Method for improving cold resistance of kandelia candel in subtropical region

Abstract

The present application relates to the method for improving the cold resistance of mangrove in subtropical region, and particularly to the method for improving the cold resistance of Kandelia candel in subtropical region by preparing the cold-proof agent through specific steps, combining specific spraying mode and planting steps, the method for enhancing the cold resistance of Kandelia candel by spraying the specially prepared cold-proof agent in different growth time according to specific spraying mode, reducing the damage of extreme low temperature to Kandelia candel and improving the survival rate of Kandelia candel in overwintering period.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for improving the cold resistance of mangrove, in particular a method for improving the cold resistance of Kandelia obovata by preparing a cold-proof agent through specific steps, combining with specific spraying mode and planting steps. BACKGROUND

[0002] Mangrove is a woody plant community growing in the intertidal zone of tropical and subtropical estuary bays. It not only has high ecological value, but also is one of the blue carbon ecosystems with high carbon sequestration capacity. In recent years, the increasing frequency and intensity of extreme low temperature events caused by climate change have become a new challenge for the overwintering of mangrove in China. Years of research and investigation have found that extreme low temperature events can cause the withering, defoliation and even death of mangrove plants in multiple regions, multiple mangrove species and multiple tree ages during the overwintering period. Therefore, it is urgent to improve the cold resistance of mangrove during the overwintering period.

[0003] Kandelia obovata is one of the mangrove plants with the widest distribution, the most northern latitude, the largest area and the strongest cold resistance in China, and is also the first choice for introduction and expansion to higher latitudes. Zhejiang Province is located in the subtropical monsoon climate zone, which is the northernmost boundary of mangrove introduction and afforestation in China. A large area of Kandelia obovata forest has been planted. In recent years, extreme low temperature (minimum temperature about -3℃) has caused large-scale death of young Kandelia obovata forest, resulting in serious ecological and economic losses. Therefore, improving the cold resistance of Kandelia obovata can effectively protect the safe overwintering of mangrove, increase the carbon sequestration capacity of mangrove and reduce the emission of carbon dioxide. SUMMARY

[0004] The technical problem to be solved by the present application is to enhance the cold resistance of Kandelia obovata, reduce the damage of extreme low temperature to Kandelia obovata and improve the survival rate of Kandelia obovata during the overwintering period by spraying a specially prepared cold-proof agent in a specific spraying mode at different growth times.

[0005] The method for improving the cold resistance of Kandelia obovata in subtropical regions solves the above technical problems and comprises the following steps:

[0006] (1) Selecting dry tidal mud and loading it into a plastic bucket;

[0007] (2) Preparing a cold-proof agent, wherein

[0008] A agent comprising the following components: 0.5-1.0 g·L -1 , 0.2-1.0 g·L -1 , 0.1-0.2 g·L -1 , 0.15-0.3 g·L -1 , 0.02-0.06 g·L -1 of potassium chloride, 0.1-0.2 g·L of boric acid, 0.15-0.3 g·L of nano zinc oxide and 0.02-0.06 g·L of L-cysteine;

[0009] B agent comprising the following components: 0.3-0.6g·L -1 Sodium alginate, glycerol 3-6ml·L -1

[0010] (3) In May and June, the hypocotyls of Kandelia candel are planted in plastic buckets, and the plastic buckets are placed in the natural environment to cultivate Kandelia candel to four leaves and one heart. During the cultivation, the Hoagland's nutrient solution with a NaCl concentration of 15‰ is poured twice to maintain a water layer of 1-2 cm in the bucket;

[0011] (4) In June and July, the plastic buckets containing the seedlings are moved into a glass greenhouse, and a cold protection agent is sprayed on the leaves: A agent is sprayed on the leaves of Kandelia candel seedlings every night until the leaves are wet, and the spraying is continuously performed for 2 days. On the second day after the spraying of A agent, B agent is sprayed on the leaves until the leaves are wet, and the cultivation in the glass greenhouse is continued for 4-5 days. Then the plastic buckets are moved to the natural environment for cultivation, and the Hoagland's nutrient solution without NaCl is supplemented in time to maintain a water layer of 1-2 cm;

[0012] (5) In July and August, when the Kandelia candel seedlings are cultivated to six leaves and one heart, the plastic buckets are moved into a glass greenhouse, and A agent is sprayed on the leaves every night until the leaves are wet, and the spraying is continuously performed for 3 days. On the third day after the spraying of A agent, B agent is sprayed on the leaves until the leaves are wet, and the cultivation in the glass greenhouse is continued for 4-5 days. Then the plastic buckets are moved to the natural environment for cultivation, and the Hoagland's nutrient solution without NaCl is supplemented in time to maintain a water layer of 1-2 cm;

[0013] (6) In August and September, when the Kandelia candel seedlings are cultivated to eight leaves and one heart, the plastic buckets are moved into a glass greenhouse, and A agent is sprayed on the leaves every morning and evening until the leaves are wet, and A agent is sprayed every other day for a total of three days. On the third day after the spraying of A agent, B agent is sprayed on the leaves until the leaves are wet, and the cultivation in the glass greenhouse is continued for 4-5 days. Then the plastic buckets are moved to the natural environment for cultivation, and the Hoagland's nutrient solution without NaCl is supplemented in time to maintain a water layer of 1-2 cm;

