Tibetan white bark pine of the same variety grafting method

Abstract

The present application relates to a Tibetan white bark pine of the same variety grafting method, and belongs to the technical field of forest tree asexual reproduction. In view of the technical problems of low survival rate of white bark pine grafting, easy microbial infection of grafting interface and slow formation of callus in high altitude environment, the method realizes efficient grafting through the synergistic optimization of pre-treatment of scion and cut processing of stock: 1-year-old branches with a diameter of 4-6mm are selected and treated by temperature and humidity regulation and composite antibacterial liquid immersion, and the cut of the stock is cut at an angle and coated with composite antibacterial liquid four times to form a sterile healing interface; 1-year-old branches are cut, and there are at least two or more bud points on the branches, two full bud points are retained, and the branches are cut between the two full bud points to obtain the scion. The method significantly improves the survival rate of white bark pine grafting and the interface stress resistance in alpine regions, is suitable for the rapid breeding and genetic property reservation of white bark pine excellent germplasm in the ecological restoration engineering of Tibet plateau, and can make more use of and protect the white bark pine shoot resources in Tibet.

Description

Technical Field

[0001] This invention relates to the field of forest tree cultivation technology. More specifically, this invention relates to a method for grafting Tibetan white pine of the same variety. Background Technology

[0002] Tibetan white pine, a rare and endangered tree species endemic to the southeastern edge of the Qinghai-Tibet Plateau and a key ecological construct, plays an irreplaceable role in the restoration of fragile high-altitude ecosystems, biodiversity conservation, and landscape construction. However, this species has weak natural regeneration capacity, and seed propagation faces many limitations, including a long cycle, low germination rate, and poor seedling resistance. Therefore, grafting propagation technology is considered an important way to achieve rapid asexual reproduction, maintain superior traits, and ex-situ conservation of germplasm resources, especially for genetic improvement and plantation establishment. Grafting propagation of Tibetan white pine faces multiple technical bottlenecks in high-altitude areas. In traditional methods, scion preservation often involves room temperature or simple low-temperature treatment, but the large diurnal temperature range and dry air environment of the plateau easily lead to moisture imbalance inside the scion, resulting in a significant decrease in activity after storage. During scion pretreatment, conventional disinfection methods are insufficient to completely eliminate low-temperature resistant microorganisms. These microorganisms can easily invade the cut during grafting, causing interface infection. Antibacterial treatment of rootstock cuts often relies on a single application of the agent, which has a short duration of efficacy in low-temperature environments and cannot effectively inhibit the continuous invasion of microorganisms along the xylem vessels, resulting in a persistently high risk of infection in the later stages of grafting. Summary of the Invention

[0003] Another objective of this invention is to provide a grafting method for Tibetan white pine of the same variety. Traditional grafting methods suffer from problems such as insufficient pretreatment of scions, poor compatibility of rootstock cuts, and weak post-grafting protection in high-altitude areas, resulting in insufficient contact area between the cambium layers of the scion and rootstock, high microbial infection rate, and long callus formation cycle.

[0004] Conventional scion preparation does not specify the number of buds and the cutting location, resulting in uneven distribution of scion vigor, imbalance of nutrient supply after grafting, and affecting the uniform differentiation of callus tissue.

[0005] Existing scion storage technologies lack gas composition and light regulation, and cannot simultaneously inhibit respiration consumption and activate callus-related genes, resulting in scion metabolic disorders and decreased stress resistance.

[0006] Traditional scion disinfection processes do not completely degrade lignin and pectin, resulting in insufficient softening of the cell walls on the wedge-shaped surface, which affects the close adhesion between the scion and the rootstock and the efficiency of fluid exchange.

[0007] Conventional antibacterial solutions do not penetrate deep enough to effectively remove microorganisms from deep within the scion, and improper ultrasonic parameter settings can easily damage the cambium cell structure.

[0008] Existing grafting interface protective materials have low antibacterial components and low mechanical strength, making them unable to adapt to drastic changes in temperature and humidity at high altitudes, leading to the failure of water and oxygen balance regulation.

[0009] Traditional nano-silver dispersion systems have poor stability, and bamboo charcoal fiber has insufficient adsorption capacity, resulting in uneven distribution of antibacterial components and low free radical scavenging efficiency.

[0010] Single antibacterial treatment of rootstock incisions is insufficient to cover the entire path of microbial invasion and lacks a time-sequential release mechanism for healing-promoting components, leading to a contradiction between early infection and later healing.

