Plant regulator and its use in improving plant resistance to viruses and / or alleviating low temperature stress in plants

Abstract

The application belongs to the technical field of plant growth regulators, and particularly relates to a plant regulator and application thereof in improving plant antiviral ability and / or relieving low-temperature stress of plants. The application enhances the stability of the plant regulator by regulating the concentration of chitosan oligosaccharide chelated zinc, is conducive to the absorption of zinc elements by roots and plants, and ensures the efficacy of chitosan oligosaccharide in improving the resistance of plants to diseases and pests and relieving low-temperature stress. Experiments show that the technical scheme provided by the application greatly improves the antioxidant enzyme activity and photosynthesis efficiency of plants in a low-temperature environment, and the inhibition rate of tobacco mosaic virus can be up to 71.43%. Therefore, the chitosan oligosaccharide chelated zinc plant regulator provided by the application can not only improve the stress resistance of plants, but also realize the high-value utilization of marine low-value products.

Description

Technical Field

[0001] This invention belongs to the field of plant growth regulator technology, specifically relating to a plant growth regulator and its application in improving plant antiviral ability and / or alleviating plant low-temperature stress. Background Technology

[0002] Low temperatures are one of the major natural disasters in agricultural production, severely affecting the normal growth, development, and yield of plants. Low temperature stress causes peroxidation of the plant plasma membrane, damaging its structure; it also leads to chlorophyll degradation, reducing photosynthetic efficiency and severely impacting plant growth and development, even causing plant death. Especially during the tobacco seedling stage and after transplanting, plants frequently suffer from low-temperature damage and viral diseases caused by late spring frosts, resulting in slow seedling growth, yellowing leaves, and delayed post-transplant development, thus affecting tobacco production.

[0003] Chitosan oligosaccharides are oligosaccharides obtained from the degradation of chitosan, mainly derived from aquatic product byproducts such as shrimp and crab shells. As a natural plant elicitor, they can enhance plants' resistance to pests, diseases, and abiotic stresses, showing broad application prospects in agricultural production. Zinc can affect plant photosynthesis by participating in auxin metabolism, thereby influencing plant growth; zinc can also act as a metal activator of enzymes, enhancing the activity of antioxidant enzymes in plants and improving plant stress resistance by participating in important metabolic processes. However, there are currently no reports on the use of chitosan oligosaccharide-chelated zinc to improve plant antiviral capabilities and / or alleviate plant low-temperature stress. Summary of the Invention

[0004] The purpose of this invention is to provide a plant regulator and its application in improving plant antiviral ability and / or alleviating plant low-temperature stress, which can improve plant antiviral ability while alleviating plant low-temperature stress.

[0005] This invention provides a plant growth regulator, the active ingredient of which includes chitosan oligosaccharide chelated zinc; the concentration of chitosan oligosaccharide chelated zinc in the plant growth regulator is 100-1000 mg / L.

[0006] Preferably, the zinc content in the chitosan oligosaccharide chelated zinc is 3% to 4% by mass.

[0007] Preferably, the plant regulator further includes a surfactant; the concentration of the surfactant in the plant regulator is 1-10 mL / L.

[0008] Preferably, the surfactant includes one or more of Tween surfactant, Span surfactant, OP surfactant, and NP surfactant.

[0009] This invention provides the application of the plant regulators described in the above technical solution in improving plant antiviral ability and / or alleviating plant low-temperature stress.

[0010] The present invention also provides a method for improving the antiviral ability of plants and / or alleviating low temperature stress in plants, comprising the following steps: soaking plant seeds in the plant regulator described in the above technical solution and / or applying it to the plants.

[0011] Preferably, the application method includes spraying and / or drenching.

[0012] Preferably, when soaking plant seeds, the concentration of the plant growth regulator is 0.1–0.5 g / L; and the soaking time is 6–8 h.

[0013] Preferably, when applied to plants, the concentration of the plant growth regulator is 0.1–1 g / L, and the application rate is 20–80 L / mu.

[0014] Preferably, the plant species include tobacco and / or wheat.

