Kandelia candel anti-cold agent, its preparation method and application method

Abstract

This invention relates to an anti-cold agent for Kandelia candel, its preparation method, and its application method. The anti-cold agent used in this invention is Agent A, comprising the following components: superphosphate 0.5-1.0 g / L. ‑1 Potassium chloride 0.2–1.0 g·L ‑1 Boric acid 0.1-0.2 g·L ‑1 Nano zinc oxide 0.15-0.3 g·L ‑1 L-cysteine ​​0.02-0.06 g·L ‑1 Agent B, comprising the following components: 0.3–0.6 g / L ‑1 Sodium alginate, glycerin 3-6 ml / L ‑1 By using specially formulated antifreeze agents, combined with specific preparation and application methods, the cold resistance of Kandelia candel can be enhanced, the damage caused by extreme low temperatures can be reduced, and the survival rate of Kandelia candel during the overwintering period can be improved.

Description

Technical Field

[0001] This invention relates to cold-resistant agents for improving the cold resistance of mangroves, their preparation methods, and application methods. Specifically, it relates to cold-resistant agents formulated for different substances in Kandelia candel, as well as corresponding preparation and spraying methods. Background Technology

[0002] Mangroves are woody plant communities growing in the intertidal zone of tropical and subtropical bays and estuaries. They not only possess extremely high ecological value but are also among the blue carbon ecosystems with a high carbon sequestration capacity. In recent years, the increased frequency and intensity of extreme low-temperature events caused by climate change have become a new challenge for mangroves in my country during the winter. Years of research and surveys have found that extreme low-temperature events can lead to yellowing, leaf drop, and even death of mangrove plants across multiple regions, species, and age groups during the wintering period. Therefore, it is urgent to improve the cold resistance of mangroves during the wintering period.

[0003] Kandelia obovata is one of the most widely distributed, northernmost, and largest-area mangrove plants in my country, and also one of the preferred mangrove plants for introduction and expansion to higher latitudes. Zhejiang Province, located in the subtropical monsoon climate zone, is the northernmost limit for mangrove forest introduction in my country, and has already planted large areas of Kandelia obovata forests. However, in recent years, extreme low temperatures (minimum temperatures of about -3℃) have caused large-scale death of young Kandelia obovata forests, resulting in serious ecological and economic losses. Therefore, improving the cold resistance of Kandelia obovata can effectively protect mangroves for safe overwintering, increase the carbon sequestration capacity of mangroves, and reduce carbon dioxide emissions. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to enhance the cold resistance of Kandelia candel by using a specially formulated antifreeze agent, reduce the damage of extreme low temperature to Kandelia candel, and improve the survival rate of Kandelia candel during the overwintering period.

[0005] To solve the above-mentioned technical problems, the cold-resistant agent for Kandelia candel used in this invention is:

[0006] Agent A includes the following components: superphosphate 0.5-1.0 g / L -1 Potassium chloride 0.2-1.0 g·L -1 Boric acid 0.1-0.2 g·L -1 Nano zinc oxide 0.15-0.3 g·L -1 L-cysteine ​​0.02-0.06 g·L -1 ;

[0007] Agent B includes the following components: 0.3-0.6 g·L -1 Sodium alginate, glycerin 3-6 ml / L -1 .

[0008] The preferred cold-resistant agent for Kandelia candel mentioned above is:

[0009] Agent A includes the following components: superphosphate 0.7 g / L -1 Potassium chloride 0.35 g·L -1 Boric acid 0.14 g·L -1 Nano zinc oxide 0.2 g·L -1 L-cysteine ​​0.05 g·L -1 ;

[0010] Agent B includes the following components: 0.5 g·L -1 Sodium alginate, glycerin 5ml / L -1 .

[0011] The beneficial effects of the Kandelia candel cold-resistant agent of the present invention are as follows: Plants require various nutrients during their growth. In this invention, Agent A contains essential plant nutrients such as P, S, Zn, B, K, and Ca. These substances directly affect plant growth and development, and appropriate supplementation of these nutrients can effectively promote plant growth under stress. Among these, nano-zinc oxide particles, with their small diameter, can serve as carriers for various nutrients to enter the plant, making them easier for plants to absorb and utilize, thus more effectively promoting plant growth and enhancing their resistance to adverse conditions. However, nano-zinc oxide also has a large specific surface area and specific surface energy, making it prone to aggregation and difficult to dissolve uniformly, which inhibits its nano-effect. L-cysteine ​​not only acts as an antioxidant, enhancing plant stress resistance, but also improves the aggregation of nano-zinc oxide, increasing the ability of various substances to dissolve uniformly. Superphosphate is not only a phosphate fertilizer but also contains sulfur and calcium, effectively promoting plant growth. Potassium chloride effectively supplements plant potassium, increasing the rigidity and strength of plant stems and branches, thus enhancing cold resistance. Boric acid participates in the formation of plant cells, effectively stabilizing chlorophyll synthesis and improving cold resistance. By preparing a mixed solution of superphosphate, potassium chloride, boric acid, nano-zinc oxide, and L-cysteine, the nutritional elements of Kandelia can be increased and the cold resistance of Kandelia can be improved, making it an effective cold-resistant agent.