[0014] (7) In September and November, when the Kandelia candel seedlings are cultivated to ten leaves and one heart, the plastic buckets are moved into a glass greenhouse, and A agent is sprayed on the leaves every morning and evening until the leaves are wet, and the spraying is continuously performed for 4 days. On the fourth day after the spraying of A agent, B agent is sprayed on the leaves until the leaves are wet, and after the spraying is completed, the cultivation in the glass greenhouse is continued for 4-5 days. Then the plastic buckets are moved to the natural environment for cultivation, and the Hoagland's nutrient solution without NaCl is supplemented in time to maintain a water layer of 1-2 cm.

[0015] The preferred method for improving the cold resistance of Kandelia candel in the subtropical region comprises the following components of A agent: superphosphate 0.7g·L -1 Potassium chloride 0.35g·L -1 Boric acid 0.14g·L -1 ​, nano zinc oxide 0.2 g / L -1 , L-cysteine 0.05 g / L -1 ; B agent comprising the following components: 0.5 g / L -1 sodium alginate, glycerol 5 ml / L -1 .

[0016] The preferred method for improving the cold resistance of Kandelia candel in subtropical regions comprises the preparation process of A agent: nano zinc oxide and L-cysteine are added into water in proportion, stirred by a magnetic stirrer, and then the solution is moved to an ultrasonic instrument for ultrasonic treatment, during which calcium superphosphate, potassium chloride and boric acid are sequentially added.

[0017] The preferred method for improving the cold resistance of Kandelia candel in subtropical regions comprises the preparation process of A agent, during which the magnetic stirrer is stirred at room temperature for 20-30 minutes.

[0018] The preferred method for improving the cold resistance of Kandelia candel in subtropical regions comprises the preparation process of A agent, during which the ultrasonic instrument is ultrasonically treated for 20-30 minutes.

[0019] The preferred method for improving the cold resistance of Kandelia candel in subtropical regions comprises the preparation process of B agent: sodium alginate is added into water, stirred by a magnetic stirrer, and then the solution is moved to an ultrasonic instrument for ultrasonic treatment, during which glycerol is added dropwise.

[0020] The preferred method for improving the cold resistance of Kandelia candel in subtropical regions comprises the preparation process of B agent, during which the magnetic stirrer is set to a temperature of 55-60°C and a rotation speed of 1200 RPM, and stirred for 3-3.5 hours.

[0021] The preferred method for improving the cold resistance of Kandelia candel in subtropical regions comprises the preparation process of B agent, during which the ultrasonic instrument is ultrasonically treated for 50-60 minutes, and set to a temperature of 20-25°C.

[0022] The preferred method for improving the cold resistance of Kandelia candel in subtropical regions comprises that B agent is sprayed 10-15 minutes after the spraying of A agent.

[0023] The method for improving the cold resistance of Kandelia candel in subtropical regions has the following beneficial effects:

[0024] Step (4) is sprayed 2 times in June and July. This is mainly because the seedlings are small and the amount of leaf spraying A agent is less. Spraying A agent for more than 2 days will cause some elements to accumulate too much, causing the plant to appear as a seedling, on the other hand, too much nano zinc oxide will inhibit the growth of seedlings. After spraying, the Kandelia candel seedlings continue to be cultivated in the glass greenhouse for 4-5 days to prevent the seedlings from being washed by rain after spraying to ensure the spraying effect. During the 4-5 days of cultivation, the nutrients in A agent enter the leaves through the nano zinc oxide carrier and are absorbed and utilized by the plant, and the best absorption time is 4-5 days. In addition, the film formed by B agent protects the nutrients in A agent from loss for the best time of 4-5 days.

[0025] Step (5) is sprayed for 2 days in July and August. With the increase in the number of Kandelia candel leaves, the plant needs more nutrients, and continuous spraying for 2 days cannot meet the nutrient needs of Kandelia candel seedlings. Because potassium, boron, calcium, zinc, and sulfur are not easily flowing nutrient elements, the nutrient needs of seedlings with six leaves and one heart increase, and nano zinc oxide is not only a carrier for nutrients to enter the plant, but also an important source of zinc fertilizer for the leaves. Therefore, A agent needs to be sprayed for 3 days, which can effectively supplement the plant growth of P, S, Zn, B, K, Ca, and other nutrient elements.

[0026] Step (6) is sprayed for 3 days in August and September when the Kandelia candel seedlings have eight leaves and one heart. The plant enters the vigorous growth period and needs more nutrients. However, the temperature of the growth environment is high, the light is strong, and the air humidity is low, which is not conducive to the plant leaf nutrients. Therefore, the plant needs to be fertilized several times. However, because of the early and late spraying, the concentration of A agent nutrient elements will accumulate a lot, but the water evaporation is also fast, so it may burn the leaves, so it needs to be sprayed every other day, and it is appropriate to spray for 3 days, a total of 5 days to complete the spraying process of A agent.

