cold stress conditions, reflecting that 1 μmol / L of 5-methoxytryptamine increased the rice's ability to synthesize and store substances under cold stress conditions.

[0056] like Figure 7 As shown, low and medium concentrations of 5-methoxytryptamine (1 μmol / L and 10 μmol / L) can increase the chlorophyll content of rice. Although the high concentration (50 μmol / L) can also increase the chlorophyll content, the effect is not as significant as that of the low concentration.

[0057] like Figure 8 As shown, a low concentration of 1 μmol / L can significantly increase the endogenous hydrogen peroxide content in rice, especially under cold stress conditions.

[0058] In this embodiment, low concentrations of 5-methoxytryptamine significantly alleviated the inhibitory effects of low temperature on root elongation and aboveground growth, enhanced photosynthetic pigment synthesis and chloroplast membrane stability, and induced an appropriate amount of hydrogen peroxide accumulation to activate cold resistance signaling pathways. The high-concentration treatment group showed less significant effects than the low-concentration group. This verifies the dual effect of 5-methoxytryptamine on rice growth; it only promotes growth within a suitable concentration range, while excessively high concentrations have an inhibitory effect. This treatment method maintains low application costs while ensuring rapid cold resistance during the seedling stage, making it suitable for early and mid-season rice production in situations requiring responses to sudden low temperatures.

[0059] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for improving the cold stress resistance of rice seedlings, characterized in that, The steps include: during the rice seedling stage, treating the rice roots in a solution containing 5-methoxytryptamine.

2. The method according to claim 1, characterized in that, The concentration of the 5-methoxytryptamine solution is 0 to 50 μmol / L.

3. The method according to claim 1, characterized in that, The concentration of the 5-methoxytryptamine solution is 0 μmol / L, 1 μmol / L, 10 μmol / L, or 50 μmol / L.

4. The method according to claim 1, characterized in that, The treatment time for the 5-methoxytryptamine solution is 2 hours.

5. The method according to claim 1, characterized in that, The cold stress treatment was performed at a temperature of 4°C for 36 hours.

6. An application of 5-methoxytryptamine treatment to improve the cold stress resistance of rice seedlings, characterized in that, The rice variety is Minghui 63 or Minghui 86.

7. The application according to claim 6, characterized in that, The treatment method involves soaking the roots in a solution during the seedling stage.

8. A method for detecting indicators to verify the resistance of rice seedlings to cold stress, characterized in that, This includes measuring the plant height, root length, fresh weight, dry weight, chlorophyll content, and hydrogen peroxide content of rice seedlings treated with 5-methoxytryptamine solution.

9. The method according to claim 8, characterized in that, The hydrogen peroxide content was detected by the titanium sulfate colorimetric method.

10. A combined scheme for improving the resistance of rice seedlings to cold stress, characterized in that, include: The roots of rice seedlings were soaked in 5-methoxytryptamine solution, combined with cold stress treatment and index detection steps, to improve and evaluate the cold stress resistance of rice.

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