[0012] In Agent B, sodium alginate acts as a thickener and gelling agent, while glycerol has strong hygroscopic properties, effectively inhibiting the formation of ice crystals from water molecules. This keeps the water in a supercooled state, lowering the temperature at which ice crystals form and reducing the severity of low-temperature damage. Agent B effectively protects against nutrient loss from Agent A, preventing nutrient loss and evaporation. Additionally, the cationic Ca in Agent A... 2+ Its presence will cause Na in the solution of agent B to... + With Ca 2+ Ion exchange reaction promotes the formation of a more stable hydrogel in the solution, which better prevents the loss of agent A.

[0013] This invention also provides a method for preparing a Kandelia candel anti-cold agent, characterized in that,

[0014] The preparation process of Agent A includes: adding nano zinc oxide and L-cysteine ​​to water in a certain proportion, stirring with a magnetic stirrer, and then transferring the solution to an ultrasonic instrument for ultrasonication. During the ultrasonication process, superphosphate, potassium chloride and boric acid are added in sequence.

[0015] The preparation process of Agent B includes: adding sodium alginate to water, stirring with a magnetic stirrer, and then transferring the solution to an ultrasonic instrument for ultrasonication, during which glycerin is added dropwise.

[0016] The above-mentioned preferred method for preparing the Kandelia candel anti-cold agent is as follows:

[0017] During the preparation of Agent A, the magnetic stirrer is used to stir for 20-30 minutes at room temperature.

[0018] The above-mentioned preferred method for preparing the Kandelia candel anti-cold agent is as follows:

[0019] During the preparation of Agent A, ultrasonic treatment is performed for 20-30 minutes.

[0020] The above-mentioned preferred method for preparing the Kandelia candel anti-cold agent is as follows:

[0021] During the preparation of Agent B, the magnetic stirrer is set to a temperature of 55-60℃ and a speed of 1200 RPM for 3-3.5 hours.

[0022] The above-mentioned preferred method for preparing the Kandelia candel anti-cold agent is as follows:

[0023] During the preparation of Agent B, ultrasonic treatment is performed for 50-60 minutes at a temperature of 20-25℃.

[0024] The beneficial effects of the method for preparing the Kandelia candel anti-cold agent of the present invention are as follows:

[0025] Agent A: Nano zinc oxide and L-cysteine ​​are added to water in a certain proportion to effectively improve the aggregation of nano zinc oxide, so that nano zinc oxide is evenly distributed in the solution, forming a light milky white solution. The ultrasonic process reduces the interaction force between solute molecules and solvent molecules in Agent A solution, increases the distance between molecules, and effectively accelerates the even distribution of various nutrients in the solution.

[0026] Agent B: Dissolving sodium alginate in water using a magnetic stirrer accelerates the dissolution process without altering its chemical properties. During the sonication process, glycerol is added dropwise to the aqueous phase until evenly distributed. This method prevents a chemical reaction with the sodium alginate while ensuring uniform distribution of glycerol within the sodium alginate solution.

[0027] The present invention provides a method for applying the Kandelia candel anti-cold agent:

[0028] Agent B should be sprayed 10-15 minutes after Agent A.

[0029] The beneficial effects of the application method of the Kandelia candel anti-cold agent provided by this invention are:

[0030] After spraying agent B, the reaction between agents A and B will be visible on the leaf surface within 15-20 minutes, forming a film. This film can effectively prevent the loss of nutrients from agent A and also provides physical protection against cold.

[0031] The present invention will be further described below with reference to specific embodiments and accompanying drawings. Attached Figure Description

[0032] Figure 1 The effects of different treatments on the net photosynthetic rate and stomatal conductance of leaves in Kandelia candel seedlings under low temperature stress.

[0033] Figure 2 The effects of different treatments on the actual photochemical efficiency of PSII and the maximum photochemical quantum yield of PSII in leaves of Kandelia candel seedlings under low temperature stress.

[0034] Figure 3 This study investigated the effects of different treatments on the cell membrane permeability of leaves in Kandelia candel seedlings under low-temperature stress.

[0035] Figure 4 The effect of different concentrations of nano zinc oxide on the net photosynthetic rate of leaves of Kandelia candel seedlings under low temperature stress.