[0027] Step (7) is sprayed for 4 days in September and November when the plant grows to ten leaves and one heart. The temperature, light, and humidity of the environment are all decreasing, which is conducive to the absorption of A agent nutrients by the leaves. Because the plant enters an environment with lower temperature, it needs to increase the plant nutrients, so it needs to be sprayed continuously for 4 days.

[0028] During the growth of plants, various nutrients are required. In the present application, some essential nutrients for plants such as P, S, Zn, B, K, Ca, etc. are contained in the A agent, which directly affects the growth and development of plants, and the appropriate supplement of the nutrients can effectively promote the growth of plants against adversity. Among them, the nano zinc oxide particles have a small diameter, can be used as a carrier for various nutrients to enter the plant body, are more easily absorbed and utilized by plants, and thus more effectively promote the growth of plants, and at the same time, can enhance the ability of plants to resist adversity. However, the nano zinc oxide has the characteristics of large specific surface area and large specific surface energy, and thus is prone to aggregation, which makes it difficult to uniformly dissolve, which will inhibit the nano effect. L-cysteine not only serves as the sulfur of the antioxidant to enhance the adversity resistance of plants, but also can improve the aggregation of nano zinc oxide and improve the ability of uniform dissolution of various substances. Superphosphate not only contains phosphorus, but also contains sulfur and calcium elements, which can effectively promote the growth of plants. Potassium chloride can effectively supplement the potassium element of plants, improve the rigidity and strength of the stems and branches of plants, and resist the cold resistance of plants. Boric acid participates in the formation of plant cells, effectively stabilizes the synthesis of chlorophyll, and improves the cold resistance of plants. By preparing a mixed solution of superphosphate, potassium chloride, boric acid, nano zinc oxide and L-cysteine, the nutrient elements of Kandg can be increased, and the cold resistance of Kandg can be improved, which is an effective cold protection agent.

[0029] The sodium alginate in the B agent can be used as a thickening agent and a gelling agent, and the glycerol has strong water absorption, which can effectively inhibit the formation of ice crystals by water molecules, can keep water in a supercooled state, reduces the temperature of ice crystal formation, and reduces the degree of low temperature damage. The B agent can effectively protect the loss of nutrients in the A agent, and can maintain the loss and evaporation of nutrients in the A agent. In addition, the presence of Ca 2+ ions in the A agent will cause the Na + and Ca 2+ ions in the B agent solution to exchange, so that the solution forms a relatively stable hydrogel, which can better prevent the loss of A agent substances.

[0030] In addition, the preparation method of the above A and B agents plays an auxiliary role, specifically:

[0031] The A agent: nano zinc oxide and L-cysteine are added to water in proportion, which effectively improves the aggregation of nano zinc oxide, makes the nano zinc oxide uniformly distributed in the solution, forms a light cream-colored solution, and reduces the interaction between the solute molecules and the solvent molecules in the A agent solution during the ultrasonic process, increases the distance between the molecules, and effectively accelerates the uniform distribution of various nutrients in the solution.

[0032] B agent: using magnetic stirrer to dissolve sodium alginate in water, can accelerate the dissolution rate of sodium alginate, but will not change its chemical properties. In the process of ultrasonic, glycerol is added to the water phase until it is evenly distributed, which will not cause chemical reaction of sodium alginate, and make glycerol evenly distributed in sodium alginate solution.

[0033] In addition, the way of spraying B agent 10-15 minutes after spraying A agent, the reaction film of A and B agent on the leaf surface can be formed within 15-20 minutes, which can effectively prevent the loss of nutrients in A agent, and also has physical cold-proof effect.

[0034] The application will be further described in combination with specific embodiments and drawings. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 Figure 1 is the effect of different treatments on the net photosynthetic rate of Kandelia candel seedling leaves under low temperature stress.

[0036] Figure 2 Figure 2 is the effect of different treatments on the actual photochemical efficiency of PSII of Kandelia candel seedling leaves under low temperature stress.

[0037] Figure 3 Figure 3 is the effect of different treatments on the membrane permeability of Kandelia candel seedling leaves under low temperature stress.

[0038] Figure 4 Figure 4 is the effect of different concentrations of nano zinc oxide on the net photosynthetic rate of Kandelia candel seedling leaves under low temperature stress.

[0039] Figure 5 Figure 5 is the effect of different concentrations of nano zinc oxide on the net photosynthetic rate of Kandelia candel seedling leaves under low temperature stress.

[0040] Figure 6 Figure 6 is the effect of different concentrations of nano zinc oxide on the actual photochemical efficiency of PSII of Kandelia candel seedling leaves under low temperature stress.

[0041] Figure 7 Figure 7 is the effect of different concentrations of nano zinc oxide on the maximum photochemical quantum yield of PSII of Kandelia candel seedling leaves under low temperature stress.