[0015] Beneficial effects:

[0016] This invention provides a plant growth regulator, the active ingredient of which includes chitosan oligosaccharide chelated zinc; the concentration of chitosan oligosaccharide chelated zinc in the plant growth regulator is 100–1000 mg / L. By using an appropriate concentration of chitosan oligosaccharide chelated zinc, the stability of the plant growth regulator is enhanced, which is beneficial for the absorption of metallic zinc by roots and plants, while ensuring that chitosan oligosaccharide improves the plant's resistance to pests and diseases and alleviates low-temperature stress. Therefore, using chitosan oligosaccharide chelated zinc plant growth regulator can not only improve the plant's stress resistance but also realize the high-value utilization of low-value marine products.

[0017] Based on the aforementioned technical advantages, this invention also provides a method for improving plant antiviral capabilities and / or alleviating low-temperature stress in plants, comprising the following steps: soaking plant seeds in the plant regulator described in the above technical solution and / or applying it to the plants. Soaking plant seeds and / or applying it to the plants helps to improve the plant's antiviral capabilities and alleviate low-temperature stress. Experiments show that the technical solution provided by this invention significantly improves the activity of antioxidant enzymes and photosynthetic efficiency of plants under low-temperature conditions, with an inhibition rate of up to 71.43% against tobacco mosaic virus. Therefore, the technical solution provided by this invention can be used to improve plant antiviral capabilities and / or alleviate low-temperature stress in plants. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0019] Figure 1Infrared spectra of the plant growth regulators prepared in Example 1 and Comparative Example 1;

[0020] Figure 2 The graph shows the effect of different treatment groups on the growth of tobacco seedlings in Application Example 3. Detailed Implementation

[0021] This invention provides a plant growth regulator, the active ingredient of which includes chitosan oligosaccharide chelated zinc; the concentration of chitosan oligosaccharide chelated zinc in the plant growth regulator is 100-1000 mg / L.

[0022] Unless otherwise specified, all raw materials and equipment used in this invention are conventionally purchased.

[0023] The concentration of chitosan oligosaccharide chelated zinc in the plant growth regulator of the present invention is 100-1000 mg / L, more preferably 100-500 mg / L or 700-1000 mg / L, and even more preferably 100 mg / L. In the present invention, the mass of zinc in the chitosan oligosaccharide chelated zinc is preferably 3%-4%, and even more preferably 3%.

[0024] The preferred method for preparing chitosan oligosaccharide chelated zinc according to the present invention includes: mixing a chitosan oligosaccharide solution and a zinc-containing compound solution and then performing a chelation reaction to obtain a chelation reaction solution; and dialysis, precipitation and drying of the chelation reaction solution to obtain the chitosan oligosaccharide chelated zinc.

[0025] In this invention, chitosan oligosaccharide is preferably dissolved in water to obtain a chitosan oligosaccharide solution. In this invention, the mass-to-volume ratio of chitosan oligosaccharide to water is preferably 10-100 mg:10-50 mL, more preferably 20 mg:10 mL; the type of water is preferably distilled water. There are no special requirements for the mixing method in this invention, as long as the chitosan oligosaccharide is completely dissolved in the water. The molecular weight of the chitosan oligosaccharide in this invention is preferably 2000-5000 Da, more preferably 3500 Da.

[0026] This invention also preferably involves dissolving a zinc-containing compound in water to obtain a zinc-containing compound solution. In this invention, the mass-to-volume ratio of the zinc-containing compound to water is preferably 10–100 mg:10–100 mL, more preferably 10 mg:10 mL; the water is preferably distilled water. The zinc-containing compound in this invention is preferably zinc sulfate. There are no special requirements for the mixing method in this invention; the goal is for the zinc-containing compound to be completely dissolved in the water.

[0027] After obtaining the chitosan oligosaccharide solution and the zinc compound solution respectively, the present invention preferably mixes the chitosan oligosaccharide solution and the zinc compound solution to carry out a chelation reaction to obtain a chelation reaction solution. In the present invention, the mixture is preferably heated in a water bath during the chelation reaction. The pH value of the mixture is preferably 5.5-7.0, more preferably 7.0; the water bath heating temperature is preferably 35-45°C, more preferably 40°C; and the water bath heating time is preferably 20-40 min, more preferably 30 min.

[0028] After obtaining the chelation reaction solution, the present invention preferably dialyzes the chelation reaction solution with distilled water. The dialyzing time of the present invention is 24-72 hours, more preferably 48 hours; the dialyzing is preferably performed using a dialysis bag capable of retaining molecules with a molecular weight cutoff of 3500 Da. The source of the dialysis bag of the present invention is not particularly required and can be purchased conventionally.