[0011] Conventional wound management does not regulate osmotic pressure and nutrient supply in stages, which cannot match the cell differentiation needs at different stages of vascular bundle regeneration, resulting in inconsistent healing quality.

[0012] Existing sustained-release technologies cannot respond to changes in the pH of the interface microenvironment, leading to a mismatch between the release rate of growth regulators and the callus process, resulting in resource waste or local toxicity.

[0013] To achieve these objectives and other advantages according to the present invention, a method for grafting Tibetan white pine of the same variety is provided, comprising the following steps:

[0014] Step 1: Select a healthy mother tree and cut one-year-old branches with a diameter of 4-6mm and at least three buds. Select two plump buds on the branch and cut the branch between the two plump buds to obtain the scion. Make a slanted cut at the bottom of the scion to form a slanted surface. Place the scion in a sealed container at a temperature of 8-12℃ and a humidity of 75-85% for 36-48 hours for pretreatment. During the pretreatment, open the container for ventilation for 8-10 minutes every 6 hours. After the pretreatment, soak the scion in a compound antibacterial solution containing 0.5%-1% chitosan and 0.1%-0.3% tea polyphenols for 10-15 minutes to obtain the pretreated scion. Before grafting, refrigerate the pretreated scion at a temperature of -5-0℃.

[0015] Step 2: Select 2-year-old seedlings with a diameter of 8-12mm as rootstock. First, make a horizontal cut to form a top cross section. Then, make a vertical cut downwards from the top cross section and a diagonal cut downwards from the outside of the rootstock. The vertical and diagonal cuts form a matching incision that matches the scion. After cutting, apply a compound antibacterial solution to the surface of the cut every 3 minutes for a total of four times.

[0016] Step 3: Insert the pretreated scion into the incision in the rootstock and tie it with a binding tape. Spray a hydrogel containing 0.05%-0.1% nano-silver particles onto the outer surface of the grafting site after binding. The amount of spraying is 0.02-0.03 ml per square centimeter of grafting surface. Keep the binding tape on until the scion and rootstock heal.

[0017] Preferably, in step one, the specific method for treating the scion in a sealed container is as follows:

[0018] A porous ceramic granule adsorbent loaded with nano zinc oxide is placed in a sealed container. The adsorbent contains 3 wt% nano zinc oxide and is coated with a 0.8% polydopamine membrane. The mass ratio of adsorbent to scion is 1:12.

[0019] During ventilation, a gas stream containing 0.3 ppm of cyclopropene carboxylate is injected into the container. The gas stream is carried by a mixture of nitrogen and carbon dioxide in a volume ratio of 20:1, and the injection time is 4-6 minutes each time ventilation is performed.

[0020] During ventilation, light control is carried out simultaneously, with red light of wavelength 660nm intermittently irradiating the wedge-shaped surface of the scion at an intensity of 10-15μmol / (m²·s), and irradiation for 2-3 minutes each time ventilation is performed;

[0021] After treatment in a sealed container, immerse the wedge-shaped surface of the scion in a mixture of 0.01% salicylic acid and 0.005% proline for 10-15 seconds, drain, refrigerate at 0-5℃ for 1 hour, and then place it in a compound antibacterial solution.

[0022] Preferably, in step one, before soaking in the compound antibacterial solution, the wedge-shaped surface of the scion is first immersed in a pretreatment solution containing 0.5-1 U / mL laccase and 0.05%-0.1% pectin lyase, and treated at 35-38℃ for 8-10 minutes.

[0023] Preferably, in step one, the chitosan in the composite antibacterial solution is a cationic nanofiber modified with succinic anhydride, the tea polyphenols are encapsulated by β-cyclodextrin to form a complex with a particle size of <10nm, and 0.005% sodium subtilis lipopeptide is added to the composite antibacterial solution.

[0024] During the soaking process with the compound antibacterial solution, low-frequency ultrasound of 40-50kHz is applied, with the sound intensity controlled at 0.5W / cm². The process is repeated for 3 minutes followed by a 2-minute interval, and then repeated 3 times. Finally, the strips are rinsed with deionized water to obtain the pretreated spikelets.

