A composite plant growth regulator and its application in improving cold tolerance of cotton seedlings

Abstract

The present invention discloses a composite plant growth regulator and its application in improving the cold tolerance of cotton seedlings. The present invention firstly compounds 2,4-epibrassinolide and prohexadione calcium, and sprays the composite growth regulator obtained by the compounding on cotton seedlings after cold stress. The results show that compared with the control, the composite growth regulator effectively increases the relative chlorophyll content, plant height, dry weight and fresh weight of stems and leaves of cotton seedlings in the recovery stage, indicating that it can quickly restore the growth of leaves and stems of cotton seedlings after cold stress, promote the accumulation of substances in cotton seedlings after cold stress, and explore the optimal ratio (EBR 0.04 mg / L+PC 30 mg / L). The composite growth regulator of the present invention eliminates the adverse effects of cold stress to a certain extent, ensures the healthy growth of cotton seedlings, effectively solves the problem of cotton agricultural production in Xinjiang, and is of great significance for ensuring high and stable yield of cotton.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composite plant growth regulator and its application in improving the cold tolerance of cotton seedlings, and in particular to a composite plant growth regulator comprising 2,4-epibrassinolide and prohexadione calcium and its application in improving the cold tolerance of cotton seedlings. Background Art

[0002] Plant growth regulators (PGRs) are substances with physiological and biological effects similar to those of plant hormones, used to artificially manipulate plant growth and development in desired directions. PGRs can be synthetic or natural products extracted from organisms. Currently, my country has a diverse and complex list of registered PGR active ingredients, totaling over 60, and this number is steadily increasing annually. However, despite their enormous potential in agricultural production, their practical application faces a number of challenges, most notably the limitations of the efficacy of single-type PGRs. Plant traits are often controlled by multiple dimensions, and the regulatory capabilities of a single PGR are therefore limited. Therefore, exploring the application of complex PGRs—that is, by combining multiple active ingredients in a scientifically formulated formula to achieve multi-level, comprehensive control of plant growth and development, thereby overcoming the efficacy bottlenecks of single-type regulators—is of great significance for agricultural production.

[0003] Xinjiang is my country's primary cotton-growing and production region, but young cotton seedlings face severe challenges. Large diurnal temperature swings and unstable spring temperatures have created a critical barrier to robust cotton seedling growth, impacting subsequent yield and quality. This has severely hampered cotton production in Xinjiang. Therefore, developing and promoting compound agents that offer cold resistance and resilience not only addresses pressing agricultural needs but also represents a key initiative to advance agricultural science and technology, promoting agricultural modernization and sustainable development. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to improve the cold tolerance of cotton in the seedling stage, so as to promote cotton growth, increase cotton yield and improve cotton quality.

[0005] In order to solve the above technical problems, the present invention first provides a composite plant growth regulator.

[0006] The active ingredients of the composite plant growth regulator provided by the invention include 2,4-epibrassinolide and prohexadione-calcium.

[0007] Furthermore, the concentration of 2,4-epibrassinolide in the composite plant growth regulator may be 0.01-0.1 mg / L or 0.01-0.04 mg / L or 0.04-0.1 mg / L, specifically 0.01 mg / L or 0.04 mg / L or 0.1 mg / L, preferably 0.04 mg / L.

[0008] The concentration of prohexadione-calcium in the composite plant growth regulator may be 20-100 mg / L or 20-30 mg / L or 30-60 mg / L or 60-100 mg / L, specifically 20 mg / L or 30 mg / L or 60 mg / L or 100 mg / L, preferably 30 mg / L.

[0009] Furthermore, the active ingredients of the composite plant growth regulator consist of 2,4-epibrassinolide and prohexadione-calcium.

[0010] In one embodiment of the present invention, the composite plant growth regulator is prepared by the following method: a 2,4-epibrassinolide solution, a prohexadione calcium solution, and water are mixed to obtain the composite plant growth regulator; the 2,4-epibrassinolide solution is prepared by dissolving 2,4-epibrassinolide in an ethanol solution to obtain a 2,4-epibrassinolide mother liquor (e.g., a 2,4-epibrassinolide mother liquor having a concentration of 1000 mg / L), and then mixing the 2,4-epibrassinolide mother liquor with water to obtain the 2,4-epibrassinolide solution; the prohexadione calcium solution is prepared by mixing prohexadione calcium (e.g., prohexadione calcium having a total active ingredient content of 15% and in the form of a suspension concentrate) with water to obtain the prohexadione calcium solution.

