A seedling raising method for enhancing cold tolerance of michelia
Patent Information
- Application Number
- CN202611051756.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-15
- Publication Date
- 2026-10-23
AI Technical Summary
[0005]鉴于此，本发明的目的是提供一种增强含笑耐寒性的育苗方法，解决传统的喷洒生长调节剂提高含笑耐寒性的方法，存在的防治不及时、持续效果差的问题
[0031]本发明采用嫁接繁殖方法进行含笑的培育，选用具有良好抗寒性的望春玉兰作为砧木，并对嫁接苗进行低温梯度驯化，同时在低温梯度驯化期间喷洒以壳聚糖和烯效唑为主要成分的调节剂进行处理，保证低温梯度驯化期间嫁接苗正常生理状况的同时，进一步诱导脯氨酸、可溶性糖等渗透调节物质的合成，提高抗氧化酶活性等，进而培育得到具有良好抗寒性的含笑嫁接苗。
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Figure CN122953876A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of Michelia figo seedling technology, and in particular to a seedling method for enhancing the cold resistance of Michelia figo. Background Technology
[0002] Michelia is a general term for plants in the genus Michelia of the family Magnoliaceae. It comprises about 50 species of evergreen shrubs or trees, widely used in landscaping, garden decoration, and potted plants due to their beautiful tree shapes, elegant fragrance, and high ornamental value. Furthermore, aromatic oils can be extracted from their flowers and leaves, making them important garden plants with both ecological and economic value.
[0003] Michelia genus originates from tropical to subtropical Asia. In China, its natural distribution is concentrated in South China, Southwest China, and southern East China. Its natural distribution areas are mostly in warm and humid environments, and its poor cold tolerance is a key factor restricting its northward expansion. In northern my country and high-altitude areas, the low winter temperatures easily cause frost damage to Michelia, resulting in frost damage to its branches and leaves, cessation of growth, or even death, reducing survival rate and growth quality, and greatly limiting the development of the Michelia industry. Current techniques for improving plant cold tolerance include physical protection, variety selection, and chemical regulation. Physical protection measures, such as winter mulching, soil mounding, trunk wrapping, and windbreaks, can alleviate low-temperature damage to some extent, but they are cumbersome, costly, and cannot fundamentally improve the plant's cold tolerance. Traditional cold-resistant variety selection relies heavily on natural variation screening or hybridization breeding, which is time-consuming and inefficient. Chemical regulation, through spraying growth regulators to activate stress resistance pathways, induces and improves plant cold tolerance, with significant effects. However, the traditional method usually involves spraying growth regulators when low temperature freezing damage occurs. Although this can quickly improve the plant's short-term cold resistance, it has problems such as untimely control and poor lasting effect. Furthermore, excessive application may also have a negative impact on plant growth.
[0004] Therefore, there is a need to find a way to enhance the cold resistance of Michelia figo and solve the problems of untimely prevention and poor sustained effect of the traditional method of spraying growth regulators to improve the cold resistance of Michelia figo. Summary of the Invention
[0005] Therefore, the purpose of this invention is to provide a seedling cultivation method that enhances the cold resistance of Michelia figo, solving the problems of untimely prevention and poor sustained effect in the traditional method of spraying growth regulators to improve the cold resistance of Michelia figo.
[0006] The present invention solves the above-mentioned technical problems through the following technical means:
[0007] A method for enhancing the cold resistance of Michelia figo, the method comprising the following steps:
[0008] (1) Grafting and cultivation of seedlings: In March of spring, two-year-old Magnolia denudata seedlings with a ground diameter of 1-3 cm are used as rootstocks, and healthy one-year-old Michelia figo branches are collected as scions. The scions are grafted onto the rootstocks to obtain grafted seedlings, which are then cultivated outdoors under natural conditions.
[0009] (2) Low temperature gradient acclimatization: The grafted seedlings were cultivated outdoors under natural conditions until the beginning of October, and then low temperature gradient acclimatization was carried out. On the 5th day of low temperature gradient acclimatization, a regulator was sprayed.
[0010] (3) Post-planting care: After the grafted seedlings have been acclimatized to low temperature gradients, they are transferred to outdoor natural conditions for cultivation to obtain cold-resistant Michelia figo seedlings. They are then transplanted and planted, and managed according to conventional methods.
[0011] Furthermore, the low-temperature gradient acclimatization operation in step (2) is as follows:
[0012] The temperature gradient acclimatization lasted for 28 days. Specifically, the temperature was 12℃ on days 1-2, 10℃ on days 3-4, 8℃ on days 5-7, 4℃ on days 8-14, 0℃ on days 15-21, -2℃ on days 22-25, and -4℃ on days 26-28.