[0029] Following dialysis, the present invention preferably uses anhydrous ethanol to perform a precipitation reaction on the retentate to obtain a precipitate. The volume ratio of anhydrous ethanol to the chelation reaction solution is preferably 3-4:1, more preferably 3:1. In the present invention, the precipitation reaction preferably includes vacuum filtration; the method of vacuum filtration is not particularly required and any technique well-known in the art can be used. The present invention preferably also includes washing the reactants obtained by vacuum filtration to obtain the precipitate; the washing reagent is preferably anhydrous ethanol, and the washing method is not particularly required and any technique well-known in the art can be used.

[0030] After obtaining the precipitate, the present invention preferably dries the precipitate to obtain chitosan oligosaccharide chelated zinc. In the present invention, the drying temperature is preferably 45–65°C, more preferably 60°C; the drying equipment is preferably a forced-air drying oven. The present invention does not have specific requirements for the drying time, which is determined by the degree of drying. Through the above method, fully chelated chitosan oligosaccharide chelated zinc can be prepared, greatly improving the stability of zinc.

[0031] In this invention, the plant regulator preferably includes a surfactant; the concentration of the surfactant in the plant regulator is 1–10 mL / L, preferably 1–3 mL / L, 3–5 mL / L, or 7–10 mL / L; more preferably 1 mL / L; the surfactant is preferably one or more of Tween surfactant, Span surfactant, OP surfactant, and NP surfactant, more preferably Tween surfactant. Adding a surfactant to the plant regulator helps reduce the surface tension of the target solution, enabling the resulting plant regulator to be used as a spray.

[0032] The preferred method for preparing the plant regulator includes: mixing and adjusting the volume of a chitosan oligosaccharide chelated zinc aqueous solution with a surfactant to obtain a plant regulator containing a surfactant. In this invention, the chitosan oligosaccharide chelated zinc is dissolved in water to obtain a chitosan oligosaccharide chelated zinc aqueous solution. The method of mixing and adjusting the volume of the chitosan oligosaccharide chelated zinc aqueous solution with the surfactant is not particularly demanding and can be achieved using techniques well-known in the art. Accurately adjusting the volume to the concentrations of the surfactant and the chitosan oligosaccharide chelated zinc not only facilitates the absorption of metallic zinc by roots and plants but also simultaneously ensures that the chitosan oligosaccharide enhances the plant's resistance to pests and diseases and alleviates low-temperature stress.

[0033] Based on the above-mentioned technical advantages, the present invention also provides a method for improving the antiviral ability of plants and / or alleviating low-temperature stress in plants, comprising the following steps: soaking plant seeds in the plant regulator described in the above technical solution and / or applying it to the plants.

[0034] Preferably, the present invention involves soaking plant seeds in the plant regulator and then applying it to the plants. The components and preparation method of the plant regulator of the present invention have been described in detail above and will not be repeated here.

[0035] In this invention, when soaking plant seeds, the concentration of the plant growth regulator is preferably 0.1–0.5 g / L, more preferably 0.1 g / L; the soaking time is preferably 6–8 hours, more preferably 6 hours. When soaking seeds, the amount of the plant growth regulator used in this invention is such that the seeds are completely submerged.

[0036] In this invention, when applied to plants, the concentration of the plant growth regulator is preferably 0.1–1 g / L, more preferably 0.1 g / L; the application method is preferably spraying and / or root irrigation, more preferably spraying; the application period is preferably the seedling stage and / or maturity stage of the plant. When applied to plants, the application rate of the plant growth regulator is preferably 20–80 L / mu, more preferably 30 L / mu or 60 L / mu. In a specific embodiment of this invention, during tobacco planting, the application rate of the plant growth regulator is 30 L / mu during the tobacco seedling stage and 60 L / mu during the tobacco maturity stage; during wheat planting, the application rate of the plant growth regulator is 30 L / mu during the tobacco seedling stage and 60 L / mu during the tobacco maturity stage.

[0037] This invention also provides the application of the plant regulators described in the above-mentioned technical solutions in improving plant antiviral capabilities and / or alleviating plant low-temperature stress. In this invention, the plant species are preferably tobacco and / or wheat, more preferably tobacco and wheat. Experiments show that using the technical solutions provided by this invention not only significantly improves the activity of antioxidant enzymes and photosynthetic efficiency of plants under low-temperature conditions, but also effectively enhances the antiviral capabilities of plants, especially achieving an inhibition rate of up to 71.43% against tobacco mosaic virus.