[0025] Preferably, in step three, the preparation method of the hydrogel containing 0.05%-0.1% nano-silver particles is as follows: sodium carboxymethyl cellulose and sodium alginate are mixed at a mass ratio of 3:2, dissolved in deionized water to prepare a 2% mixed solution, nano-silver particles, 0.3% bamboo charcoal fiber and 0.15% polyethylene glycol are added; the mixture is stirred at 60℃ and 200r / min for 2 hours, cooled to room temperature, and then 1% calcium chloride solution is added to crosslink and form a hydrogel.

[0026] The spraying is done in two stages. The first spraying is completed within 10 minutes after grafting, and the second spraying is done 30 minutes after the first spraying. After the second spraying, the grafting is irradiated with 365nm ultraviolet light for 10-15 seconds.

[0027] Preferably, the nano-silver particles are pretreated by means of: dispersing the nano-silver particles in deionized water containing 0.2% polyvinylpyrrolidone, ultrasonically treating for 20 min, then adding 0.5% of propyl gallate by mass of nano-silver for surface modification, stirring at 40°C for 1 h, and centrifuging to obtain nano-silver particles with an antioxidant coating on the surface.

[0028] The bamboo charcoal fiber is pretreated by soaking it in a 3% hydrogen peroxide solution for 30 minutes, then loading 3% by mass of nano zinc oxide particles onto the surface of the bamboo charcoal fiber, and forming a coating layer through dopamine polymerization.

[0029] Preferably, in step two, before the first application of the composite antibacterial liquid, a layer of extract is applied first. The extract includes 2% by mass of aloe polysaccharide, 0.01% by mass of indolebutyric acid, and 0.3% by mass of nano-diatomaceous earth.

[0030] After a 3-minute interval, apply the composite antibacterial solution again. After applying the last layer of composite antibacterial solution, after a 10-minute interval, apply a protective solution. The protective solution includes 0.1% lysozyme, 0.1% houttuynia cordata extract, and 0.003% silver nitrate.

[0031] Preferably, in step three, after grafting, the cultivation method is as follows:

[0032] On the 3rd to 5th day after grafting, spray a healing-promoting solution around the binding band every day. The healing-promoting solution includes 0.3% chitosan oligosaccharide, 0.05% proline and 0.01% zinc sulfate by mass, and adjust the osmotic pressure to 1250 mOsm / kg.

[0033] From the 6th to the 12th day after grafting, spray the vascular bundle induction solution around the binding tape every day. The vascular bundle induction solution includes 0.8% potassium dihydrogen phosphate, 0.003% sodium naphthaleneacetate and 0.1% nano zinc oxide by mass fraction, and the osmotic pressure is increased to 350-450 mOsm / kg. The spraying frequency is adjusted to once every 2 days.

[0034] From the 13th day after grafting until the scion and rootstock have healed, spray the plant with a balanced nutrient solution every three days. The balanced nutrient solution includes 0.3% potassium dihydrogen phosphate, 0.1% calcium nitrate, and 0.05% zinc sulfate. At the same time, in the evening, alternately irradiate the grafting site with 450nm blue light and 660nm red light, with a light intensity of 8-12μmol / (m²・s), and each type of light irradiation for 15-20 minutes.

[0035] Preferably, 0.03% of pH-responsive sustained-release microspheres are added to both the healing-promoting solution and the vascular bundle-inducing solution. The pH-responsive sustained-release microspheres have a three-layer structure: the outer layer is a porous pH-sensitive membrane composed of a chitosan-sodium alginate complex; the middle layer is a nanofiber network loaded with γ-aminobutyric acid and silicate, wherein the nanofibers are formed by cross-linking polylactic acid-glycolic acid copolymer and sodium lignosulfonate, and the surface of the nanofibers is grafted with 0.1-0.5% catalase by mass; the core is a sustained-release unit containing a nanoscale montmorillonite carrier, which adsorbs zinc ions and borate ions through ion exchange, and 5-8% of 2,4-epibrassinolide is intercalated between the montmorillonite layers.

[0036] The present invention has at least the following beneficial effects:

[0037] First, this invention significantly enhances scion cell activity and inhibits the accumulation of metabolic byproducts through gradient temperature and humidity control and gas environment optimization; it precisely matches the geometric parameters of the scion wedge surface and the rootstock incision to enhance the cambium contact tightness, laying the foundation for efficient callus formation. Combining enzymatic pretreatment and ultrasonic-assisted penetration technology, it deeply softens the scion cell wall structure, promotes the directional migration of antibacterial components, and constructs a synergistic biocompatible interface; this invention uses Tibetan white pine grafted onto Tibetan white pine, and grafting with the same variety avoids rejection reactions, improves graft survival rate, and maintains the inherent excellent shape of Tibetan white pine.