[0013] Furthermore, the regulator in step (2) includes the following raw materials:
[0014] Sodium alginate, sodium lignosulfonate, calcium chloride, chitosan, uniconazole, zein, propylene glycol, polylactic acid, nano calcium carbonate, Tween-80.
[0015] Furthermore, the nano-calcium carbonate has a particle size of 5–20 nm; the polylactic acid has a molecular weight of 30–40 kDa.
[0016] Furthermore, the method for preparing the regulator is as follows:
[0017] A: Chitosan was added to 10 times its mass of a 1% acetic acid solution and stirred at room temperature to dissolve, thus obtaining a chitosan solution as the inner aqueous phase W1. Uniconazole was added to 100 times its mass of ethyl acetate and stirred to dissolve, thus obtaining an uniconazole solution as the oil phase O. Sodium alginate was dissolved in water to prepare a 2wt% sodium alginate solution, and then sodium lignosulfonate and Tween-80 were added and stirred to mix and dissolve, thus obtaining the outer aqueous phase W2.
[0018] B: Add the inner aqueous phase W1 dropwise to the oil phase O at a rate of 1 mL / min, and stir continuously at 3000-5000 rpm for 20-40 min to obtain a primary emulsion. Add the primary emulsion dropwise to the outer aqueous phase W2 at a rate of 2 mL / min, and stir continuously at 500-800 rpm for 20-40 min to obtain a composite emulsion. Then, slowly add the composite emulsion dropwise to a 1 wt% calcium chloride solution, and stir continuously at 150-300 rpm to solidify for 1-3 h. After filtering to remove the filtrate, wash twice with water and dry at 35-45℃ to obtain drug-loaded microcapsules.
[0019] C: Add zein to propylene glycol and stir to dissolve. Then add drug-loaded microcapsules and stir to disperse to obtain a microcapsule dispersion. Add polylactic acid to 20 times its mass of dichloromethane and stir to dissolve. Then add 1% of the mass of polylactic acid nano-calcium carbonate and stir to disperse to obtain a polylactic acid suspension. Add the microcapsule dispersion dropwise to the polylactic acid suspension stirred at 300-500 rpm at a rate of 1-3 mL / min. After the dropwise addition is complete, continue stirring at 1000-2000 rpm for 5-10 min. Then remove the dichloromethane by rotary evaporation at 35°C. Wash with water and dry at 40°C for 20-30 min to obtain the regulator.
[0020] Furthermore, in step A, the mass ratio of sodium alginate solution, sodium lignosulfonate, and Tween-80 is 10:(0.03-0.04):(0.05-0.2).
[0021] Furthermore, in step B, the mass ratio of the internal aqueous phase W1 to the oil phase O is 1:(2-5).
[0022] Furthermore, in step B, the mass ratio of the primary emulsion to the external aqueous phase W2 is 1:(4-6).
[0023] Furthermore, in step C, the mass ratio of propylene glycol, zein, and drug-loaded microcapsules is 1:(0.08-0.1):(0.3-0.5).
[0024] Furthermore, in step C, the mass ratio of the microcapsule dispersion to the polylactic acid suspension is 1:(3-8).
[0025] This invention involves preparing a regulator that is sprayed during the low-temperature gradient acclimatization of grafted Michelia figo seedlings. The active ingredients in the regulator, chitosan and uniconazole, can be combined with low-temperature acclimatization treatment to induce and improve the cold resistance of the grafted Michelia figo seedlings. Specifically, after uniconazole and chitosan enter the grafted seedlings, they can promote the synthesis of osmotic regulators such as proline, betaine, and soluble sugars in the cells, increase the activity of antioxidant enzymes, stabilize the cell membrane structure, and scavenge free radicals generated by cells under low-temperature stress, thereby inducing and improving their own stress resistance and cultivating Michelia figo grafted seedlings with good cold resistance.
[0026] However, when using traditional methods to directly spray chitosan and uniconazole solutions, the solution is easily lost and wasted, requiring repeated spraying during the low-temperature acclimatization period, increasing costs, and also easily leading to over-spraying. Furthermore, if the solution is sprayed directly, when the low-temperature gradient acclimatization temperature of this invention is below 0°C, the water in the solution easily cools and freezes, forming ice crystals that cause physical damage to the grafted seedlings. Therefore, this invention further prepares chitosan and uniconazole into a regulator for spraying to solve the existing problems.
[0027] Specifically, in this invention, chitosan is dissolved to form a solution as the inner aqueous phase, uniconazole is dissolved to form the oil phase, sodium lignosulfonate and Tween-80 are added to sodium alginate solution as the outer aqueous phase, and then a water-in-oil-in-water emulsion is prepared and dried to obtain drug-loaded microparticles; the drug-loaded microparticles are then dispersed in propylene glycol containing dissolved zein to obtain a dispersion, and then added dropwise to polylactic acid suspension for re-encapsulation to prepare a regulator.