[0038] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a plant regulator provided by the present invention and its application in improving plant antiviral ability and / or alleviating plant low-temperature stress, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the methods used in the embodiments, comparative examples and application examples of this invention are conventional methods in the art.

[0040] Example 1

[0041] The preparation of plant growth regulators consists of the following steps:

[0042] 1) Weigh 20 mg of chitosan oligosaccharide (purchased from Qingdao Hehai Biotechnology Co., Ltd., average molecular weight 3500 Da, degree of deacetylation ≥85%) and 10 mg of zinc sulfate, and place them separately into two beakers. Add 10 mL of distilled water to each beaker and stir until dissolved. Then mix the chitosan oligosaccharide solution and zinc sulfate solution and stir thoroughly to obtain a mixture. Adjust the pH of the mixture with a 1% ammonia solution, slowly raising it to 7. Then place the beaker containing the mixture in a 40℃ water bath and perform a chelation reaction for 30 min. Dialyze the mixture for 48 h using a dialysis bag with a molecular weight cutoff of 3500 Da. Then slowly add three volumes of anhydrous ethanol to the dialysate, filter, and wash with anhydrous ethanol. Finally, dry the precipitate in a 60℃ oven. The resulting yellow solid is chitosan oligosaccharide chelated zinc. The test results showed that the zinc content in the chitosan oligosaccharide chelated zinc prepared in this manner was 10% (zinc sulfate was purchased from Sinopharm Group Pharmaceutical Co., Ltd., analytical grade).

[0043] 2) Weigh 10 mg of chitosan oligosaccharide chelated zinc from step 1) and dissolve it in distilled water. Then add 100 μL of Tween-80 and stir well. After adjusting the volume to 100 mL and stirring well, plant growth regulator B is obtained (the concentration of chitosan oligosaccharide chelated zinc in plant growth regulator B is 100 mg / L).

[0044] Comparative Example 1

[0045] The preparation of plant growth regulators consists of the following steps:

[0046] Weigh 10 mg of chitosan oligosaccharide from step 1) of Example 1 and dissolve it in 80 mL of distilled water. Then add 100 μL of Tween-80 and stir until homogeneous. Make up to 100 mL and stir until homogeneous to obtain plant growth regulator A (the concentration of chitosan oligosaccharide chelated zinc in plant growth regulator B is 100 mg / L).

[0047] Results and Analysis

[0048] The plant growth regulators A and B prepared in Comparative Example 1 and Example 1, respectively, were analyzed by infrared spectroscopy. The results are shown in the figure. Figure 1 (COS represents plant growth regulator A prepared in Comparative Example 1; COS-Zn represents plant growth regulator B prepared in Example 1).

[0049] Depend on Figure 1 It can be seen that the wavenumber is approximately 556.6 cm⁻¹ -1 The -NH2 absorption peak at this location is significantly weakened, indicating that the -NH2 in the chitosan oligosaccharide molecule participates in the chelation reaction; the peak is located at a wavenumber of approximately 1514.6 cm⁻¹. -1 The absorption peak of the -C=O bond around 100°C is significantly weakened, indicating that the -C=O double bond in the chitosan oligosaccharide molecule has chelated with zinc ions to form -CO-Zn. Infrared spectroscopy results show that chitosan oligosaccharide and zinc ions have chelated to form a stable chelate.

[0050] Application Example 1

[0051] Antiviral experiment: The inhibitory effects of the plant growth regulators in Example 1 and Comparative Example 1 on tobacco mosaic virus (TMV) were determined by pot experiment and half-leaf necrotic spot method (Reference: Liu Xiaowei, Qin Yuanxia, ​​Yuan Lianlian et al. Study on biocontrol of two rhizosphere growth-promoting bacteria against TMV [J]. China Tobacco Science, 2018, 24(06):78-85.DOI:10.16472 / j.chinatobacco.2018.080).