[0038] Secondly, the composite antibacterial system of this invention forms a dynamic protective barrier through a triple action of physical barrier, chemical inhibition, and biological antagonism, effectively inhibiting the microbial infection pathway. The synergistic effect of nano-silver hydrogel and functionalized bamboo charcoal fiber constructs a humidity-adaptive network at the grafting interface, simultaneously achieving mechanical strength enhancement and free radical scavenging, ensuring the long-term stability of the healing microenvironment.

[0039] Third, this invention designs a time-sequential nutrient supply program based on the differentiation patterns of callus tissue, precisely matching the energy and signal requirements of different stages of vascular bundle regeneration. The intelligent responsive sustained-release system triggers the on-demand release of active ingredients through environmental sensing, and optimizes cell polarity distribution by coupling light signal modulation technology, significantly improving the synchronicity of the lignification process and resource utilization efficiency, ultimately achieving high-quality callus formation.

[0040] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of scion acquisition in one of the technical solutions of the present invention, wherein the gray lines indicate the cutting positions;

[0042] Figure 2 In one embodiment of the present invention, the scion and rootstock are joined together, wherein A is the scion and B is the rootstock. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0044] <Example 1>

[0045] The implementation steps for grafting Tibetan white pine of the same variety are as follows:

[0046] Step 1: Scion Pretreatment

[0047] Select a healthy mother tree and cut one-year-old branches with a diameter of 4-6mm, ensuring each branch has at least three buds. Select two plump buds on the branch (remove the rest), and cut the branch between the two plump buds (e.g., ...). Figure 1 As shown), scions are obtained (one branch can yield two scions).

[0048] The scions were pretreated in a sealed container at 10°C and 80% humidity for 48 hours, during which the container was opened for ventilation for 10 minutes every 6 hours.

[0049] After the pretreatment, the scion is immersed in a compound antibacterial solution (1% chitosan + 0.3% tea polyphenols + the remainder is water) for 15 minutes. During the immersion, the buds should not be submerged and should be above the immersion solution.

[0050] After soaking, place the scions in a refrigerator at -5 to 0℃ until use.

[0051] Step 2: Rootstock Treatment

[0052] Select two-year-old seedlings with a ground diameter of 8-12mm as rootstock. First, make a horizontal cut to form a top cross section. Then, make a vertical cut downward from the top cross section and a downward oblique cut on the outside of the rootstock. The vertical and oblique cuts form a matching incision that matches the scion.

[0053] After cutting, apply a compound antibacterial solution (1% chitosan + 0.3% tea polyphenols + the remainder is water) to the cut surface every 3 minutes for a total of four applications.

[0054] Step 3: Grafting and Protection

[0055] Embed the pretreated scion into the rootstock incision (e.g.) Figure 2 (As shown), and use a binding tape to secure it, leaving the binding tape in place until the scion and rootstock have healed;

[0056] Spray a hydrogel containing 0.1% nano-silver particles onto the outer surface of the grafted area after binding, with a spraying amount of 0.02-0.03 ml per square centimeter of grafting surface;

[0057] After grafting is complete (after spraying the hydrogel), place the plastic bag with the opening facing down over the scion and tie the opening of the plastic bag tightly to the rootstock. Then, place it under shade (using a shade net) for one month before removing the shade net. On the 7th day after putting on the plastic bag, cut an opening (the diameter of the opening can be 5-8mm). Cut it every two days for a total of three times. Remove the plastic bag on the 20th day after putting on the bag.

[0058] <Example 2>

[0059] The implementation steps of grafting Tibetan white pine of the same variety are the same as in Example 1, except that:

[0060] Step 1: Scion Pretreatment

[0061] Select a healthy mother tree and cut one-year-old branches with a diameter of 4-6mm, ensuring each branch has at least three buds. Select two plump buds on the branch (remove the rest), and cut the branch between the two plump buds (e.g., ...). Figure 1 As shown), scions are obtained (one branch can yield two scions).

[0062] The specific method for treating the scions in a sealed container (temperature 10℃, humidity 80%) for 48 hours is as follows:

[0063] A porous ceramic granule adsorbent loaded with nano zinc oxide is placed in a sealed container. The adsorbent contains 3 wt% nano zinc oxide and is coated with a 0.8% polydopamine membrane. The mass ratio of adsorbent to scion is 1:12.