[0036] Figure 5 The effect of different concentrations of nano zinc oxide on the net photosynthetic rate of leaves of Kandelia candel seedlings under low temperature stress.

[0037] Figure 6 The effect of different concentrations of nano zinc oxide on the actual photochemical efficiency of PSII in leaves of Kandelia candel seedlings under low temperature stress.

[0038] Figure 7 The effect of different concentrations of nano-zinc oxide on the maximum photochemical quantum yield of PSII in leaves of Kandelia candel seedlings under low temperature stress.

[0039] Figure 8 This study investigated the effects of different concentrations of nano-zinc oxide on the malondialdehyde content in the leaves of Kandelia candel seedlings under low-temperature stress. Specific Implementation

[0040] I. Experimental Materials and Preparation

[0041] Hypocotyls of *Kandelia candel* were collected from the coastlines of southern Zhejiang or northern Fujian in May and June. Disease-free, healthy, and mature hypocotyls were selected and planted in plastic buckets (20 cm in diameter and 25 cm in height) containing 3 kg of dry coastal mud (without stones). Three or four plants were placed in each bucket and cultivated in a natural environment. Nutrient solution was applied and the plants were cultivated until they had four leaves and one bud. Then, the various experimental treatments in Part II were carried out.

[0042] The specific preparation process of the above nutrient solution is as follows:

[0043] A: pH = 6.0, calcium nitrate 82.07 g / L or calcium nitrate tetrahydrate 118.08 g / L -1 ;

[0044] B: pH = 8.0, potassium nitrate 50.55 g / L -1 Potassium dihydrogen phosphate 13.61 g·L -1 Magnesium sulfate 24.07 g·L -1 Or magnesium sulfate heptahydrate 49.03 g·L -1 ;

[0045] C: pH = 5.5, Nat-EDTA 7.46 g·L -1 5.56 g·L⁻¹ ferrous sulfate heptahydrate -1 ;

[0046] D: Boric acid 2.86 g·L -1 Magnesium chloride tetrahydrate 1.81 g·L -1 Zinc sulfate heptahydrate 0.22 g·L -1 Copper sulfate pentahydrate 0.08 g·L -1 0.02 g·L molybdic acid -1 ;

[0047] Then take 10ml each of A and B, and 1ml each of C and D, mix them and bring the volume to 1L to make Hoagland's nutrient solution.

[0048] During the nutrient solution irrigation period when the hypocotyl is cultivated to the stage of four leaves and one bud, it is necessary to irrigate twice with Hoagland's nutrient solution containing 15‰ NaCl, and maintain a water level of 1-2 cm in the container.

[0049] In all subsequent experimental treatments of the four-leaf clover, it is necessary to supplement the water with Hoagland's nutrient solution that does not contain NaCl to maintain a water layer of 1-2 cm.

[0050] Additionally, when the highest temperature in the natural environment exceeds 33℃, the plastic containers used for cultivating seedlings need to be moved to areas with cooling facilities, such as glass greenhouses, to ensure that the seedling roots are not damaged by high temperatures. When the temperature drops below 33℃, the seedlings can be moved back to the natural environment.

[0051] The specific preparation process for the cold protection agent is as follows:

[0052] The preparation process of Agent A is as follows: Nano zinc oxide and L-cysteine ​​are added to water in a certain proportion and stirred at room temperature for 20-30 minutes using a magnetic stirrer. This effectively improves the aggregation of nano zinc oxide, ensuring that the nano zinc oxide is evenly distributed in the solution, forming a light milky white solution. After stirring, the solution is transferred to an ultrasonic instrument. During the ultrasonication process, weighed superphosphate, potassium chloride, and boric acid are added sequentially. After the solution is ultrasonicated for 20-30 minutes, this ultrasonic process reduces the interaction forces between solute and solvent molecules in the Agent A solution, increases the distance between molecules, and effectively accelerates the uniform distribution of various nutrients in the solution, forming Agent A solution.

[0053] The preparation process of Agent B is as follows: Sodium alginate is dissolved in water using a magnetic stirrer at a temperature of 55-60℃ and a stirring speed of 1200 RPM to accelerate the dissolution rate of sodium alginate without altering its chemical properties. After stirring for 3-3.5 hours, the sodium alginate is fully dissolved, and no clumps of sodium alginate powder are visible in the solution, which becomes slightly viscous. After stirring, the resulting slightly viscous solution is transferred to an ultrasonic instrument and sonicated. Glycerin is then added dropwise to the aqueous phase until it is evenly distributed. The temperature is controlled at 20-25℃ during the dropwise addition. After the oil dropwise addition is complete, the solution is sonicated for another 50-60 minutes. This method prevents a chemical reaction of sodium alginate and ensures that glycerin is evenly distributed in the sodium alginate solution, forming Agent B, a protective solution that prevents nutrient loss and evaporation after Agent A is applied. Additionally, the cation Ca in Agent A... 2+ Its presence will cause Na in the solution of agent B to... + With Ca 2+ Ion exchange reaction promotes the formation of a more stable hydrogel in the solution, which better prevents the loss of agent A.