[0052] The experiment consists of the following steps:

[0053] 1) Plant growth regulators: Dilute plant growth regulator A in Comparative Example 1 and plant growth regulator B in Example 1 by 100 times respectively, and set aside for later use;

[0054] Control reagent 1: Prepare zinc sulfate reagent separately by dissolving 100 mg of zinc sulfate in 100 mL of distilled water to prepare a solution with a final concentration of 1000 mg / L for later use;

[0055] Control reagent 2: Mix 100 mg chitosan oligosaccharide directly with 1000 mg zinc sulfate reagent, and bring the volume to 1 L for later use;

[0056] Control reagent 3: Commercially available product: Chitosan oligosaccharide zinc (purchased from Shandong Lvlong Biotechnology Co., Ltd.)

[0057] 2) Test crop: Nicotiana scabra. Nicotiana scabra was planted in sterilized seedling material and placed in an insect-proof greenhouse for cultivation. It was used when the Nicotiana scabra had grown to the 7-8 leaf stage (planting and cultivation can be carried out according to local planting and management practices).

[0058] 3) Tobacco mosaic virus (TMV): The variety was Yunyan 87, which was preserved on tobacco plants in an insect-proof greenhouse.

[0059] 4) Preparation of test virus inoculum: Take 1g of TMV-infected leaves, grind them into a homogenate in a mortar, filter with gauze, transfer the filtrate to a centrifuge tube and mix with 10mL of water for later use.

[0060] 5) Treatment group setup: Take 1 mL of the diluted plant growth regulator A from step 1) and mix it with 1 mL of the test virus inoculation solution from step 4) (referred to as mixture COS). Take 1 mL of the diluted plant growth regulator B from step 1) and mix it with 1 mL of the test virus inoculation solution from step 4) (referred to as mixture COS-Zn). Place the mixtures in 2 mL centrifuge tubes and let them stand for 15 min. Then, use the half-leaf method to inoculate the mixtures onto the test tobacco leaves (the test tobacco variety is Zhongyan 100). Mixture COS was inoculated onto the right half of tobacco leaf 1, and mixture COS-Zn was inoculated onto the right half of tobacco leaf 2.

[0061] Control group setup: Mix 1 mL of water with 1 mL of the test virus inoculation solution from step 4) (referred to as mixture CK). Mixture CK was then applied by rubbing the mixture onto the untreated left half of tobacco leaf 1 (i.e., COS was inoculated on the right half of a tobacco leaf and CK was inoculated on the left half) and the untreated left half of tobacco leaf 2 (i.e., COS-Zn was inoculated on the right half of a tobacco leaf and CK was inoculated on the left half) using the half-leaf method.

[0062] Control reagents 1 to 3 were inoculated using the same half-leaf method; each treatment was repeated three times in parallel, and the inoculated tobacco was cultured in a greenhouse.

[0063] Results and Analysis

[0064] The formula for calculating the disease prevention effect is as follows:

[0065] Disease prevention effect = (Number of necrotic spots on the treated half-leaf - Number of necrotic spots on the control half-leaf) / Number of necrotic spots on the control half-leaf × 100%

[0066] After the appearance of typical necrotic spots at 48h and 72h, the number of necrotic spots was counted. The results are shown in Table 1. COS represents tobacco leaves with the left half inoculated with CK and the right half inoculated with COS; COS-Zn represents tobacco leaves with the left half inoculated with CK and the right half inoculated with COS-Zn; ZnSO4 represents tobacco leaves with the left half inoculated with CK and the right half inoculated with ZnSO4; COS+ZnSO4 represents tobacco leaves with the left half inoculated with CK and the right half inoculated with COS+ZnSO4; and commercial products represent tobacco leaves with the left half inoculated with CK and the right half inoculated with commercial products. The inhibitory effect of chitosan oligosaccharide and its chelates on tobacco mosaic virus (TMV) was studied.

[0067] Table 1. Inhibitory effects of chitosan oligosaccharide and chitosan oligosaccharide chelated zinc on tobacco mosaic virus.

[0068]

[0069] As shown in Table 1, compared with the control, all treatment groups showed inhibitory effects on tobacco mosaic virus; the inhibition rates of chitosan oligosaccharide or zinc sulfate alone were comparable. However, the inhibition rate of chitosan oligosaccharide combined with zinc sulfate was improved; chitosan oligosaccharide chelated with zinc showed the best inhibitory effect on tobacco mosaic virus, with an inhibition rate as high as 71.43%, which was superior to commercially available products.