[0042] Figure 8 Figure 8 is the effect of different concentrations of nano zinc oxide on the malondialdehyde content of Kandelia candel seedling leaves under low temperature stress. DETAILED DESCRIPTION

[0043] I. Test materials and preparation

[0044] 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.

[0045] The specific preparation process of the above nutrient solution is as follows:

[0046] A: pH = 6.0, calcium nitrate 82.07 g / L or calcium nitrate tetrahydrate 118.08 g / L -1 ;

[0047] 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 ;

[0048] C: pH = 5.5, Nat-EDTA 7.46 g·L -1 5.56 g·L⁻¹ ferrous sulfate heptahydrate -1 ;

[0049] 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 ;

[0050] 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.

[0051] 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.

[0052] In all subsequent experimental treatments of the four-leaf bud, it is necessary to supplement with NaCl-free Hoagland's nutrient solution to maintain a water layer of 1-2 cm.

[0053] 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.

[0054] The specific configuration process of the cold-resistant agent is as follows:

[0055] The preparation process of the A agent is as follows: the nano zinc oxide and L-cysteine are added into water in proportion, and a magnetic stirrer is used for stirring at room temperature for 20-30 min, so as to effectively improve the agglomeration of the nano zinc oxide, make the nano zinc oxide uniformly distributed in the solution, and form a light milky white solution. After the stirring is completed, the solution is moved to an ultrasonic instrument, and the weighed calcium superphosphate, potassium chloride and boric acid are sequentially added during the ultrasonic process. After the solution is ultrasonically treated for 20-30 min, the ultrasonic process reduces the interaction force between the solute molecules and the solvent molecules in the A agent solution, increases the distance between the molecules, and effectively accelerates the uniform distribution of the various nutrient elements in the solution, so as to form the A agent solution.

[0056] The preparation process of the B agent is as follows: the sodium alginate is dissolved in water by using a magnetic stirrer, the temperature is set to 55-60℃, and the stirring speed is 1200 RPM, so as to accelerate the dissolution speed of the sodium alginate without changing the chemical properties of the sodium alginate. After the sodium alginate is fully dissolved after being stirred for 3-3.5 hours, the solution is in a slightly viscous state without visible lumps formed by the adhesion of the sodium alginate powder. After the stirring is completed, the obtained slightly viscous solution is moved to an ultrasonic instrument, glycerol is added dropwise into the water phase until the glycerol is uniformly distributed, the temperature is controlled to be 20-25℃ during the dropwise addition process, and the ultrasonic treatment is further performed for 50-60 min after the dropwise addition of the dry oil is completed. This way does not cause the chemical reaction of the sodium alginate, and makes the glycerol uniformly distributed in the sodium alginate solution, so as to form the protective liquid B agent which can prevent the loss and evaporation of the nutrients after the A agent is sprayed. In addition, the cation Ca 2+ exists in the A agent, which can cause the ion exchange reaction between Na + and Ca 2+ in the B agent solution, so as to form a relatively stable hydrogel, and better play the role of preventing the loss of the A agent.

[0057] II. Test treatment scheme

[0058] Scheme 1 (normal cultivation of seedlings, set as a control, abbreviated as CK)

[0059] In June and July, when the Kandelia ocellata grows to four leaves and one heart, the seedlings are kept in the natural environment for conventional cultivation until they grow to ten leaves and one heart.

[0060] Scheme 2 (the minimum concentration of the cold-resistant agent is used to study the cold resistance of the Kandelia ocellata seedlings, set as a low-concentration cold-resistant agent treatment, abbreviated as LKHJ)

[0061] (1) In June and July, when the Kandelia ocellata grows to four leaves and one heart, the seedlings are moved to a glass greenhouse, and the leaves are sprayed with the cold-resistant agent. Because the seedlings are small, the amount of the cold-resistant agent sprayed is small. (A agent: calcium superphosphate 0.5 g·L -1, potassium 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 ; B agent: sodium alginate 0.3 g·L -1 , glycerol 3 ml·L -1. )

[0062] The specific spraying method is: at 7 o'clock every night, A agent is sprayed on the leaves of the kandelia candel seedlings, and the leaves are wetted during spraying, and the spraying is continuously conducted for 2 days, B agent is sprayed after the second day of A agent spraying, and B agent is sprayed 10-15 minutes after A agent spraying (if there is no special situation, B agent spraying described below is also 10-15 minutes after A agent spraying), and the leaves are wetted again. After spraying, the kandelia candel seedlings continue to be cultivated in the glass greenhouse for 4-5 days, and then the seedlings are moved to the natural environment.

[0063] (2) When the kandelia candel seedlings are cultivated to six leaves per heart in July and August, the seedlings are moved to the glass greenhouse, the leaves are sprayed with A agent at 7 o'clock every night, and the spraying is continuously conducted for 3 days. B agent is sprayed after A agent spraying for 3 days. After the spraying is completed, the seedlings continue to be placed in the glass greenhouse for 4-5 days, and then are moved to the natural environment for cultivation.