[0064] During ventilation, a gas stream containing 0.3 ppm of ethyl cyclopropene carboxylate is injected into the container. The gas stream is carried by a nitrogen and carbon dioxide mixture with a volume ratio of 20:1, and the injection time is 6 minutes each time ventilation is performed.

[0065] During ventilation, light control was carried out simultaneously, with red light of wavelength 660nm intermittently irradiating the wedge-shaped surface of the scion at an intensity of 15μmol / (m²·s), and irradiation for 2-3 minutes each time ventilation was carried out;

[0066] After treatment in a sealed container, immerse the container in a mixture containing 0.01% salicylic acid and 0.005% proline (the remainder being water) for 10-15 seconds, drain, and then refrigerate at 0-5°C for 1 hour.

[0067] After the pretreatment, the scion is immersed in a compound antibacterial solution (1% chitosan + 0.3% tea polyphenols + the remainder is water) for 15 minutes. During the immersion, the buds should not be submerged and should be above the immersion solution.

[0068] Steps two and three are the same as in Example 1.

[0069] <Example 3>

[0070] The implementation steps of grafting Tibetan white pine of the same variety are the same as in Example 2, except that:

[0071] Step 1: Scion Pretreatment

[0072] Before soaking in the compound antibacterial solution, the scions are first immersed in a pretreatment solution containing 0.5 U / mL laccase and 0.075% pectin lyase, and treated at 35-38℃ for 8-10 minutes.

[0073] The chitosan in the composite antibacterial solution is a cationic nanofiber modified with succinic anhydride, and the tea polyphenols are encapsulated by β-cyclodextrin to form a complex with a particle size of <10nm. 0.005% sodium subtilis lipopeptide is added to the composite antibacterial solution.

[0074] During the soaking process with the compound antibacterial solution, apply low-frequency ultrasound at 40-50kHz with a sound intensity of 0.5W / cm² for 3 minutes, followed by a 2-minute interval. Repeat this cycle 3 times. Finally, rinse with deionized water to obtain pretreated panicles. During soaking, the buds should not be submerged and should be above the soaking solution.

[0075] Steps two and three are the same as in Example 2.

[0076] <Example 4>

[0077] The implementation steps of grafting Tibetan white pine of the same variety are the same as in Example 3, except that:

[0078] Step 3: Grafting and Protection

[0079] The pretreated scion is embedded into the rootstock incision and tied with binding tape;

[0080] A hydrogel containing 0.1% silver nanoparticles was sprayed onto the outer surface of the grafted area after binding. The preparation method of the hydrogel containing 0.1% silver nanoparticles is as follows: sodium carboxymethyl cellulose and sodium alginate were mixed at a mass ratio of 3:2 and dissolved in deionized water to prepare a 2% mixed solution. Silver nanoparticles, 0.3% bamboo charcoal fiber and 0.15% polyethylene glycol were added. The mixture was stirred at 60℃ and 200r / min for 2 hours. After cooling to room temperature, 1% calcium chloride solution was added to crosslink and form a hydrogel.

[0081] The spraying is done in two stages. The first spraying is completed within 10 minutes after grafting, and the second spraying is done 30 minutes after the first spraying. After the second spraying, the graft is irradiated with 365nm ultraviolet light for 10-15 seconds.

[0082] The nano-silver particles were pretreated by dispersing them in deionized water containing 0.2% polyvinylpyrrolidone and sonicating for 20 min. Then, 0.5% of propyl gallate by mass of the nano-silver particles was added for surface modification. After stirring at 40°C for 1 h, the particles were centrifuged to obtain nano-silver particles with an antioxidant coating on the surface.

[0083] The bamboo charcoal fiber is pretreated by soaking it in a 3% hydrogen peroxide solution for 30 minutes, then loading 3% by mass of nano zinc oxide particles onto the surface of the bamboo charcoal fiber, and forming a coating layer through dopamine polymerization.

[0084] After grafting is completed (after spraying hydrogel), place the plastic bag with the opening facing down on the scion, and tie the opening of the plastic bag tightly to the rootstock. Then, place it under shade (using a shade net) for one month before removing the shade net. On the 7th day after putting on the plastic bag, cut an opening (the diameter of the opening can be 5-8mm). Cut it every two days for three times. Remove the plastic bag on the 20th day after putting on the bag.