[0028] On the 5th day of low-temperature gradient acclimatization, a growth regulator was sprayed. At this time, the grafted seedlings were still at an acclimatization temperature of 8℃, under mild low-temperature stress. Chitosan and uniconazole in the drug-loaded microparticles of the growth regulator were slowly released into propylene glycol. Since propylene glycol is a low-volatility substance, it remained in liquid form after the growth regulator dried for a short time. Then, the chitosan and uniconazole released from the drug-loaded microparticles were slowly released through the outermost polylactic acid membrane layer along with the propylene glycol, thus continuously and slowly providing the effective components chitosan and uniconazole. Combined with mild low-temperature stress, this began to induce and improve the cold resistance of the grafted seedlings. Later, when the acclimatization temperature dropped to around 0℃, moderate stress was introduced, and low molecular weight... Polylactic acid (PLA) becomes brittle and cracks under low temperature and the induction of nano-calcium carbonate, leading to structural damage and the release of residual propylene glycol and drug-loaded microparticles. Because the zein dissolved in propylene glycol has good film-forming properties, it can form a hydrophobic film on the plant surface, effectively protecting the plant from physical damage caused by ice crystals. At the same time, after the structural damage of the outer PLA layer of the drug-loaded microparticles is directly released, the internal chitosan and uniconazole are released and act directly on the grafted seedlings without the need for secondary release through the PLA membrane. Therefore, it can provide a higher concentration of chitosan and uniconazole under moderate stress, and better synergistically induce the cold resistance of grafted seedlings with low temperature stress.
[0029] By preparing a slow-release regulator from chitosan and uniconazole according to the method of the present invention, spraying operations can be effectively reduced, and the loss and waste of the liquid can be prevented, thereby reducing operating costs and material costs. It can also avoid the problem of physical damage to plants caused by water crystallization under conventional spraying methods. The hydrophobic film layer formed by the regulator can also effectively protect the plants and prevent the problem of physical damage to the plants caused by the cooling and crystallization of water on the plant surface.
[0030] Beneficial effects:
[0031] This invention employs a grafting propagation method to cultivate Michelia figo. Magnolia sprengeri, which exhibits good cold resistance, is selected as the rootstock. The grafted seedlings undergo low-temperature gradient acclimatization. During this acclimatization period, a regulator primarily composed of chitosan and uniconazole is sprayed to ensure the grafted seedlings maintain normal physiological conditions while further inducing the synthesis of osmotic regulators such as proline and soluble sugars, and enhancing antioxidant enzyme activity. This process results in the cultivation of Michelia figo grafted seedlings with excellent cold resistance. Attached Figure Description
[0032] Figure 1 These are images showing the growth of grafted seedlings during the early stages of low-temperature acclimatization.
[0033] Figure 2 : These are pictures of the outdoor growth of grafted seedlings in experimental group 1 of this invention after low-temperature acclimatization;
[0034] Figure 3 : These are images showing the outdoor growth of grafted seedlings after low-temperature acclimatization, which is the blank control of this invention. Detailed Implementation
[0035] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings:
[0036] This invention discloses a seedling cultivation method to enhance the cold resistance of Michelia figo. When cultivating Michelia figo seedlings, it is necessary to prepare a regulator, wherein the nano-calcium carbonate used has a particle size of about 10 nm and the polylactic acid used has a molecular weight of 35 kDa.
[0037] Example 1: Preparation of regulator
[0038] A: Chitosan was added to 10 times its mass of a 1% (v / v) acetic acid solution and stirred at room temperature to dissolve, thus obtaining a chitosan solution, which served as the inner aqueous phase W1. Uniconazole was added to 100 times its mass of ethyl acetate and stirred to dissolve, thus obtaining an uniconazole solution, which served as the oil phase O. Sodium alginate was dissolved in water to prepare a 2wt% sodium alginate solution. Then, sodium alginate solution, sodium lignin sulfonate, and Tween-80 were added to the sodium alginate solution in a mass ratio of 10:0.035:0.1, and stirred to mix and dissolve, thus obtaining the outer aqueous phase W2.
[0039] B: The internal aqueous phase W1 was added dropwise to the oil phase O at a rate of 1 mL / min according to a mass ratio of internal aqueous phase W1: oil phase O = 1:4, and the mixture was stirred continuously at 4000 rpm for 30 min to obtain a primary emulsion. The primary emulsion was added dropwise to the external aqueous phase W2 at a rate of 2 mL / min according to a mass ratio of primary emulsion: external aqueous phase W2 = 1:5, and the mixture was stirred continuously at 600 rpm for 30 min to obtain a composite emulsion. The composite emulsion was then slowly added dropwise to a 1 wt% calcium chloride solution, and the mixture was stirred continuously at 200 rpm for 2 h to solidify. After filtering to remove the filtrate, the mixture was washed twice with water and dried at 40℃ to obtain drug-loaded microcapsules.