[0064] (3) When the kandelia candel seedlings are cultivated to eight leaves per heart in August and September, the seedlings are moved to the glass greenhouse, the leaves are sprayed with A agent at 7 o'clock every morning and evening, the spraying is conducted every other day, the spraying is conducted for a total of 3 days, and the A agent spraying process is completed in a total of 5 days. B agent is sprayed after A agent spraying for 3 days. After the spraying is completed, the seedlings continue to be placed in the glass greenhouse for 4-5 days, and then are moved to the natural environment for cultivation.

[0065] (4) When the kandelia candel seedlings are cultivated to ten leaves per heart in September and November, the seedlings are moved to the glass greenhouse, the leaves are sprayed with A agent at 7 o'clock every morning and evening, and the spraying is continuously conducted for 4 days. B agent is sprayed after A agent spraying for 4 days.

[0066] Scheme 3 (the frost resistance of the kandelia candel seedlings is studied by applying the highest concentration of the frost resistance agent, which is referred to as high-concentration frost resistance agent treatment, abbreviated as HKHJ)

[0067] (1) When the kandelia candel grows to four leaves per heart in June and July, the seedlings are moved to the glass greenhouse, and the leaves are sprayed with the frost resistance agent. Because the seedlings are small, the amount of the frost resistance agent sprayed on the leaves is small. (A agent: superphosphoric acid calcium 1.0 g·L -1 , potassium chloride 1.0 g·L -1 , boric acid 0.2 g·L -1 , nano zinc oxide 0.3 g·L -1 , L-cysteine 0.06 g·L -1; B agent: 0.6 g·L -1 Sodium alginate, glycerol 6 ml·L -1 )

[0068] Specific spraying method: at 7 o'clock every night, spray A agent on the leaves of Kandelia candel seedlings, pay attention to leaf wetting during spraying, spray for 2 consecutive days, spray B agent after 2 days of A agent spraying, also pay attention to leaf wetting. After spraying, continue to cultivate Kandelia candel seedlings in the glass greenhouse for 4-5 days, and then move the seedlings to the natural environment.

[0069] (2) Cultivate Kandelia candel seedlings to six leaves per heart in July and August, move the seedlings into the glass greenhouse, and spray A agent on the leaves at 7 o'clock every night, spray for 3 consecutive days. Spray B agent after 3 days of A agent spraying. After spraying, continue to place in the glass greenhouse for 4-5 days, and then move to the natural environment for cultivation.

[0070] (3) Cultivate Kandelia candel seedlings to eight leaves per heart in August and September, move the seedlings into the glass greenhouse, and spray A agent on the leaves at 7 o'clock every morning and evening, spray every other day, spray for a total of 3 days, and complete the A agent spraying process in a total of 5 days. Spray B agent after 3 days of A agent spraying. After spraying, continue to place in the glass greenhouse for 4-5 days, and then move to the natural environment for cultivation.

[0071] (4) Cultivate Kandelia candel seedlings to ten leaves per heart in September and November, move the seedlings into the glass greenhouse, and spray A agent at 7 o'clock every morning and evening, spray for 4 consecutive days. Spray B agent after 4 days of A agent spraying.

[0072] Scheme 4 (Study the cold resistance of Kandelia candel seedlings by applying cold resistance agent of appropriate concentration, set as appropriate concentration cold resistance agent treatment, abbreviated as SKHJ)

[0073] (1) When Kandelia candel grows to four leaves per heart in June and July, move the seedlings into the glass greenhouse, and spray cold resistance agent on the leaves. Because the seedlings are small, the amount of antifreeze agent sprayed is small. (A agent: 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 ; B agent: 0.5 g·L -1 Sodium alginate, glycerol 5 ml·L -1 )

[0074] Specific spraying method: at 7 o'clock every night, spray A agent on the leaves of Kandelia candel seedlings, pay attention to leaf wetting during spraying, spray continuously for 2 days, spray B agent after 2 days of A agent spraying, also pay attention to leaf wetting. After spraying, continue to cultivate Kandelia candel seedlings in the glass greenhouse for 4-5 days, and then move the seedlings to the natural environment.

[0075] (2) Cultivate Kandelia candel seedlings to six leaves per heart in July-August, move the seedlings into the glass greenhouse, spray A agent on the leaves at 7 o'clock every night, and spray continuously for 3 days. Spray B agent after 3 days of A agent spraying. After spraying, continue to place in the glass greenhouse for 4-5 days, and then move to the natural environment for cultivation.

[0076] (3) Cultivate Kandelia candel seedlings to eight leaves per heart in August-September, move the seedlings into the glass greenhouse, spray A agent on the leaves at 7 o'clock every morning and evening, spray every other day, spray for a total of 3 days, and complete the A agent spraying process in a total of 5 days. Spray B agent after 3 days of A agent spraying. After spraying, continue to place in the glass greenhouse for 4-5 days, and then move to the natural environment for cultivation.

[0077] (4) Cultivate Kandelia candel seedlings to ten leaves per heart in September-November, move the seedlings into the glass greenhouse, spray A agent on the leaves at 7 o'clock every morning and evening, and spray continuously for 4 days. Spray B agent after 4 days of A agent spraying.