[0070] Application Example 2

[0071] Low-temperature resistance experiment of wheat: The plant growth regulators prepared in Comparative Example 1 and Example 1 were sprayed on wheat seedlings in the seedling stage. The specific experiments are as follows:

[0072] 1) Plant growth regulators: Take the plant growth regulator A, plant growth regulator B, control reagent 1 and control reagent 2 prepared by step 1) in application example 1, diluted 100 times, and set aside for use;

[0073] After adjusting the volume of 100 μL of Tween 80 surfactant to 100 mL, water containing the surfactant was prepared for later use.

[0074] 2) Test crop: Wheat (variety: Jimai 22). After disinfection, soaking, and germination of wheat seeds, once the seeds just began to sprout, plump and uniformly sized seeds were selected and sown into petri dishes, 30 seeds per dish. The seeds were cultured in a light incubator using Hoagland nutrient solution. The culture conditions were: 25 / 20℃ (day / night), light intensity 800 μmol / m². 2 / s, light cycle 14 / 10h (day / night), relative humidity 65±5%;

[0075] Prepare for use when the wheat seedlings have grown to the 2-leaf-1-heart stage;

[0076] 3) Treatment group setup: The diluted plant growth regulator A, diluted plant growth regulator B, control reagent 1 and control reagent 2 from step 1) were sprayed on the leaves of wheat seedlings in step 2) respectively (the leaves were completely wetted, but the liquid did not run off), and were respectively recorded as COS treatment group, COS-Zn treatment group, ZnSO4 treatment group and COS+ZnSO4 mixture group.

[0077] Negative control group 1: Sprayed with water containing surfactant prepared in step 1) (until the leaves are completely wet but the liquid does not drip), treated for 24 hours and then cultured at room temperature (around 25°C).

[0078] Negative control group 2: Spray with the water containing surfactant prepared in step 1) (until the leaves are completely wetted but the liquid does not run off);

[0079] Comparison with commercially available products: Chitosan oligosaccharide zinc (purchased from Shandong Lvlong Biotechnology Co., Ltd.), spray according to the product instructions.

[0080] Each treatment had 3 replicates, with 30 wheat seedlings per replicate.

[0081] 4) After treating the treatment group, negative control group 2 and commercial product control group in step 3) for 24 hours, they were placed in a low temperature stress of 4℃ for 48 hours. Then, the wheat was placed in room temperature (around 25℃) to recover for 48 hours. Various indicators of wheat seedling leaves in the treatment group and negative control group were measured respectively. The results are shown in Tables 2 to 5.

[0082] Results and Analysis

[0083] Table 2 Effects of different treatment groups on wheat seedling growth parameters

[0084]

[0085] As shown in Table 2, wheat seedling growth was inhibited under low temperature stress, with significant reductions in seedling height, root length, fresh weight, and dry weight. Compared with the negative control group 2 (low temperature stress group), the wheat seedling height, root length, fresh weight, and dry weight increased by 24.6%, 15.3%, 35.9%, and 56.8%, respectively, after treatment with the COS-Zn group provided in Example 1 of this invention. The wheat seedling height, root length, fresh weight, and dry weight increased by 39.0%, 21.1%, 43.1%, and 81.1%, respectively. When zinc sulfate was applied alone, wheat seedling height, root length, fresh weight, and dry weight increased by 22.2%, 11.9%, 22.5%, and 32.4%, respectively. When chitosan oligosaccharide was mixed with zinc sulfate, wheat seedling height, root length, fresh weight, and dry weight increased by 28.4%, 16.4%, 40.2%, and 64.9%, respectively. When commercially available products were applied, wheat seedling height, root length, fresh weight, and dry weight increased by 28.48%, 15.9%, 37.9%, and 67.6%, respectively. Therefore, the plant regulator prepared in this invention can promote the accumulation of plant seedling biomass under low-temperature stress, and its effect is superior to applying chitosan oligosaccharide or zinc sulfate alone, as well as to a simple combination of chitosan oligosaccharide and zinc sulfate, and also superior to commercially available products.