[0040] C: According to the mass ratio of propylene glycol: zein: drug-loaded microcapsules = 1:0.09:0.4, zein was added to propylene glycol and stirred to dissolve. Then, drug-loaded microcapsules were added and stirred to disperse, resulting in a microcapsule dispersion. Polylactic acid was added to 20 times its mass of dichloromethane and stirred to dissolve. Then, 1% of nano-calcium carbonate (by mass of polylactic acid) was added and stirred to disperse, resulting in a polylactic acid suspension. According to the mass ratio of microcapsule dispersion to polylactic acid suspension = 1:5, the microcapsule dispersion was added dropwise to the polylactic acid suspension being stirred at 400 rpm at a rate of 2 mL / min. After the addition was completed, the mixture was stirred continuously at 1500 rpm for 8 min. Then, the dichloromethane was removed by rotary evaporation at 35 °C. After washing with water, the mixture was dried at 40 °C for 25 min to obtain the regulator.
[0041] Example 2: Preparation of the regulator
[0042] A: Chitosan was added to 10 times its mass of a 1% (v / v) acetic acid solution and stirred at room temperature to dissolve, thus obtaining a chitosan solution, which served as the inner aqueous phase W1. Uniconazole was added to 100 times its mass of ethyl acetate and stirred to dissolve, thus obtaining an uniconazole solution, which served as the oil phase O. Sodium alginate was dissolved in water to prepare a 2wt% sodium alginate solution. Then, sodium alginate solution, sodium lignin sulfonate, and Tween-80 were added to the sodium alginate solution in a mass ratio of 10:0.03:0.05 and stirred to mix and dissolve, thus obtaining the outer aqueous phase W2.
[0043] B: The internal aqueous phase W1 was added dropwise to the oil phase O at a rate of 1 mL / min according to the mass ratio of internal aqueous phase W1: oil phase O = 1:2, and the mixture was stirred continuously at 3000 rpm for 40 min to obtain the primary emulsion. The primary emulsion was added dropwise to the external aqueous phase W2 at a rate of 2 mL / min according to the mass ratio of primary emulsion: external aqueous phase W2 = 1:4, and the mixture was stirred continuously at 500 rpm for 40 min to obtain the composite emulsion. The composite emulsion was then slowly added dropwise to a 1 wt% calcium chloride solution, and the mixture was stirred continuously at 150 rpm for 3 h to solidify. After filtering to remove the filtrate, the mixture was washed twice with water and dried at 35°C to obtain drug-loaded microcapsules.
[0044] C: According to the mass ratio of propylene glycol: zein: drug-loaded microcapsules = 1:0.08:0.3, zein was added to propylene glycol and stirred to dissolve. Then, drug-loaded microcapsules were added and stirred to disperse, resulting in a microcapsule dispersion. Polylactic acid was added to 20 times its mass of dichloromethane and stirred to dissolve. Then, 1% of nano-calcium carbonate (by mass of polylactic acid) was added and stirred to disperse, resulting in a polylactic acid suspension. According to the mass ratio of microcapsule dispersion to polylactic acid suspension = 1:3, the microcapsule dispersion was added dropwise to the polylactic acid suspension being stirred at 300 rpm at a rate of 1 mL / min. After the addition was completed, the mixture was stirred continuously at 1000 rpm for 10 min. Then, the dichloromethane was removed by rotary evaporation at 35 °C. After washing with water, the mixture was dried at 40 °C for 20 min to obtain the regulator.
[0045] Example 3: Preparation of Regulator
[0046] A: Chitosan was added to 10 times its mass of a 1% (v / v) acetic acid solution and stirred at room temperature to dissolve, thus obtaining a chitosan solution, which served as the inner aqueous phase W1. Uniconazole was added to 100 times its mass of ethyl acetate and stirred to dissolve, thus obtaining an uniconazole solution, which served as the oil phase O. Sodium alginate was dissolved in water to prepare a 2wt% sodium alginate solution. Then, sodium alginate solution, sodium lignin sulfonate, and Tween-80 were added to the sodium alginate solution in a mass ratio of 10:0.04:0.2 and stirred to mix and dissolve, thus obtaining the outer aqueous phase W2.