[0078] Scheme 5 (Effect of nano-zinc oxide of different concentrations on the cold resistance of Kandelia candel seedlings under low temperature stress)

[0079] Move Kandelia candel seedlings growing to ten leaves per heart into the glass greenhouse, spray nano-zinc oxide of different concentrations on the leaves at 7 o'clock every morning and evening, and spray continuously for 3 days. The specific spraying method includes the following contents: (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.

[0080] Three, temperature treatment

[0081] (1) Effect of normal temperature on the growth of Kandelia candel seedlings

[0082] The seedlings treated in the above scheme 1 were moved into a 25℃ (day) / 22℃ (night) light incubator, with 12 hours of light per day, a relative humidity of 50%-60%, and a light intensity of no less than 400 μmol·m -2 ·s -1 , and maintained for 4 days.

[0083] (2) Effect of low temperature stress on the growth of Kandelia candel seedlings treated with different cold protection agents

[0084] The seedlings obtained by the above scheme 1, scheme 2, scheme 3 and scheme 4 were directly placed in a cold light source incubator with a temperature of 8℃ (day) / -3℃ (night) for low temperature stress for 4 days, wherein the seedlings treated in scheme 1 were low temperature stress (LTS). The relative humidity of the incubator was controlled at 50%-60%, and the light intensity was no less than 400 μmol·m -2 ·s -1 , with 12 hours of light per day.

[0085] (3) Effect of low temperature stress on the growth of Kandelia candel seedlings treated with different concentrations of nano zinc oxide

[0086] The seedlings obtained in the above scheme 5 were moved into a cold light source incubator with a temperature of 8℃ (day) / -3℃ (night) for low temperature stress for 4 days, with a relative humidity of 50%-60% and a light intensity of no less than 400 μmol·m -2 ·s -1 , with 12 hours of light per day.

[0087] IV. Determination items and methods

[0088] (1) Observation of leaf color

[0089] The changes in leaf color of each treatment after low temperature for 4 days were observed.

[0090] (2) Determination of photosynthetic parameters

[0091] Kandelia candel was recovered for 30 minutes under natural light conditions, and the photosynthetic parameters were determined. The net photosynthetic rate and stomatal conductance of the third pair of leaves counted from the top were determined using a Li-6400 portable photosynthesis instrument from 10:00 to 11:30.

[0092] (3) Determination of chlorophyll fluorescence parameters

[0093] Kandelia candel was recovered for 30 minutes under natural light conditions, and the PSII actual photochemical efficiency and PSII reaction center energy capture efficiency were determined using an FMS-2 portable modulated chlorophyll fluorescence instrument.

[0094] (4) Determination of cell membrane permeability

[0095] The 0.3 g fresh leaf fragments were placed in 10 ml deionized water, and after 15 minutes of air extraction with an air extractor, they were left to stand for 1 hour. The exosmotic electrolyte (S1) was determined using a DDS-307 type conductivity meter, and then the conductivity (S2) was determined after boiling in boiling water for 10 minutes. The cell membrane permeability was calculated according to the following formula: cell membrane permeability = S1 / S2 x 100%.

[0096] (5) Method for determining the content of malondialdehyde

[0097] 1 g of fresh leaf fragments was weighed, 2 ml of 10% TCA and a small amount of quartz sand were added, and grinding was performed until homogenate was obtained. Then 8 ml of TCA was added for grinding, and homogenate was obtained by centrifugation at 4000 r·min -1 for 10 minutes. The supernatant was the sample extract. 2 ml of the supernatant after centrifugation (2 ml of distilled water was added to the control) was taken, 2 ml of 0.6% TBA solution was added, and the mixture was mixed and reacted in a boiling water bath for 15 minutes. After rapid cooling, it was centrifuged again. The extinction at 532, 600 and 450 nm was determined. The content of malondialdehyde was calculated according to the following formula:

[0098] Malondialdehyde content (μmol·g -1 ) = [MDA concentration (μmol·L -1 ) x extract volume (mL)] x [sample weight (g) x 1000] -1 .

[0099] (6) Data processing

[0100] Single factor variance analysis (One-way ANOVA) was performed by SPSS 21.0 statistical software for data comparison and analysis. The data in the figure are mean + standard deviation, and the drawing was made by SigmaPlot 10.0 drawing software.

[0101] V. Results and analysis

[0102] 5.1 Effect of different cold resistance components on the growth of Kandelia candel seedlings

[0103] (1) Change in leaf color

[0104] Under normal circumstances, the leaves of the control (CK) treated Kandelia candel seedlings were green. After 4 days of low temperature stress, about 65% of the leaves of the Kandelia candel seedlings treated with low temperature stress (LTS) turned black and brown, about 35% of the leaves of the Kandelia candel seedlings treated with low concentration of cold resistance agent (LKHJ) turned black and brown, about 32% of the leaves of the Kandelia candel seedlings treated with high concentration of cold resistance agent (HKHJ) turned black and brown, and 10% of the leaves of the Kandelia candel seedlings treated with suitable concentration of cold resistance agent (SKHJ) turned black and brown. This indicates that the cold resistance ability is in the order of: SKHJ > HKHJ > LKHJ > LTS.