[0085] After grafting, the cultivation method (the process from grafting to healing between the scion and rootstock) is as follows:

[0086] On the 3rd to 5th day after grafting, spray a healing-promoting solution around the binding tape every day (temporarily loosen the plastic bag at the connection point with the rootstock, roll the plastic bag upwards to expose the grafted part, and tie the plastic bag tightly to the rootstock after spraying). The healing-promoting solution includes 0.3% chitosan oligosaccharide, 0.05% proline and 0.01% zinc sulfate by mass, and adjust the osmotic pressure to 1250 mOsm / kg.

[0087] From the 6th to the 12th day after grafting, spray the vascular bundle induction solution around the binding tape every day (the treatment method for plastic bags is the same as for the bottom 3-5 days). The vascular bundle induction solution includes 0.8% potassium dihydrogen phosphate, 0.003% sodium naphthaleneacetate and 0.1% nano zinc oxide by mass fraction, and the osmotic pressure is increased to 350 mOsm / kg. The spraying frequency is adjusted to once every 2 days.

[0088] From the 13th day after grafting until the scion and rootstock have healed, spray the plant with a balanced nutrient solution every three days (at which point the grafted part is exposed). The balanced nutrient solution includes 0.3% potassium dihydrogen phosphate, 0.1% calcium nitrate, and 0.05% zinc sulfate. At the same time, in the evening, alternately irradiate the grafting site with 450nm blue light and 660nm red light at a light intensity of 12μmol / (m²・s) for 15-20 minutes with each type of light.

[0089] Both the healing-promoting solution and the vascular bundle-inducing solution contain 0.03% pH-responsive sustained-release microspheres. The pH-responsive sustained-release microspheres have a three-layer structure: the outer layer is a porous pH-sensitive membrane composed of chitosan-sodium alginate complex; the middle layer is a nanofiber network loaded with γ-aminobutyric acid and silicate; the nanofibers are formed by cross-linking polylactic acid-glycolic acid copolymer and sodium lignosulfonate; the surface of the nanofibers is grafted with 0.1-0.5% catalase; and the core is a sustained-release unit containing nanoscale montmorillonite carrier, which adsorbs zinc ions and borate ions through ion exchange. Furthermore, 5-8% of 2,4-epibrassinolide is intercalated between the montmorillonite layers.

[0090] Steps one and two are the same as in Example 3.

[0091] <Example 5>

[0092] The implementation steps of grafting Tibetan white pine of the same variety are the same as in Example 4, except that:

[0093] Step 2: Rootstock Treatment

[0094] Select two-year-old seedlings with a ground diameter of 8-12mm as rootstock. Make a horizontal cut to form a top cross section. Make a vertical cut downward from the top cross section and a downward oblique cut on the outside of the rootstock. The vertical and oblique cuts form a matching incision that matches the scion.

[0095] After cutting, apply a compound antibacterial solution (1% chitosan + 0.3% tea polyphenols + the remainder is water) to the cut surface every 3 minutes for a total of four times;

[0096] Before applying the first layer of the compound antibacterial liquid, apply a layer of extract, which includes 2% by mass of aloe polysaccharide, 0.01% by mass of indolebutyric acid and 0.3% by mass of nano diatomaceous earth, with the remainder being water.

[0097] Steps one and three are the same as in Example 4.

[0098] <Comparative Example 1>

[0099] Grafting was performed using the method described in Example 5, except that neither the nano-silver particles nor the bamboo charcoal fiber underwent pretreatment.

[0100] The remaining steps are the same as in Example 5.

[0101] <Comparative Example 2>

[0102] Grafting was performed using the method described in Example 5, except that pH-responsive sustained-release microspheres were not added.

[0103] The remaining steps are the same as in Example 5.

[0104] <Comparative Example 3>

[0105] Grafting was performed using the method described in Example 5, except that the chitosan in the composite antibacterial solution was modified with succinic anhydride, and no 0.005% sodium subtilis lipopeptide was added to the composite antibacterial solution.

[0106] <Experimental Characterization>

[0107] 1. Sample grouping

[0108] Grafting was carried out according to the methods of Examples 1-5 and Comparative Examples 1-3, for a total of eight groups, with 100 grafted seedlings prepared in each group.