[0047] B: The internal aqueous phase W1 was added dropwise to the oil phase O at a rate of 1 mL / min according to a mass ratio of internal aqueous phase W1: oil phase O = 1:5, and the mixture was stirred continuously at 5000 rpm for 20 min to obtain a primary emulsion. The primary emulsion was added dropwise to the external aqueous phase W2 at a rate of 2 mL / min according to a mass ratio of primary emulsion: external aqueous phase W2 = 1:6, and the mixture was stirred continuously at 800 rpm for 20 min to obtain a composite emulsion. The composite emulsion was then slowly added dropwise to a 1 wt% calcium chloride solution, and the mixture was stirred continuously at 300 rpm for 1 h to solidify. After filtering to remove the filtrate, the mixture was washed twice with water and dried at 45℃ to obtain drug-loaded microcapsules.
[0048] C: Add zein to propylene glycol at a mass ratio of 1:0.1:0.5 and stir to dissolve. Then add the drug-loaded microcapsules and stir to disperse to obtain a microcapsule dispersion. Add polylactic acid to 20 times its mass of dichloromethane and stir to dissolve. Then add 1% of nano-calcium carbonate (by mass of polylactic acid) and stir to disperse to obtain a polylactic acid suspension. Add the microcapsule dispersion to the polylactic acid suspension at a mass ratio of 1:8 at a rate of 3 mL / min to the polylactic acid suspension being stirred at 500 rpm. After the addition is complete, continue stirring at 2000 rpm for 5 min. Then remove the dichloromethane by rotary evaporation at 35 °C. Wash with water and dry at 40 °C for 30 min to obtain the regulator.
[0049] Comparative Example 1: Preparation of Regulator
[0050] Compared with Example 1, the only difference is that sodium lignosulfonate was not added in step A of the preparation of the regulator in Comparative Example 1. All other steps are the same as in Example 1, as detailed below:
[0051] A: Chitosan was added to 10 times its mass of a 1% (v / v) acetic acid solution and stirred at room temperature to dissolve, thus obtaining a chitosan solution, which served as the inner aqueous phase W1. Uniconazole was added to 100 times its mass of ethyl acetate and stirred to dissolve, thus obtaining an uniconazole solution, which served as the oil phase O. Sodium alginate was dissolved in water to prepare a 2wt% sodium alginate solution. Then, Tween-80 was added to the sodium alginate solution at a mass ratio of sodium alginate solution: sodium lignosulfonate: Tween-80 = 10:0.1 and stirred to mix and dissolve, thus obtaining the outer aqueous phase W2.
[0052] B~C: Same as in Example 1.
[0053] Comparative Example 2: Preparation of Regulator
[0054] Compared with Example 1, the only difference is that zein was not added in step C during the preparation of the regulator in Comparative Example 2. All other steps are the same as in Example 1, as detailed below:
[0055] A-B: Same as in Example 1;
[0056] C: Add drug-loaded microcapsules to propylene glycol at a mass ratio of 1:0.4 and stir to disperse, obtaining a microcapsule dispersion. Add polylactic acid to 20 times its mass of dichloromethane and stir to dissolve. Then add 1% of nano-calcium carbonate (by mass of polylactic acid) and stir to disperse, obtaining a polylactic acid suspension. Add the microcapsule dispersion to the polylactic acid suspension at a mass ratio of 1:5 at a rate of 2 mL / min to the polylactic acid suspension being stirred at 400 rpm. After the addition is complete, continue stirring at 1500 rpm for 8 min. Then, remove the dichloromethane by rotary evaporation at 35 °C. Wash with water and dry at 40 °C for 25 min to obtain the regulator.
[0057] Comparative Example 3: Preparation of Regulator
[0058] In contrast to Example 1, the only difference is that in Comparative Example 3, water was used instead of propylene glycol in step C during the preparation of the regulator, as detailed below:
[0059] A~B:
[0060] C: Add zein to propylene glycol at a mass ratio of water:zein:drug-loaded microcapsules = 1:0.09:0.4 and stir to disperse. Then add the drug-loaded microcapsules and stir to disperse to obtain a microcapsule dispersion. Add polylactic acid to 20 times its mass of dichloromethane and stir to dissolve. Then add 1% of nano-calcium carbonate (by mass of polylactic acid) and stir to disperse to obtain a polylactic acid suspension. Add the microcapsule dispersion to the polylactic acid suspension at a mass ratio of microcapsule dispersion:polylactic acid suspension = 1:5 at a rate of 2 mL / min to the polylactic acid suspension being stirred at 400 rpm. After the addition is complete, continue stirring at 1500 rpm for 8 min. Then remove the dichloromethane by rotary evaporation at 35 °C. Wash with water and dry at 40 °C for 25 min to obtain the regulator.