[0054] II. Test Treatment Plan

[0055] Option 1 (Normal seedling cultivation, designated as control, abbreviated as CK)

[0056] When the autumn eggplant has grown to four leaves and one bud in June or July, the seedlings are kept in a natural environment for conventional cultivation until they grow to ten leaves and one bud.

[0057] Scheme 2 (Studying the cold resistance of Kandelia candel seedlings by applying the lowest concentration of the cold-resistant agent, designated as the low-concentration cold-resistant agent treatment, abbreviated as LKHJ)

[0058] (1) When the Kandelia candel has grown to four leaves and one bud in June or July, transplant the seedlings into a glass greenhouse and spray the leaves with a frost protectant. Because the seedlings are small, the amount of frost protectant sprayed should be small. (Agent A: 0.5 g / L superphosphate) -1Potassium chloride 0.2 g·L -1 Boric acid 0.1 g·L -1 Nano zinc oxide 0.15 g·L -1 L-cysteine ​​0.02 g·L -1 Agent B: Sodium alginate 0.3 g / L -1 3 ml / L of glycerin -1. )

[0059] The specific spraying method is as follows: Spray Agent A on the leaves of the Kandelia candel seedlings at 7 pm every day, ensuring the leaves are moist. Spray for two consecutive days. On the second day after spraying Agent A, spray Agent B 10-15 minutes after spraying Agent A (unless otherwise specified, Agent B should be sprayed 10-15 minutes after Agent A), again ensuring the leaves are moist. After spraying, continue to cultivate the Kandelia candel seedlings in the glass greenhouse for 4-5 days, then transplant them to the natural environment.

[0060] (2) When the seedlings of Kandelia candel reach the stage of six leaves and one bud in July and August, move them to a glass greenhouse. Spray the leaves with agent A every evening at 7 pm for 3 consecutive days. Spray agent B after the 3 days of agent A spraying. After spraying, keep the seedlings in the glass greenhouse for another 4-5 days, and then move them to the natural environment for further cultivation.

[0061] (3) When the seedlings of Kandelia candel reach the stage of eight leaves and one bud in August or September, they should be moved into a glass greenhouse. Spray the leaves with Agent A every morning at 7:00 AM and evening at 7:00 PM, and spray every other day for a total of 3 days, completing the Agent A spraying process in a total of 5 days. Agent B should be sprayed 3 days after Agent A. After spraying, continue to keep the seedlings in the glass greenhouse for 4-5 days, and then move them to the natural environment for continued cultivation.

[0062] (4) When the seedlings of Kandelia candel have ten leaves and one heart from September to November, move them into a glass greenhouse and spray the leaves with Agent A at 7 am and 7 pm every day for 4 consecutive days. Agent B should be sprayed 4 days after Agent A.

[0063] Scheme 3 (Studying the cold resistance of Kandelia candel seedlings by applying the highest concentration of the cold-resistant agent, designated as the high-concentration cold-resistant agent treatment, abbreviated as HKHJ)

[0064] (1) When the Kandelia candel has grown to four leaves and one bud in June or July, transplant the seedlings into a glass greenhouse and spray the leaves with a frost protectant. Because the seedlings are small, the amount of frost protectant sprayed on the leaves should be small. (Agent A: 1.0 g·L⁻¹ superphosphate) -1 1.0 g·L⁻¹ potassium chloride -1 Boric acid 0.2 g·L -1 Nano zinc oxide 0.3 g·L -1 L-cysteine ​​0.06 g·L -1Agent B: 0.6 g / L -1 Sodium alginate, glycerin 6ml / L -1 )

[0065] Specific spraying method: Spray agent A on the leaves of the Kandelia candel seedlings at 7 pm every day, ensuring the leaves are moist. Spray for two consecutive days. Apply agent B the day after spraying agent A, also ensuring the leaves are moist. After spraying, continue to cultivate the Kandelia candel seedlings in the glass greenhouse for 4-5 days, and then move the seedlings to the natural environment.

[0066] (2) When the seedlings of Kandelia candel reach the stage of six leaves and one bud in July and August, transplant them into a glass greenhouse. Spray the leaves with agent A every evening at 7 pm for 3 consecutive days. Spray agent B after the 3 days of agent A spraying. After spraying, continue to keep them in the glass greenhouse for 4-5 days, and then move them to the natural environment for cultivation.