[0109] 2. Experimental Testing

[0110] 2.1 Callus formation time

[0111] Methods: Interface tissue was collected every 5 days, paraffin sections were prepared and stained with safranin-fast green, and the time of dedifferentiation of thin-walled cells was counted under a microscope (based on the appearance of callus cells in 50% of samples). The results are shown in Table 1.

[0112] 2.2 Grafting survival rate statistics

[0113] Sixty days after grafting, the number of surviving grafted samples in each group was counted, and the grafting survival rate was calculated using the formula: Grafting survival rate = (Number of surviving samples ÷ Total number of samples) × 100%. The results are shown in Table 1.

[0114] 2.3 Measurement of new shoot growth

[0115] Ninety days after grafting, the length of new shoots of surviving samples in each group was measured, and the average value was taken as the indicator of new shoot growth. The results are shown in Table 1.

[0116] 2.4 Survival rate after transplanting

[0117] The surviving plants were transplanted to a simulated nursery in the Tibetan native habitat at an altitude of 3200m (soil pH 6.8±0.2, organic matter content 1.2%±0.1%). After transplanting, they were managed uniformly (watered once a day, shading rate 50%, and weeded regularly).

[0118] Fifty surviving seedlings were selected from each group and transplanted. The number of surviving seedlings was recorded 90 days after transplanting. The results are shown in Table 1.

[0119] Table 1 Experimental Data

[0120] Callus formation time (days) Grafting survival rate New shoot growth (cm) Survival rate of transplanted seedlings Example 1 10.2±0.8 64% 8.0±0.6 32 Example 2 8.7±0.6 68% 9.2±0.4 33 Example 3 7.6±0.5 71% 10.5±0.3 35 Example 4 7.2±0.4 78% 12.2±0.3 38 Example 5 6.1±0.3 96% 13.8±0.2 48 Comparative Example 1 6.4±0.4 90% 10.0±0.3 44 Comparative Example 2 6.7±0.5 85% 10.3±0.3 43 Comparative Example 3 6.9±0.5 81% 9.8±0.3 41

[0121] Comparative analysis of callus formation time in Table 1 shows that the callus formation time in Examples 1-5 decreased sequentially, with Example 5 taking only 6.1 days, and Comparative Examples 1-3 taking between 6.4 and 6.9 days. This indicates that Examples 2-5, through a closed environment of 8-12℃ low temperature and 75-85% humidity, reduced the scion respiration rate, and the nano-zinc oxide ceramic adsorbent (containing 3wt% nano-zinc oxide) effectively adsorbed ethylene, reducing the ethylene content in the sealed box and delaying cell senescence. Examples 2-5, by injecting an airflow containing 0.3ppm cyclopropene carboxylate during ventilation, blocked ethylene signal transduction. Examples 2-5, by irradiating the wedge-shaped surface of the scion with 10-15μmol / (m²・s) red light during ventilation, activated phytochrome receptors, upregulated the expression of key callus genes, and increased cell dedifferentiation rate. Examples 3-5, through 0.5-1U / mL laccase + 0.05%... Pectin lyase treatment enhances the softening of the cell wall on the wedge-shaped surface. Combined with 40kHz low-frequency ultrasound, the antibacterial solution penetrates from the epidermis to the cambium, removing contaminants and reducing callus initiation obstacles. In Example 5, 2% aloe polysaccharide and 0.01% indolebutyric acid extract were first applied to the rootstock cut. The aloe polysaccharide formed a nanoscale moisturizing film, preventing cut dehydration caused by the dry environment at high altitudes and improving cell survival rate. Indolebutyric acid diffused into the cambium of the rootstock, increasing the cell division index.

[0122] Comparative analysis of the grafting survival rates in Table 1 shows that the survival rate of Examples 1-5 increased from 64% to 96%, while that of Comparative Examples 1-3 was 81%-90%. The sodium carboxymethyl cellulose-sodium alginate hydrogel (containing 0.1% nano-silver) in Examples 4-5, through double-layer spraying (rapid film formation within 10 minutes for the first layer, and filling gaps after 30 minutes for the second layer), formed a continuous barrier of 0.3-0.5 mm thickness, reducing microbial penetration. Succinic anhydride-modified chitosan carries a positive charge and electrostatically adsorbs onto the bacterial cell membrane (negative charge), increasing the antibacterial rate compared to ordinary chitosan. β-cyclodextrin encapsulates tea polyphenols (particle size <10nm), slowly releasing active ingredients and prolonging the duration of antioxidant capacity. Sodium subtilis lipopeptide (Example 5) inserts into the fungal cell membrane to form pores, inhibiting the growth of dominant pathogens in high-altitude areas. In Example 5, 2% sodium subtilis lipopeptide was first applied to the rootstock cut. Aloe polysaccharides, with their molecular structure, competitively bind to receptors on the surface of pathogens, reducing the chance of colonization; the subsequent application of 0.1% lysozyme protective solution hydrolyzes bacterial cell wall peptidoglycan, forming a dual defense line of "biological occupation + enzymatic killing"; in Example 5, through modification with polyvinylpyrrolidone and propyl gallate, an antioxidant layer is formed on the surface of nano-silver; the bamboo charcoal fiber in Example 5, after being coated with dopamine polymer, has enhanced free radical adsorption capacity, inhibiting soil pathogens.