[0105] (2) The effects of different treatments on the net photosynthetic rate and stomatal conductance of Kandelia candel seedlings

[0106] Reference to the attached Figure 1 It can be seen that, under low temperature stress for 4 days, the net photosynthetic rate and stomatal conductance of Kandelia candel seedlings treated with low-concentration cold protection agent (LKHJ) were significantly higher than those of untreated plants (LTS) (P<0.05). This was similar to the results of high-concentration cold protection agent (HKHJ) treatment. Compared with LKHJ and HKHJ treatments, the net photosynthetic rate and stomatal conductance of Kandelia candel seedlings treated with a suitable concentration of cold protection agent (SKHJ) were 1.64 and 1.59 times, and 1.79 and 1.67 times those of LKHJ and HKHJ treatments, respectively, with significant differences (P<0.05). This indicates that cold protection agent can significantly improve the cold resistance of Kandelia candel seedlings, and the effect is better with a suitable concentration of cold protection agent.

[0107] (3) The effects of different treatments on the actual photochemical efficiency of PSII and the maximum photochemical quantum yield of PSII of Kandelia candel seedlings

[0108] Reference to the attached Figure 2 It can be seen that, under low temperature stress for 4 days, the actual photochemical efficiency of PSII of Kandelia candel seedlings treated with low-concentration cold protection agent (LKHJ) was about 1.68 and 1.74 times that of untreated plants (LTS), and the maximum photochemical quantum yield of PSII of Kandelia candel seedlings treated with high-concentration cold protection agent (HKHJ) was about 2.12 and 2.07 times that of untreated plants (LTS), with significant differences (P<0.05). The actual photochemical efficiency of PSII and the maximum photochemical quantum yield of PSII of Kandelia candel seedlings treated with a suitable concentration of cold protection agent (SKHJ) were about 1.34 and 1.30 times, and 1.22 and 1.24 times those of LKHJ and HKHJ treatments, respectively, with significant differences (P<0.05).

[0109] (4) The effects of different treatments on the cell membrane permeability of Kandelia candel seedlings

[0110] Reference to the attached Figure 3 It can be seen that, under low temperature stress for 4 days, the cell membrane permeability of Kandelia candel seedlings treated with low-concentration cold protection agent (LKHJ) was about 22.1% lower than that of untreated plants (LTS), and the cell membrane permeability of Kandelia candel seedlings treated with high-concentration cold protection agent (HKHJ) was about 18.9% lower than that of untreated plants (LTS), with significant differences (P<0.05). The cell membrane permeability of Kandelia candel seedlings treated with a suitable concentration of cold protection agent (SKHJ) was about 13.5% and 15.5% lower than that of LKHJ and HKHJ treatments, respectively, with significant differences (P<0.05).

[0111] 5.2 Effects of different antifreeze components on the growth of Kandelia candel seedlings

[0112] (1) Effects of different nano-zinc oxides on the net photosynthetic rate of leaves of Kandelia candel seedlings

[0113] 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).

[0114] (2) Effects of different nano-zinc oxides on stomatal conductance of leaves of Kandelia candel seedlings

[0115] 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.

[0116] (3) Effects of different nano-zinc oxides on the actual photochemical efficiency of PSII in leaves of Kandelia candel seedlings

[0117] 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.

[0118] (4) Effects of different nano-zinc oxides on the maximum photochemical quantum yield of PSII in leaves of Kandelia candel seedlings

[0119] Reference Appendix Figure 7It can be seen that under the condition of low temperature stress for 4 days, with the increase of nano zinc oxide concentration, the leaf stomatal conductance of Kandelia candel seedlings showed a trend of first increasing and then decreasing, and the maximum was reached at the concentration of 0.2 g·L -1 Under the treatment of NZ0.15, NZ0.2 and NZ0.3, the leaf stomatal conductance of Kandelia candel seedlings increased by about 1.5, 1.7 and 1.4 times respectively compared with no nano zinc oxide treatment, and reached significant difference (P<0.05), and the maximum was reached at NZ0.2.

[0120] (5), the effect of different nano zinc oxide on the malondialdehyde content of Kandelia candel seedlings

[0121] Reference to the attached Figure 8 It can be seen that under the condition of low temperature stress for 4 days, with the increase of nano zinc oxide concentration, the leaf malondialdehyde content of Kandelia candel seedlings showed a trend of first decreasing and then increasing, and the minimum was reached at the concentration of 0.2 g·L -1 Under the treatment of NZ0.15, NZ0.2 and NZ0.3, the leaf malondialdehyde content of Kandelia candel seedlings decreased by about 24.8%, 27.4% and 13.6% respectively compared with no nano zinc oxide treatment, and reached significant difference (P<0.05).