[0067] (3) When the seedlings of Kandelia candel reach the stage of eight leaves and one bud in August or September, they should be moved into a glass greenhouse. Spray the leaves with Agent A every morning at 7:00 AM and evening at 7:00 PM, and spray every other day for a total of 3 days, completing the Agent A spraying process in a total of 5 days. Agent B should be sprayed 3 days after Agent A. After spraying, continue to keep the seedlings in the glass greenhouse for 4-5 days, and then move them to the natural environment for continued cultivation.

[0068] (4) When the seedlings of Kandelia candel have ten leaves and one heart from September to November, move them into a glass greenhouse and spray Agent A at 7 am and 7 pm every day for 4 consecutive days. Agent B should be sprayed 4 days after Agent A.

[0069] Scheme 4 (Study the cold resistance of Kandelia candel seedlings by applying a cold-resistant agent of appropriate concentration, referred to as the appropriate concentration cold-resistant agent treatment, abbreviated as SKHJ)

[0070] (1) When the Kandelia candel has grown to four leaves and one bud in June or July, transplant the seedlings into a glass greenhouse and spray the leaves with a frost protectant. Because the seedlings are small, the amount of frost protectant sprayed is small. (Agent A: 0.7 g / L superphosphate) -1 Potassium chloride 0.35 g·L -1 Boric acid 0.14 g·L -1 Nano zinc oxide 0.2 g·L -1 L-cysteine ​​0.05 g·L -1 Agent B: 0.5 g / L -1 Sodium alginate, glycerin 5ml / L -1 )

[0071] Specific spraying method: Spray agent A on the leaves of the Kandelia candel seedlings at 7 pm every day, ensuring the leaves are moist. Spray for two consecutive days. Apply agent B the day after spraying agent A, also ensuring the leaves are moist. After spraying, continue to cultivate the Kandelia candel seedlings in the glass greenhouse for 4-5 days, and then move the seedlings to the natural environment.

[0072] (2) When the seedlings of Kandelia candel reach the stage of six leaves and one bud in July and August, transplant them into a glass greenhouse. Spray the leaves with agent A every evening at 7 pm for 3 consecutive days. Spray agent B after the 3 days of agent A spraying. After spraying, continue to keep them in the glass greenhouse for 4-5 days, and then move them to the natural environment for cultivation.

[0073] (3) When the seedlings of Kandelia candel reach the stage of eight leaves and one bud in August or September, they should be moved into a glass greenhouse. Spray the leaves with Agent A every morning at 7:00 AM and evening at 7:00 PM, and spray every other day for a total of 3 days, completing the Agent A spraying process in a total of 5 days. Agent B should be sprayed 3 days after Agent A. After spraying, continue to keep the seedlings in the glass greenhouse for 4-5 days, and then move them to the natural environment for continued cultivation.

[0074] (4) When the seedlings of Kandelia candel have ten leaves and one heart from September to November, move them into a glass greenhouse and spray the leaves with Agent A at 7 am and 7 pm every day for 4 consecutive days. Agent B should be sprayed 4 days after Agent A.

[0075] Scheme 5 (Effect of different concentrations of nano-zinc oxide on the cold resistance of Kandelia candel seedlings under low temperature stress)

[0076] Transplant the seedlings of Kandelia candel with ten leaves and one bud into a glass greenhouse. Spray the leaves with different concentrations of nano zinc oxide at 7 am and 7 pm every day for three consecutive days. The specific spraying method includes the following: (1) Spray 0 g·L -1 Nano zinc oxide, abbreviated as NZ0; (2) Spray 0.05 g·L -1 Nano zinc oxide, abbreviated as NZ0.05; (3) Spray 0.1 g·L -1 Nano zinc oxide, abbreviated as NZ0.1; (4) Spray 0.15 g·L -1 Nano zinc oxide, abbreviated as NZ0.15; (5) Spray 0.2 g·L -1 Nano zinc oxide, abbreviated as NZ0.2; (6) Spray 0.3 g·L -1 Nano zinc oxide, abbreviated as NZ0.3; (7) Spray 0.4 g·L -1 Nano zinc oxide, abbreviated as NZ0.4.

[0077] III. Temperature Treatment

[0078] (1) The effect of normal temperature on the growth of Kandelia candel seedlings

[0079] The seedlings treated with scheme 1 were transferred to a 25℃ (daytime) / 22℃ (nighttime) light incubator, with 12 hours of light per day, relative humidity controlled at 50%-60%, and light intensity not lower than 400 μmol·m⁻¹. -2 ·s -1 Keep it for 4 days.