[0061] Comparative Example 4: Preparation of Regulator
[0062] Compared with Example 1, the only difference is that nano-calcium carbonate was not added in step C during the preparation of the regulator in Comparative Example 4. All other steps are the same as in Example 1, as detailed below:
[0063] A~B:
[0064] C: Following a mass ratio of propylene glycol: zein: drug-loaded microcapsules = 1:0.09:0.4, zein was dissolved in propylene glycol by stirring. Then, drug-loaded microcapsules were added and dispersed by stirring to obtain a microcapsule dispersion. Polylactic acid (PLA) was dissolved in 20 times its mass of dichloromethane by stirring to obtain a PLA mixed solution. The microcapsule dispersion was added dropwise to the PLA mixed solution being stirred at 400 rpm at a mass ratio of 1:5. After the addition was complete, the mixture was stirred continuously at 1500 rpm for 8 minutes. Then, the dichloromethane was removed by rotary evaporation at 35°C. The mixture was then washed with water and dried at 40°C for 25 minutes to obtain the regulator.
[0065] Comparative Example 5: Preparation of Regulator
[0066] Compared with Example 1, the only difference is that in Comparative Example 5, steps A and B were omitted during the preparation of the regulator; that is, chitosan and uniconazole were not prepared into drug-loaded microcapsules, as shown below:
[0067] Zeatin was added to propylene glycol at a mass ratio of 1:0.09 and stirred until dissolved. Then, chitosan and uniconazole, in the same amounts as in Example 1, were added and stirred to disperse the mixture, resulting in a microcapsule dispersion. Polylactic acid was added to 20 times its mass of dichloromethane and stirred until dissolved. Then, 1% of the mass of polylactic acid nano-calcium carbonate was added and stirred to disperse the mixture, resulting in a polylactic acid suspension. The microcapsule dispersion was added dropwise to the polylactic acid suspension at a mass ratio of 1:5 at a rate of 2 mL / min at a stirring speed of 400 rpm. After the addition was complete, the mixture was stirred continuously at 1500 rpm for 8 min. Then, the dichloromethane was removed by rotary evaporation at 35 °C. The mixture was then washed with water and dried at 40 °C for 25 min to obtain the regulator.
[0068] Comparative Example 6: Preparation of Regulator
[0069] Compared with Example 1, the only difference is that in Comparative Example 6, the original step C was missing in the preparation of the regulator, that is, the drug-loaded microcapsules prepared directly from steps A and B are the regulator.
[0070] Example 4: Seedling cultivation method to enhance the cold resistance of Michelia champaca
[0071] (1) Grafting and cultivation of seedlings: In March of spring, two-year-old Magnolia denudata seedlings with a ground diameter of about 2cm are used as rootstocks. One-year-old healthy and disease-free Michelia champaca branches are collected as scions. The scions are grafted onto the rootstocks using the branch grafting method to obtain grafted seedlings, which are then cultivated outdoors under natural conditions.
[0072] (2) Low temperature gradient acclimatization: The grafted seedlings were cultivated outdoors under natural conditions until the beginning of October, and then low temperature gradient acclimatization was carried out. Specifically, the low temperature gradient acclimatization lasted for 28 days. Specifically, the temperature was 12℃ on the 1st to 2nd day, 10℃ on the 3rd to 4th day, 8℃ on the 5th to 7th day, 4℃ on the 8th to 14th day, 0℃ on the 15th to 21st day, -2℃ on the 22nd to 25th day, and -4℃ on the 26th to 28th day. On the 5th day of low temperature gradient acclimatization, the regulator of Example 2 was sprayed. The spraying amount was 10g / plant. The spraying method was to add the regulator to 50 times the mass of water, stir and disperse it before spraying.
[0073] (3) Post-planting care: After the grafted seedlings have been acclimatized to low temperature gradients, they are transferred to outdoor natural conditions for cultivation to obtain cold-resistant Michelia figo seedlings. They can then be transplanted and planted. Watering, fertilization, and pest and disease control can be carried out in accordance with conventional methods.
[0074] Experiment: Seedling cultivation methods to enhance the cold resistance of Michelia champaca
[0075] A seedling cultivation experiment of Michelia figo was conducted at Xi'an Botanical Garden, Shaanxi Province. The experiment was divided into 10 groups: experimental group 1, control groups 1-8, and blank control group. Experimental group 1 used the regulator of Example 1 and the seedling cultivation method of Example 4; control groups 1-6 used the regulators of Comparative Examples 1-6 and the seedling cultivation method of Example 4, respectively; control group 7 used the regulator of Example 1, but did not undergo low-temperature gradient acclimatization treatment, and was cultivated at room temperature under the conditions of 20°C during the day and 10°C at night. The time and method of spraying the regulator were the same as in Example 4; control group 8 was not treated with the regulator, but was sprayed with an equal amount of water; the blank control group was not sprayed with the regulator, but was sprayed with an equal amount of water at the same time, and did not undergo low-temperature gradient acclimatization treatment, but was cultivated at room temperature under the conditions of 20°C during the day and 10°C at night. The remaining steps were carried out according to the seedling cultivation method of Example 4.