[0086] Table 3 Effects of different treatment groups on physiological indicators of wheat seedlings

[0087]

[0088] Table 3 shows that under low-temperature stress, the malondialdehyde (MDA) content in wheat seedling leaves increased significantly. Compared with the negative control group 2, the MDA content decreased by 34.4%, 28.9%, 34.1%, and 62.0% in the treatment groups treated with COS alone, zinc sulfate, a mixture of chitosan oligosaccharide and zinc sulfate, and COS-Zn (Example 1), respectively. This indicates that all treatments can alleviate the peroxidation of the plant plasma membrane under low-temperature stress and protect its structure, with the COS-Zn treatment group showing significantly better results than the other groups. Furthermore, COS-Zn can also increase the content of proline and soluble sugars in plants under low-temperature stress, maintain osmotic pressure balance in plant cells, and ensure normal physiological functions.

[0089] Furthermore, low-temperature stress causes chlorophyll degradation in wheat seedling leaves; however, treatments such as chitosan oligosaccharide can alleviate chlorophyll degradation caused by low temperature. Compared with control group 2, the chlorophyll content increased by 60.8%, 41.9%, 59.5%, and 63.5% respectively in the treatment groups treated with COS alone, zinc sulfate, a mixture of chitosan oligosaccharide and zinc sulfate, and the COS-Zn treatment group in Example 1. This demonstrates that chitosan oligosaccharide and its chelates can alleviate chlorophyll degradation in plants under low-temperature stress and improve photosynthetic efficiency, especially the plant regulator provided in this invention, which uses chitosan oligosaccharide chelated zinc as its main component, exhibiting even more significant effects.

[0090] Table 4. Effects of different treatment groups on antioxidant enzyme activity in wheat leaves.

[0091]

[0092] Table 4 shows that the activity of antioxidant enzymes in wheat seedling leaves was enhanced under low-temperature stress; different COS treatment groups further enhanced the activity of antioxidant enzymes in the plant. Compared with control group 2, the activities of wheat SOD, POD, and CAT increased by 20.0%, 18.8%, and 56.6% in the COS treatment group, respectively; while the activities of wheat SOD, POD, and CAT increased by 34.0%, 22.7%, and 68.9% in the COS-Zn treatment group, respectively; when zinc sulfate was applied alone, the activities of wheat SOD, POD, and CAT increased by 15.8%, 9.4%, and 41.8%; when chitosan oligosaccharide was combined with zinc sulfate, the activities of wheat SOD, POD, and CAT increased by 27.4%, 17.1%, and 61.8%; when commercially available products were applied, the activities of wheat SOD, POD, and CAT increased by 20.0%, 18.2%, and 65.0%, respectively. Therefore, although different treatments with chitosan oligosaccharide and zinc can enhance the activity of antioxidant enzymes in plants under low temperature stress, remove excess reactive oxygen species induced by low temperature stress, protect the plant plasma membrane structure, and improve the plant's ability to resist low temperature stress, the effect of chitosan oligosaccharide chelating zinc is better and superior to commercial products.

[0093] Application Example 3

[0094] Tobacco Low Temperature Resistance Experiment: Taking tobacco as an example, this invention sprayed tobacco seedlings with the plant growth regulators prepared in Comparative Example 1 and Example 1. The specific experiments are as follows:

[0095] 1) Plant growth regulator; same as step 1) in application example 2;

[0096] 2) Test crop: Tobacco, specifically Zhongyan 100. Four-leaf stage tobacco seedlings were cultured in a light-controlled incubator under the following conditions: 25 / 20℃ (day / night) and light intensity 800 μmol / m². 2 / s, light cycle 14 / 10h (day / night), relative humidity 65±5%; ready for use when tobacco seedlings grow to the 6-leaf stage;

[0097] 3) Treatment group setup: The tobacco seedling leaves in step 2) were sprayed with diluted plant growth regulator A, diluted plant growth regulator B, control reagent 1 and control reagent 2 respectively (until the leaves were completely wet but the liquid did not run off), and were respectively recorded as COS treatment group, COS-Zn treatment group, ZnSO4 treatment group and COS+ZnSO4 mixed group.

[0098] Negative control group 1: Sprayed with water containing surfactant prepared in step 1) (until the leaves are completely wet but the liquid does not drip), treated for 24 hours and then cultured at room temperature (around 25°C).

[0099] Negative control group 2: Sprayed with water containing surfactant prepared in step 1) (until the leaves are completely wetted but the liquid does not drip), and cultured at low temperature (4°C).

[0100] Each treatment had 3 replicates, and each replicate had 8 tobacco seedlings.