[0080] (2) Effects of low temperature stress on the growth of Kandelia candel seedlings treated with different antifreeze agents

[0081] Seedlings treated according to schemes 1, 2, 3, and 4 were all placed directly in a cold light incubator at a temperature of 8℃ (daytime) / –3℃ (nighttime) for 4 days of low-temperature stress. Seedlings treated with scheme 1 underwent low-temperature stress (LTS). The relative humidity of the incubator was controlled at 50%-60%, and the light intensity was not lower than 400 μmol·m⁻¹. -2 ·s -1 It receives 12 hours of sunlight every day.

[0082] (3) Effects of low temperature stress on the growth of Kandelia candel seedlings under different concentrations of nano zinc oxide treatment

[0083] The seedlings obtained from scheme 5 were transferred to a cold light source incubator with a temperature of 8℃ (daytime) / –3℃ (nighttime) for 4 days of low-temperature stress, with relative humidity controlled at 50%-60% and light intensity not lower than 400 μmol·m⁻¹. -2 ·s -1 It receives 12 hours of sunlight every day.

[0084] IV. Measurement Items and Methods

[0085] (1) Observation of leaf color

[0086] Observe the changes in leaf color in each treatment after 4 days of low temperature.

[0087] (2) Measurement of photosynthetic parameters

[0088] After restoring the Kandelia candel to natural light for 30 minutes, photosynthetic parameters were measured. The net photosynthetic rate and stomatal conductance of the third pair of leaves counting downwards from the apex were measured using a Li-6400 portable photosynthesis system from 10:00 to 11:30.

[0089] (3) Chlorophyll fluorescence parameter measurement

[0090] After restoring the Kandelia candel to natural light for 30 minutes, the actual photochemical efficiency of PSII and the energy capture efficiency of the PSII reaction center were determined using an FMS-2 portable modulated chlorophyll fluorometer.

[0091] (4) Measurement of cell membrane permeability

[0092] Place 0.3 g of fresh leaf fragments in 10 ml of deionized water, evacuate the water for 15 minutes using a vacuum pump, and let it stand for 1 hour. Measure the extravasated electrolyte (S1) using a DDS-307 conductivity meter. Then, boil the mixture in boiling water for 10 minutes and measure the conductivity (S2). Calculate the cell membrane permeability using the following formula: Cell membrane permeability = S1 / S2 × 100%.

[0093] (5) Method for determining malondialdehyde content

[0094] Weigh 1 gram of chopped fresh leaves, add 2 ml of 10% TCA and a small amount of quartz sand, grind until homogeneous, then add 8 ml of TCA and grind again at 4000 rpm. -1 Centrifuge for 10 minutes; the supernatant is the sample extract. Take 2 mL of the centrifuged supernatant (add 2 mL of distilled water for the control), add 2 mL of 0.6% TBA solution, mix well, and react in a boiling water bath for 15 minutes. Cool rapidly and centrifuge again. Measure the extinction at wavelengths of 532, 600, and 450 nm using the following formula:

[0095] Malondialdehyde content (μmol·g) -1 ) = [MDA concentration (μmol·L)] -1 [(×extraction volume (mL))·[sample weight (g)×1000]] -1 .

[0096] (6) Data processing

[0097] One-way ANOVA was performed using SPSS 21.0 statistical software for data comparison analysis. All data in the figures are mean plus standard deviation, and graphs were created using SigmaPlot 10.0 plotting software.

[0098] V. Results and Analysis

[0099] 5.1 Effects of different antifreeze components on the growth of Kandelia candel seedlings

[0100] (1) Leaf color change

[0101] Under normal conditions, the leaves of Kandelia candel seedlings treated with the control (CK) are green. After 4 days of low-temperature stress, approximately 65% ​​of the leaves of Kandelia candel seedlings treated with low-temperature stress (LTS) turned blackish-brown, approximately 35% of the leaves of seedlings treated with low-concentration cold-resistant agent (LKHJ) turned blackish-brown, approximately 32% of the leaves of seedlings treated with high-concentration cold-resistant agent (HKHJ) turned blackish-brown, and only 10% of the leaves of seedlings treated with an appropriate concentration of cold-resistant agent (SKHJ) turned blackish-brown. This indicates that the cold resistance is in the following order: SKHJ > HKHJ > LKHJ > LTS.

[0102] (2) Effects of different treatments on net photosynthetic rate and stomatal conductance of leaves of Kandelia candel seedlings

[0103] Reference Appendix Figure 1 It was found that after 4 days of low-temperature stress, the net photosynthetic rate and stomatal conductance of Kandelia candel seedlings treated with a low concentration of antifreeze (LKHJ) were significantly increased compared to untreated plants (LTS) (P<0.05). This is similar to the results of the high concentration antifreeze (HKHJ) treatment. Compared with LKHJ and HKHJ treatments, the net photosynthetic rate and stomatal conductance of Kandelia candel seedlings treated with a more suitable concentration of antifreeze (SKHJ) were approximately 1.64 and 1.59 times, and 1.79 and 1.67 times, respectively, compared to the LKHJ and HKHJ treatments, all with significant differences (P<0.05). This indicates that antifreeze can significantly improve the cold resistance of Kandelia candel seedlings, especially with suitable concentrations.