[0076] Two-year-old Magnolia denudata with a ground diameter of 2cm and uniform growth status were used as rootstocks and scions for grafting. One-year-old healthy Michelia alba with uniform growth status were also collected as scions. After grafting using the branch grafting method, the grafted seedlings were naturally cultivated outdoors and managed in the same way. After the grafted seedlings were cultivated to early October, they were transferred to the artificial climate chamber of Xi'an Botanical Garden in Shaanxi Province for low-temperature gradient acclimatization treatment. The acclimatization time and cooling method were the same as in Example 4. After the low-temperature gradient acclimatization was completed, the grafted seedlings of each group were transferred to outdoor natural conditions for further cultivation for 20 days. The low-temperature half-lethal temperature of the Michelia alba grafted seedlings of each group was measured. Three replicate experiments were conducted, and the average data are shown in Table 1.
[0077] Table 1
[0078]
[0079] Based on the data analysis in Table 1, we can conclude that:
[0080] (1) The half-lethal temperature of the seedlings in experimental group 1 was -13.14℃, while the half-lethal temperature of the seedlings in the blank control was only -8.69℃. This indicates that the method of the present invention can effectively induce and improve the cold resistance of Michelia figo grafted seedlings by performing low temperature gradient acclimatization treatment and regulator treatment, and cultivate Michelia figo seedlings with good cold resistance.
[0081] (2) In control group 7, no low-temperature gradient acclimatization treatment was performed during the seedling cultivation of Michelia figo. Instead, the seedlings were treated with regulators at room temperature. The low-temperature semi-lethal temperature of the seedlings was significantly lower than that of the seedlings in experimental group 1. In control group 8, no regulators were sprayed during the seedling cultivation of Michelia figo. Only low-temperature gradient acclimatization treatment was performed. The low-temperature semi-lethal temperature of the seedlings was significantly lower than that of experimental group 1. This indicates that the regulators and low-temperature gradient acclimatization of the present invention can work together to more effectively induce the cold resistance of Michelia figo seedlings.
[0082] (3) In control group 1, sodium lignosulfonate was not added during the preparation of the regulator, resulting in poor stability of the drug-loaded microparticles and reduced adhesion to the plant surface, leading to low utilization of the active ingredient and reduced efficacy of the regulator. In experimental group 1, sodium lignosulfonate was added to the regulator, which improved the structural stability of the drug-loaded microparticles. After the zein dissolved in propylene glycol was released, it formed a film on the plant surface. The drug-loaded microparticles bound to the film through non-covalent interactions such as hydrophobic interactions, thereby improving the adhesion of the drug-loaded microparticles to the surface and increasing drug utilization. The control group 2 achieved better induction effects; in the preparation of the regulator, water was used instead of zein, which failed to form a hydrophobic film on the plant surface, thus reducing the effectiveness of the regulator; in the preparation of the regulator, water was used instead of propylene glycol, which firstly failed to effectively dissolve the film-forming material zein, resulting in the zein failing to form a film on the plant surface, and secondly, propylene glycol itself has the effect of lowering the water crystallization temperature, which can better protect the plant. Without the use of propylene glycol, the cold resistance induction effect of the seedlings in control group 3 was poor.
[0083] (4) In control group 4, no nano-calcium carbonate was added during the preparation of the regulator, which failed to induce the polylactic acid membrane layer to become brittle and cracked under low temperature conditions, thus failing to release the internal components efficiently, resulting in a reduction in the effect of the regulator. In control group 5, chitosan and uniconazole were not prepared into drug-loaded microcapsules during the preparation of the regulator. In control group 6, step C was missing during the preparation of the regulator, and only drug-loaded microcapsules were prepared. The release time of the effective components in the regulators of control groups 5 and 6 was short, which reduced the subsequent cold resistance induction effect and significantly reduced the degree of increase in low-temperature semi-lethal temperature.
[0084] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.