[0011] In practical applications, according to actual needs, the composite plant growth regulator may further contain other ingredients, such as synergists, cosolvents, etc.

[0012] In order to solve the above technical problems, the present invention also provides a new use of the above composite plant growth regulator.

[0013] The present invention provides application of the composite plant growth regulator in improving the cold tolerance of plants.

[0014] The present invention also provides the use of the composite plant growth regulator in preparing a product for improving the cold tolerance of plants.

[0015] In order to solve the above technical problems, the present invention finally provides a method for improving the cold tolerance of plants.

[0016] The method for improving the cold tolerance of plants provided by the present invention comprises the step of spraying the above-mentioned composite plant growth regulator on the plants.

[0017] Furthermore, the spraying site is leaves.

[0018] Furthermore, the spraying dosage is 2 ml / plant.

[0019] In any of the above-mentioned uses or methods, the cold tolerance is cold tolerance at the seedling stage.

[0020] The improvement of plant cold tolerance is embodied in any one of the following A1) to A6):

[0021] A1) Increase the chlorophyll content (e.g. relative chlorophyll content) in plant leaves after cold stress treatment;

[0022] A2) Increase plant height after cold stress treatment;

[0023] A3) Increase the fresh weight of plant stems after cold stress treatment;

[0024] A4) Increase the fresh weight of plant leaves after cold stress treatment;

[0025] A5) Increased stem dry weight of plants after cold stress treatment;

[0026] A6) Increase the dry weight of plant leaves after cold stress treatment.

[0027] In any of the above uses or methods, the plant is any one of the following C1) to C4):

[0028] C1) Monocots or dicots;

[0029] C2) Malvaceae;

[0030] C3) cotton plants;

[0031] C4) Cotton (such as medium cotton 113).

[0032] In any of the above applications or methods, the chemical name of the 2,4-epibrassinolide (EBR) is (22R,23R,24R)-2A,3A,22,23-tetrahydroxy-β-homo-7-oxa-5A-ergoster-6-one, and the molecular formula is C 28 H 48 O6, CAS number is 78821-43-9.

[0033] In any of the above applications or methods, the chemical name of the prohexadione calcium (PC) is 3-oxo-5-oxo-4-propionylcyclohex-3-enecarboxylate calcium, and the molecular formula is C 10 H 10 CaO5, CAS number is 127277-53-6.

[0034] This study, for the first time, combined 2,4-epibrassinolide and prohexadione-calcium and sprayed the resulting composite growth regulator on cotton seedlings after cold stress. The results showed that compared with the control, the composite growth regulator effectively increased the relative chlorophyll content, plant height, and stem and leaf dry and fresh weight of cotton seedlings during the recovery phase. This indicates that the composite growth regulator can rapidly restore leaf and stem growth and promote biomass accumulation in cotton seedlings after cold stress. The optimal ratio (EBR 0.04 mg / L + PC 30 mg / L) was also identified. The composite growth regulator of this invention eliminates the adverse effects of cold stress to a certain extent, ensuring the healthy growth of cotton seedlings, effectively resolving agricultural production issues in Xinjiang, and is of great significance for ensuring high and stable cotton yields. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Cold stress inhibits the growth of cotton seedlings.

[0036] Figure 2 This is the phenotype of cotton seedlings 3 days after treatment with the combination agent.

[0037] Figure 3 The compound agent treatment promotes the accumulation of substances during the recovery period. DETAILED DESCRIPTION

[0038] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0039] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0040] The chemical name of 2,4-epibrassinolide (EBR) in the following examples is (22R,23R,24R)-2A,3A,22,23-tetrahydroxy-β-homo-7-oxa-5A-ergoster-6-one, and the molecular formula is C 28 H 48 O6, CAS No. 78821-43-9. 2,4-Epibrassinolide in the present invention is a product of Shanghai Yuanye Biotechnology Co., Ltd., with the product number S18014-100mg.