[0104] (3) Effects of different treatments on the actual photochemical efficiency of PSII and the maximum photochemical quantum yield of PSII in Kandelia candel seedlings

[0105] Reference Appendix Figure 2 It was found that after 4 days of low-temperature stress, the actual photochemical efficiency of PSII in the leaves of Kandelia candel seedlings treated with low-concentration antifreeze (LKHJ) was approximately 1.68 times and 1.74 times that of untreated plants (LTS), respectively. The maximum photochemical quantum yield of leaves in Kandelia candel seedlings treated with high-concentration antifreeze (HKHJ) was approximately 2.12 times and 2.07 times that of untreated plants (LTS), respectively, both showing significant differences (P<0.05). The actual photochemical efficiency of PSII and the maximum photochemical quantum yield of PSII in the leaves of Kandelia candel seedlings treated with a suitable concentration of antifreeze (SKHJ) were approximately 1.34 and 1.30 times, and 1.22 and 1.24 times, respectively, of the LKHJ and HKHJ-treated plants, respectively, both showing significant differences (P<0.05).

[0106] (4) Effects of different treatments on cell membrane permeability of leaves of Kandelia candel seedlings

[0107] Reference Appendix Figure 3 It was found that after 4 days of low-temperature stress, the permeability of PSII cell membranes in the leaves of Kandelia candel seedlings treated with a low concentration of antifreeze (LKHJ) was reduced by approximately 22.1% compared to untreated plants (LTS), while the permeability of the leaves of Kandelia candel seedlings treated with a high concentration of antifreeze (HKHJ) was reduced by approximately 18.9% compared to untreated plants (LTS), both showing significant differences (P<0.05). The permeability of the leaves of Kandelia candel seedlings treated with a more suitable concentration of antifreeze (SKHJ) was reduced by approximately 13.5% and 15.5% compared to LKHJ and HKHJ-treated plants, respectively, both showing significant differences (P<0.05).

[0108] 5.2 Effects of different antifreeze components on the growth of Kandelia candel seedlings

[0109] (1) Effects of different nano-zinc oxides on the net photosynthetic rate of leaves of Kandelia candel seedlings

[0110] Reference Appendix Figure 4 It can be seen that under 4 days of low temperature stress, the net photosynthetic rate of Kandelia candel seedling leaves first increased and then decreased with increasing nano zinc oxide concentration, especially at nano zinc oxide concentrations of 0.15, 0.2, and 0.3 g·L⁻¹. -1 The net photosynthetic rates of Kandelia candel seedlings treated with NZ0.15, NZ0.2, and NZ0.3 were all positive and significantly higher than those of plants without nano-zinc oxide treatment (P<0.05). Furthermore, the net photosynthetic rate of seedlings treated with NZ0.2 reached its maximum, increasing by approximately 30.9% and 21.5% compared to the NZ0.15 and NZ0.3 treatments, respectively, all of which were statistically significant (P<0.05).

[0111] (2) Effects of different nano-zinc oxides on stomatal conductance of leaves of Kandelia candel seedlings

[0112] Reference Appendix Figure 5 It can be seen that under 4 days of low-temperature stress, the stomatal conductance of Kandelia candel seedlings first increased and then decreased with increasing nano zinc oxide concentration, especially at nano zinc oxide concentrations of 0.15, 0.2, and 0.3 g·L⁻¹. -1 The stomatal conductance of Kandelia candel seedlings treated with (NZ0.15, NZ0.2, and NZ0.3) increased by approximately 1.6, 2.3, and 1.7 times, respectively, compared with the treatment without nano zinc oxide, all of which showed significant differences (P<0.05), with the NZ0.2 treatment showing the greatest increase.

[0113] (3) Effects of different nano-zinc oxides on the actual photochemical efficiency of PSII in leaves of Kandelia candel seedlings

[0114] Reference Appendix Figure 6 It can be seen that under 4 days of low temperature stress, the actual photochemical efficiency of PSII in Kandelia candel seedling leaves first increased and then decreased with increasing nano zinc oxide concentration. Specifically, the efficiency decreased at nano zinc oxide concentrations of 0.15, 0.2, and 0.3 g·L⁻¹. -1 The stomatal conductance of Kandelia candel seedlings treated with (NZ0.15, NZ0.2, and NZ0.3) increased by approximately 1.4, 2.0, and 1.7 times, respectively, compared with the treatment without nano zinc oxide, all of which showed significant differences (P<0.05), with the NZ0.2 treatment showing the greatest increase.