[0101] 4) After 24 hours of treatment in step 3), the tobacco seedlings in the treatment group and the negative control group were subjected to low-temperature stress at 4℃ for 48 hours, and then restored to room temperature (around 25℃) for 48 hours. Various indicators of the leaves of the tobacco seedlings in both the treatment and control groups were measured. The results are shown in Table 5. Figure 2 .

[0102] Results and Analysis

[0103] Table 5 Effects of different treatment groups on growth parameters of tobacco seedlings

[0104]

[0105]

[0106] From Table 5 and Figure 2It can be seen that under low temperature stress, the growth of tobacco seedlings was inhibited, and leaf area, aboveground dry weight, root length, and root dry weight were all significantly reduced. Compared with the negative control group 2, the leaf area, aboveground fresh weight, root length, and root dry weight of tobacco seedlings increased by 17.9%, 36.8%, 13.0%, and 72.2%, respectively, after treatment with COS. The leaf area, aboveground fresh weight, root length, and root dry weight of tobacco seedlings increased by 34.0%, 68.4%, 27.0%, and 94.4%, respectively, after treatment with COS-Zn. The effects of applying zinc sulfate alone or simply combining chitosan oligosaccharide with zinc sulfate on the low temperature resistance of tobacco seedlings were generally limited because inorganic zinc was severely lost and could not be well absorbed and utilized by the plants. Therefore, both chitosan oligosaccharide and chitosan oligosaccharide chelated zinc can promote the accumulation of plant seedling biomass under low temperature stress, but the effect of chitosan oligosaccharide chelated zinc is better than that of chitosan oligosaccharide. This indicates that chitosan oligosaccharide chelated zinc not only exerts the stress-resistant and growth-promoting effects of chitosan oligosaccharide, but also converts zinc into an organic form, thus better exerting its growth-promoting effect.

[0107] In summary, the application of the plant regulator provided by this invention not only significantly improves the activity of antioxidant enzymes and photosynthetic efficiency in plants under low-temperature conditions, but also significantly inhibits viral diseases in plants. Therefore, the chitosan oligosaccharide chelated zinc plant regulator provided by this invention can enhance plant stress resistance and lay the foundation for the high-value utilization of low-value marine products.

[0108] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a plant growth regulator in alleviating low-temperature stress in plants; The plant species is tobacco; The low temperature is 4°C; The active ingredient of the plant growth regulator includes chitosan oligosaccharide chelated zinc; the concentration of chitosan oligosaccharide chelated zinc in the plant growth regulator is 100-1000 mg / L; The zinc content in the chitosan oligosaccharide chelated zinc is 3% to 4% by mass; The chitosan oligosaccharide chelated zinc was obtained by mixing a chitosan oligosaccharide solution and a zinc-containing compound solution, followed by chelation reaction and then dialyzing with a dialysis bag with a molecular weight cutoff of 3500 Da. The zinc-containing compound is zinc sulfate.

2. The application according to claim 1, characterized in that, The plant growth regulator also includes a surfactant; the concentration of the surfactant in the plant growth regulator is 1–10 mL / L.

3. The application according to claim 2, characterized in that, The surfactant includes one or more of Tween surfactant, Span surfactant, OP surfactant, and NP surfactant.

4. A method for alleviating low-temperature stress in plants, characterized in that, Includes the following steps: Soaking plant seeds in plant growth regulators and / or applying them to plants; The plant species is tobacco; The active ingredient of the plant growth regulator includes chitosan oligosaccharide chelated zinc; the concentration of chitosan oligosaccharide chelated zinc in the plant growth regulator is 100-1000 mg / L; The zinc content in the chitosan oligosaccharide chelated zinc is 3% to 4% by mass; The chitosan oligosaccharide chelated zinc was obtained by mixing a chitosan oligosaccharide solution and a zinc-containing compound solution, followed by chelation reaction and then dialyzing with a dialysis bag with a molecular weight cutoff of 3500 Da. The zinc-containing compound is zinc sulfate.

5. The method according to claim 4, characterized in that, The methods of application include: spraying and / or drenching.

6. The method according to claim 4, characterized in that, When soaking plant seeds, the concentration of the plant growth regulator is 0.1–0.5 g / L; the soaking time is 6–8 h.

7. The method according to claim 4, characterized in that, When applied to plants, the concentration of the plant regulator is 0.1–1 g / L, and the application rate is 20–80 L / mu.

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