[0041] The chemical name of prohexadione calcium (PC) in the following examples is calcium 3-oxo-5-oxo-4-propionylcyclohex-3-enecarboxylate, and its molecular formula is C 10 H10 CaO5, CAS No. 127277-53-6. The prohexadione calcium (with a total active ingredient content of 15% and a suspension concentrate) in the present invention is a product of Heli Technology Co., Ltd.

[0042] The cotton variety "Zhongmian 113" in the following examples is recorded in the literature "Wang L, Lin M, Han Z, et al. Simulating the effects of drought stress timing and the amount irrigation on cotton yield using the CSM-CROPGRO-cotton model[J]. Agronomy, 2023, 14(1):14." and "Yan P, Han Q, Feng Y, et al. Estimating lai for cotton usingmultisource uav data and a modified universal model[J]. Remote Sensing, 2022,14(17): 4272.".

[0043] Example 1: A composite plant growth regulator and its application in improving the cold tolerance of cotton

[0044] 1. Cultivation of cotton seedlings

[0045] 1. Select plump and uniform cotton seeds of the variety "Zhongmian 113", add 75% ethanol and shake to mix for 15 minutes. After standing, pour out the upper layer of ethanol.

[0046] 2. Add sterile water, invert to mix, remove suspended impurities and sterile water, repeat 5 times, and soak the seeds in sterile water for 6 hours after rinsing to achieve seed swelling.

[0047] 3. After soaking the seeds, dry the moisture from the seeds appropriately and place them on sterile filter paper for cultivation. Place them in a constant temperature box (28°C) for germination for 1 day.

[0048] 4. Select white seeds and sow them in soil (peat soil: vermiculite = 2:1), place them in a light incubator (28℃ light for 12h, 25℃ dark for 12h per day) and culture for 7 days to obtain cotton seedlings with uniform growth.

[0049] 2. Experimental treatment

[0050] 1. Select seedlings with consistent growth for cold stress treatment and place them in a light incubator (6°C light for 12 h, 4°C dark for 12 h per day) for 3 days (simulating the cold stress environment of Xinjiang cotton seedlings) to obtain cold-stressed cotton seedlings.

[0051] At the same time, normal cotton seedlings cultured in a normal environment (28°C light for 12 h, 25°C dark for 12 h per day) without cold stress for 3 days were used as controls.

[0052] 2. After the cold stress ended, the leaves of the cold-stressed cotton seedlings were sprayed with the drug at a dosage of 2 ml per plant to ensure that every leaf was exposed to the drug solution. After spraying, the seedlings were placed in a normal environment (28°C light for 12 hours, 25°C dark for 12 hours per day) for recovery. According to the different spraying treatment methods, the seedlings were divided into the following groups:

[0053] CK group: 2 ml of water was sprayed on normal cotton seedlings.

[0054] Group A1: 2 ml of water was sprayed on the cold-stressed cotton seedlings.

[0055] Group A2: 2 ml of 0.04 mg / L EBR solution was sprayed on cold-stressed cotton seedlings.

[0056] Group A3: 2 ml of 60 mg / L PC solution was sprayed on cold-stressed cotton seedlings.

[0057] Group A4: 2 ml of the compound solution was sprayed onto the cold-stressed cotton seedlings. The EBR concentration in the compound solution was 0.04 mg / L and the PC concentration was 60 mg / L.

[0058] Group A5: 2 ml of the compound solution was sprayed onto the cold-stressed cotton seedlings. The EBR concentration in the compound solution was 0.04 mg / L and the PC concentration was 30 mg / L.

[0059] Group A6: 2 ml of the compound solution was sprayed onto the cold-stressed cotton seedlings. The EBR concentration in the compound solution was 0.1 mg / L and the PC concentration was 60 mg / L.

[0060] Group A7: 2 ml of the compound solution was sprayed onto the cold-stressed cotton seedlings. The EBR concentration in the compound solution was 0.1 mg / L and the PC concentration was 30 mg / L.