Claims

1. A method for improving the cold tolerance of Kandelia candel in subtropical regions, characterized in that It comprises the following steps: (1) Select dry mudflat sea mud into plastic buckets; (2) Configure cold-proof agent, wherein A agent 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 ; B agent comprising the following components: 0.3-0.6 g·L -1 Sodium alginate, glycerol 3-6 ml·L -1 ; (3) In May and June, pick up Kandelia candel hypocotyls and plant them in plastic buckets, and place the plastic buckets in a natural environment to cultivate Kandelia candel to four leaves and one heart, during which pour Hoagland's nutrient solution with a NaCl concentration of 15 ‰ twice to maintain a water layer of 1-2 cm in the buckets; (4) In June and July, move the plastic buckets containing the seedlings into a glass greenhouse, and spray the leaves with the cold-proof agent: every night, spray A agent on the leaves of the Kandelia candel seedlings until the leaves are wet, continuously spray for 2 days, and then spray B agent on the leaves until the leaves are wet on the second day of A agent spraying, and continue to cultivate in the glass greenhouse for 4-5 days, and then move the plastic buckets to a natural environment for cultivation, and timely supplement Hoagland's nutrient solution without NaCl to maintain a water layer of 1-2 cm; (5) In July and August, cultivate the Kandelia candel seedlings to six leaves and one heart, move the plastic buckets into a glass greenhouse, and spray A agent on the leaves every night until the leaves are wet, continuously spray for 3 days, and then spray B agent on the leaves until the leaves are wet on the third day of A agent spraying, and continue to cultivate in the glass greenhouse for 4-5 days, and then move the plastic buckets to a natural environment for cultivation, and timely supplement Hoagland's nutrient solution without NaCl to maintain a water layer of 1-2 cm; (6) In August and September, cultivate the Kandelia candel seedlings to eight leaves and one heart, move the plastic buckets into a glass greenhouse, and spray A agent on the leaves every morning and evening until the leaves are wet, spray A agent every other day, a total of three days of A agent spraying, and then spray B agent on the leaves until the leaves are wet on the third day of A agent spraying, and continue to cultivate in the glass greenhouse for 4-5 days, and then move the plastic buckets to a natural environment for cultivation, and timely supplement Hoagland's nutrient solution without NaCl to maintain a water layer of 1-2 cm; (7) In September and November, cultivate the Kandelia candel seedlings to ten leaves and one heart, move the plastic buckets into a glass greenhouse, and spray A agent on the leaves every morning and evening until the leaves are wet, continuously spray for 4 days, and then spray B agent on the leaves until the leaves are wet on the fourth day of A agent spraying, and continue to cultivate in the glass greenhouse for 4-5 days after the spraying is completed, and then move the plastic buckets to a natural environment for cultivation, and timely supplement Hoagland's nutrient solution without NaCl to maintain a water layer of 1-2 cm.

2. The method for improving the cold resistance of Kandelia candel in subtropical regions according to claim 1, characterized in that A agent comprising the following components: superphosphate of lime 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 ; B agent comprising the following components: 0.5 g·L -1 Sodium alginate, glycerol 5 ml·L -1 .

3. The method of claim 1 or 2 for improving cold tolerance of Kandelia candel in subtropical regions, characterized in that The A agent preparation process comprises: Add nano-zinc oxide and L-cysteine into water in proportion, stir with a magnetic stirrer, then move the solution to an ultrasonic instrument for ultrasonic treatment, and add calcium superphosphate, potassium chloride and boric acid in sequence during the ultrasonic treatment.

4. The method for improving the cold resistance of Kandelia candel in subtropical regions according to claim 3, characterized in that, The magnetic stirrer is stirred at room temperature for 20-30 minutes during the A agent preparation process.

5. The method for improving the cold resistance of Kandelia candel in subtropical regions according to claim 3, characterized in that, The ultrasonic instrument is ultrasonically treated for 20-30 minutes during the A agent preparation process.

6. The method of claim 1 or 2, wherein the Kandelia candel is grown in a subtropical region. The B agent preparation process comprises: add sodium alginate into water, stir with a magnetic stirrer, then move the solution to an ultrasonic instrument for ultrasonic treatment, and drop glycerol during the ultrasonic treatment.

7. The method of claim 6, wherein the Kandelia candel is grown in a subtropical region. The magnetic stirrer is set to a temperature of 55-60℃ and a rotation speed of 1200 RPM, and is stirred for 3-3.5 hours during the B agent preparation process.

8. The method of claim 6, wherein the Kandelia candel is grown in a subtropical region. The ultrasonic instrument is ultrasonically treated for 50-60 minutes during the B agent preparation process, and is set to a temperature of 20-25℃.

9. The method of claim 1 or 2 for increasing the cold tolerance of Kandelia candel in subtropical regions, characterized in that, B is sprayed 10-15 minutes after spraying of A.

10. The method of claim 1 or 2 for increasing the cold tolerance of Kandelia candel in subtropical regions, characterized in that, 3-4 seedlings of Kandelia candel hypocotyls are planted in a single plastic bucket.

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