Claims
1. A seedling cultivation method for enhancing the cold resistance of Michelia figo, characterized in that, The method includes the following steps: (1) Grafting and cultivation of seedlings: In March of spring, two-year-old Magnolia denudata seedlings with a ground diameter of 1-3 cm are used as rootstocks, and healthy one-year-old Michelia figo branches are collected as scions. The scions are grafted onto the rootstocks to obtain grafted seedlings, which are then cultivated outdoors under natural conditions. (2) Low temperature gradient acclimatization: The grafted seedlings were cultivated outdoors under natural conditions until the beginning of October, and then low temperature gradient acclimatization was carried out. On the 5th day of low temperature gradient acclimatization, a regulator was sprayed. (3) Post-planting care: After the grafted seedlings have been acclimatized to low temperature gradients, they are transferred to outdoor natural conditions for cultivation to obtain cold-resistant Michelia figo seedlings. They are then transplanted and planted, and managed according to conventional methods.
2. The seedling cultivation method for enhancing the cold resistance of Michelia champaca according to claim 1, characterized in that, The low-temperature gradient acclimatization operation in step (2) is as follows: The temperature gradient acclimatization lasted for 28 days. Specifically, the temperature was 12℃ on days 1-2, 10℃ on days 3-4, 8℃ on days 5-7, 4℃ on days 8-14, 0℃ on days 15-21, -2℃ on days 22-25, and -4℃ on days 26-28.
3. The seedling cultivation method for enhancing the cold resistance of Michelia champaca according to claim 2, characterized in that, The regulator in step (2) includes the following raw materials: Sodium alginate, sodium lignosulfonate, calcium chloride, chitosan, uniconazole, zein, propylene glycol, polylactic acid, nano calcium carbonate, Tween-80.
4. The seedling cultivation method for enhancing the cold resistance of Michelia champaca according to claim 3, characterized in that, The nano-calcium carbonate has a particle size of 5–20 nm; the polylactic acid has a molecular weight of 30–40 kDa.
5. The seedling cultivation method for enhancing the cold resistance of Michelia champaca according to claim 4, characterized in that, The method for preparing the regulator is as follows: A: Chitosan was added to 10 times its mass of a 1% acetic acid solution and stirred at room temperature to dissolve, thus obtaining a chitosan solution as the inner aqueous phase W1. Uniconazole was added to 100 times its mass of ethyl acetate and stirred to dissolve, thus obtaining an uniconazole solution as the oil phase O. Sodium alginate was dissolved in water to prepare a 2wt% sodium alginate solution, and then sodium lignosulfonate and Tween-80 were added and stirred to mix and dissolve, thus obtaining the outer aqueous phase W2. B: Add the inner aqueous phase W1 dropwise to the oil phase O at a rate of 1 mL / min, and stir continuously at 3000-5000 rpm for 20-40 min to obtain the primary emulsion. Add the primary emulsion dropwise to the outer aqueous phase W2 at a rate of 2 mL / min, and stir continuously at 500-800 rpm for 20-40 min to obtain the composite emulsion. Then, slowly add the composite emulsion dropwise to a 1 wt% calcium chloride solution, and stir continuously at 150-300 rpm to solidify for 1-3 h. After filtering to remove the filtrate, wash twice with water and dry at 35-45℃ to obtain drug-loaded microcapsules. C: Add zein to propylene glycol and stir to dissolve. Then add drug-loaded microcapsules and stir to disperse to obtain a microcapsule dispersion. Add polylactic acid to 20 times its mass of dichloromethane and stir to dissolve. Then add 1% of the mass of polylactic acid nano-calcium carbonate and stir to disperse to obtain a polylactic acid suspension. Add the microcapsule dispersion dropwise to the polylactic acid suspension stirred at 300-500 rpm at a rate of 1-3 mL / min. After the dropwise addition is complete, continue stirring at 1000-2000 rpm for 5-10 min. Then remove the dichloromethane by rotary evaporation at 35°C. Wash with water and dry at 40°C for 20-30 min to obtain the regulator.
6. The seedling cultivation method for enhancing the cold resistance of Michelia champaca according to claim 5, characterized in that, In step A, the mass ratio of sodium alginate solution, sodium lignosulfonate, and Tween-80 is 10:(0.03-0.04):(0.05-0.2).
7. The seedling cultivation method for enhancing the cold resistance of Michelia champaca according to claim 6, characterized in that, In step B, the mass ratio of the aqueous phase W1 to the oil phase O is 1:(2-5).
8. A method for enhancing the cold resistance of Michelia champaca according to claim 7, characterized in that, In step B, the mass ratio of the primary emulsion to the external aqueous phase W2 is 1:(4-6).
9. A seedling cultivation method for enhancing the cold resistance of Michelia champaca according to claim 8, characterized in that, In step C, the mass ratio of propylene glycol, zein, and drug-loaded microcapsules is 1:(0.08-0.1):(0.3-0.5).
10. A method for enhancing the cold resistance of Michelia champaca according to claim 9, characterized in that, In step C, the mass ratio of the microcapsule dispersion to the polylactic acid suspension is 1:(3-8).