[0061] The EBR solution was prepared as follows: EBR was dissolved in an ethanol solution to obtain an EBR mother solution with a concentration of 1000 mg / L, and then the EBR mother solution was diluted with water to a desired concentration to obtain an EBR solution.

[0062] The PC solution is prepared as follows: prohexadione calcium (total active ingredient content of 15%, dosage form is suspension) is diluted with water to a desired concentration to obtain a PC solution.

[0063] The preparation method of the composite solution is as follows: the EBR solution and the PC solution are mixed with water, and the EBR and PC are diluted with water to the required concentration to obtain the composite solution.

[0064] 3. After three days of recovery, phenotypic analysis and agronomic trait measurement were performed to determine the extent of recovery. Agronomic traits measured included SPAD (relative chlorophyll content), plant height, stem fresh weight, stem dry weight, leaf fresh weight, and leaf dry weight. SPAD (relative chlorophyll content) was measured in live leaves using a SPAD chlorophyll meter (SPAD-502, Minolta Camera Co., Ltd., Osaka, Japan). Five measurements were taken on the same leaf, and the average value was used to represent the relative chlorophyll content of that leaf.

[0065] 3. Experimental Results

[0066] 1. Cold stress inhibits the growth of cotton seedlings

[0067] After 3 days of cold stress treatment, the growth of cotton seedlings was severely inhibited ( Figure 1 Compared to the control group, cold stress caused the cotton seedlings to develop bent stems, wilted leaves, and virtually stopped growing. Before the application of plant growth regulators, the cold-stressed cotton seedlings grew uniformly.

[0068] 2. Treatment with compound plant growth regulators restored the growth of cotton seedling leaves and stems

[0069] After 3 days of treatment with various concentrations of plant growth regulators, phenotypic acquisition and agronomic trait determination were performed. The phenotypes showed that cold stress inhibited the growth of cotton seedlings. After 3 days of recovery, there was still a large gap between the growth of cotton seedlings and normal growth ( Figure 2 However, spraying plant growth regulators restored the inhibitory effect of cold stress to a certain extent, promoted leaf growth, and increased plant height.

[0070] SPAD (relative chlorophyll content) and plant height data showed that cold stress caused SPAD and plant height to decrease by 21.9% and 53.4%, respectively. The average SPAD (relative chlorophyll content) of the CK, A1, A2, A3, A4, A5, A6, and A7 groups were 41.7, 32.5, 34.8, 33.1, 35.3, 37.2, 36.8, and 35.6, respectively. The average plant height of the CK, A1, A2, A3, A4, A5, A6, and A7 groups was 16.4 cm, 7.7 cm, 10.0 cm, 8.8 cm, 10.4 cm, 11.4 cm, 10.9 cm, and 9.6 cm, respectively.

[0071] Compared with individual cold stress treatments, all concentrations of plant growth regulators were able to restore the inhibitory effects of cold stress to a certain extent. The combination treatment A5 (EBR 0.04 mg / L + PC 30 mg / L) significantly increased SPAD values ​​and plant height by 14.3% and 49.3%, respectively. Furthermore, the combination treatments (A4, A5, A6, and A7) were more effective than the individual treatments (A2 and A3, commercial concentrations). Specifically, compared with the individual treatment A2 (EBR 0.04 mg / L), the combination treatment A5 (EBR 0.04 mg / L + PC 30 mg / L) significantly increased SPAD values ​​and plant height by 14.1% and 7.0%, respectively. Compared with treatment A3 (PC 60 mg / L), the combined treatment A5 (EBR 0.04 mg / L + PC 30 mg / L) significantly increased SPAD values ​​and plant height by 29.6% and 12.4%, respectively. These results indicate that the combined EBR and PC exhibit synergistic effects, effectively restoring the inhibitory effects of cold stress. Treatment A5 (EBR 0.04 mg / L + PC 30 mg / L) exhibited the best effect.