[0115] (4) Effects of different nano-zinc oxides on the maximum photochemical quantum yield of PSII in leaves of Kandelia candel seedlings

[0116] Reference Appendix Figure 7It can be seen that under 4 days of low-temperature stress, the stomatal conductance of Kandelia candel seedlings first increased and then decreased with increasing nano zinc oxide concentration, especially at nano zinc oxide concentrations of 0.15, 0.2, and 0.3 g·L⁻¹. -1 The stomatal conductance of Kandelia candel seedlings treated with (NZ0.15, NZ0.2, and NZ0.3) increased by approximately 1.5, 1.7, and 1.4 times, respectively, compared with the treatment without nano zinc oxide, all of which showed significant differences (P<0.05), with the NZ0.2 treatment showing the greatest increase.

[0117] (5) Effects of different nano-zinc oxides on malondialdehyde content in leaves of Kandelia candel seedlings

[0118] Reference Appendix Figure 8 It can be seen that under 4 days of low-temperature stress, the malondialdehyde content in the leaves of Kandelia candel seedlings showed a trend of first decreasing and then increasing with the increase of nano zinc oxide concentration, especially at nano zinc oxide concentrations of 0.15, 0.2, and 0.3 g·L⁻¹. -1 The malondialdehyde (MDA) content in the leaves of Kandelia candel seedlings treated with NZ0.15, NZ0.2, and NZ0.3 was reduced by approximately 24.8%, 27.4%, and 13.6% respectively compared with the treatment without nano zinc oxide, all of which showed significant differences (P<0.05).

Claims

1. Eggplant (Agaricus regia) Kandelia obovata The cold-resistant agent is characterized by, Agent A includes the following components: superphosphate 0.5-1.0 g / L -1 Potassium chloride 0.2-1.0 g·L -1 Boric acid 0.1-0.2 g·L -1 Nano zinc oxide 0.15-0.3 g·L -1 L-cysteine ​​0.02-0.06 g·L -1 ; Agent B includes the following components: 0.3-0.6 g·L -1 Sodium alginate, glycerin 3-6 ml / L -1 .

2. The Kandelia candel anti-cold agent according to claim 1, characterized in that, Agent A includes the following components: superphosphate 0.7 g / L -1 Potassium chloride 0.35 g·L -1 Boric acid 0.14 g·L -1 Nano zinc oxide 0.2 g·L -1 L-cysteine ​​0.05 g·L -1 ; Agent B includes the following components: 0.5 g·L -1 Sodium alginate, glycerin 5ml / L -1 .

3. The method for preparing the Kandelia candel anti-cold agent according to claim 1, characterized in that, The preparation process of Agent A includes: adding nano zinc oxide and L-cysteine ​​to water in a certain proportion, stirring with a magnetic stirrer, and then transferring the solution to an ultrasonic instrument for ultrasonication. During the ultrasonication process, superphosphate, potassium chloride and boric acid are added in sequence. The preparation process of Agent B includes: adding sodium alginate to water, stirring with a magnetic stirrer, and then transferring the solution to an ultrasonic instrument for ultrasonication, during which glycerin is added dropwise.

4. The method for preparing the Kandelia candel anti-cold agent according to claim 3, characterized in that, During the preparation of Agent A, the magnetic stirrer is used to stir for 20-30 minutes at room temperature.

5. The method for preparing the Kandelia candel anti-cold agent according to claim 3 or 4, characterized in that, During the preparation of Agent A, ultrasonic treatment is performed for 20-30 minutes.

6. The method for preparing the Kandelia candel anti-cold agent according to claim 3 or 4, characterized in that, During the preparation of Agent B, the magnetic stirrer is set to a temperature of 55-60℃ and a speed of 1200 RPM for 3-3.5 hours.

7. The method for preparing the Kandelia candel anti-cold agent according to claim 5, characterized in that, During the preparation of Agent B, the magnetic stirrer is set to a temperature of 55-60℃ and a speed of 1200 RPM for 3-3.5 hours.

8. The method for preparing the Kandelia candel anti-cold agent according to claim 3 or 4, characterized in that, During the preparation of Agent B, ultrasonic treatment is performed for 50-60 minutes at a temperature of 20-25℃.

9. The method for preparing the Kandelia candel anti-cold agent according to claim 5, characterized in that, During the preparation of Agent B, ultrasonic treatment is performed for 50-60 minutes at a temperature of 20-25℃.

10. The method of applying the Kandelia candel anti-cold agent according to claim 1, characterized in that, Agent B should be sprayed 10-15 minutes after Agent A.

Images