[0072] 3. Treatment with compound plant growth regulators promotes substance accumulation in cotton seedlings after cold stress

[0073] Consistent with the above trends in chlorophyll content and plant height, cold stress severely inhibited the growth of stems and leaves, making it difficult to recover to normal levels. The average stem fresh weights of the CK, A1, A2, A3, A4, A5, A6 and A7 groups were 0.8303 g, 0.3105 g, 0.3975 g, 0.3517 g, 0.4106 g, 0.4790 g, 0.4699 g and 0.4428 g, respectively. The average leaf fresh weights of the CK, A1, A2, A3, A4, A5, A6 and A7 groups were 1.1624 g, 0.4668 g, 0.5826 g, 0.5166 g, 0.6470 g, 0.7012 g, 0.6701 g and 0.6233 g, respectively. The average stem dry weights of the CK, A1, A2, A3, A4, A5, A6 and A7 groups were 0.0804 g, 0. The average leaf dry weights of CK group, A1 group, A2 group, A3 group, A4 group, A5 group, A6 group and A7 group were 0.1269 g, 0.0507 g, 0.0639 g, 0.0560 g, 0.0661 g, 0.0754 g, 0.0733 g and 0.0672 g, respectively.

[0074] Compared with individual cold stress treatments, all concentrations of plant growth regulators were able to restore the inhibitory effects of cold stress to a certain extent. Furthermore, the combined treatments demonstrated greater efficacy than the individual treatments. Specifically, compared with individual treatment A2 (EBR 0.04 mg / L), combined treatment A5 (EBR 0.04 mg / L + PC 30 mg / L) significantly increased stem fresh weight, leaf fresh weight, stem dry weight, and leaf dry weight by 20.5%, 20.4%, 20.9%, and 18.0%, respectively. Compared with individual treatment A3 (PC 60 mg / L), combined treatment A5 (EBR 0.04 mg / L + PC 30 mg / L) significantly increased stem fresh weight, leaf fresh weight, stem dry weight, and leaf dry weight by 36.2%, 35.7%, 31.2%, and 34.6%, respectively. Therefore, the combined treatment group had better effects than the single treatment group, among which the combined treatment group A5 (EBR 0.04 mg / L + PC 30 mg / L) had the best treatment effect.

[0075] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. Application of compound plant growth regulators in improving plant cold tolerance or preparing products that improve plant cold tolerance; The active ingredients of the composite plant growth regulator are composed of 2,4-epibrassinolide and prohexadione calcium; The concentration of 2,4-epibrassinolide in the composite plant growth regulator is 0.04 mg / L; The concentration of prohexadione-calcium in the composite plant growth regulator is 30 mg / L.

2. The use according to claim 1, characterized in that: The cold tolerance is cold tolerance at the seedling stage.

3. The use according to claim 1, characterized in that: The improvement of plant cold tolerance is embodied in any one of the following A1) to A6): A1) Increase the chlorophyll content in plant leaves after cold stress treatment; A2) Increase plant height after cold stress treatment; A3) Increase the fresh weight of plant stems after cold stress treatment; A4) Increase the fresh weight of plant leaves after cold stress treatment; A5) Increased stem dry weight of plants after cold stress treatment; A6) Increase the dry weight of plant leaves after cold stress treatment.

4. The use according to any one of claims 1 to 3, characterized in that: The plant is cotton.

5. A method for improving the cold tolerance of plants, comprising the step of spraying a composite plant growth regulator on the plants; The active ingredients of the composite plant growth regulator are composed of 2,4-epibrassinolide and prohexadione calcium; The concentration of 2,4-epibrassinolide in the composite plant growth regulator is 0.04 mg / L; The concentration of prohexadione-calcium in the composite plant growth regulator is 30 mg / L.

6. The method according to claim 5, characterized in that: The cold tolerance is cold tolerance at the seedling stage.

7. The method according to claim 5, characterized in that: The improvement of plant cold tolerance is embodied in any one of the following A1) to A6): A1) Increase the chlorophyll content in plant leaves after cold stress treatment; A2) Increase plant height after cold stress treatment; A3) Increase the fresh weight of plant stems after cold stress treatment; A4) Increase the fresh weight of plant leaves after cold stress treatment; A5) Increased stem dry weight of plants after cold stress treatment; A6) Increase the dry weight of plant leaves after cold stress treatment.

8. The method according to any one of claims 5 to 7, characterized in that: The plant is cotton.

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