[0123] Comparative analysis of the new shoot growth in Table 1 shows that the new shoot growth in Examples 1-5 increased progressively, reaching 13.8 cm in Example 5, a 72.5% increase compared to the baseline group. Comparative examples 1-3 showed growth of 9.8-10.3 cm. Examples 4-5 involved spraying with a 1250 mOsm / kg hypertonic healing solution (containing 0.3% chitosan oligosaccharide + 0.05% proline), which induced the synthesis of osmolysin in the scion through high osmotic pressure, increasing proline content. A 350 mOsm / kg hypotonic induction solution (containing 0.8% potassium dihydrogen phosphate + 0.003% sodium naphthaleneacetate) promoted cell vacuolation. Combined with 450nm blue light irradiation (enhancing microtubule polymerization), nutrient transport efficiency was improved. Red light irradiation + 0.1% calcium nitrate nutrient solution promoted calmodulin synthesis, improving the transport of photosynthetic products to the apex. The inner montmorillonite carrier slowly released 2,4-epibrassin lactone, enhancing the stability of the thylakoid membrane in chloroplasts. The outer chitosan- Sodium alginate membranes dissolve in the callus environment, releasing γ-aminobutyric acid (GABA) and promoting lateral cell expansion.

[0124] Comparative analysis of the transplant survival rates in Table 1 shows that the survival rate of transplanted plants in Examples 1-5 increased from 32 to 48, while in Comparative Examples 1-3 it was 41-44. In Examples 2-5, soaking the wedge-shaped surface of the scion in 0.01% salicylic acid + 0.005% proline induced the expression of the DREB1A stress-resistance gene, significantly enhancing its dehydration resistance. In Example 5, applying 0.01% indolebutyric acid to the rootstock cut surface induced ARF gene expression via polar transport to the roots, significantly enhancing its adaptability to arid highland soils. The hydrogel in Example 5 continuously released Ag within 15 days after transplanting. + It inhibits rhizosphere pathogens and reduces root disease incidence.

[0125] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and embodiments shown and described herein.

Claims

1. A grafting method for Tibetan white pine of the same variety, characterized in that, Includes the following steps: Step 1: Select a healthy mother tree and cut one-year-old branches with a diameter of 4-6mm and at least three buds. Select two plump buds on the branch and cut the branch between the two plump buds to obtain the scion. Make a slanted cut at the bottom of the scion to form a slanted surface. Place the scion in a sealed container at a temperature of 8-12℃ and a humidity of 75-85% for 36-48 hours for pretreatment. During the pretreatment, open the container for ventilation for 8-10 minutes every 6 hours. After the pretreatment, soak the scion in a compound antibacterial solution containing 0.5%-1% chitosan and 0.1%-0.3% tea polyphenols for 10-15 minutes to obtain the pretreated scion. Before grafting, refrigerate the pretreated scion at a temperature of -5-0℃. Step 2: Select 2-year-old seedlings with a diameter of 8-12mm as rootstock. Make a horizontal cut to form a top cross section. Make a vertical cut downward from the top cross section and a diagonal cut downward from the outside of the rootstock. The vertical and diagonal cuts form a matching incision that matches the scion. After the cutting is completed, apply a compound antibacterial solution to the surface of the cut every 3 minutes. Apply the solution four times. Step 3: Insert the pretreated scion into the rootstock incision and tie it with a binding tape. Spray a hydrogel containing 0.05%-0.1% nano silver particles onto the outer surface of the tied grafting site. The amount of spraying is 0.02-0.03 ml per square centimeter of grafting surface. Keep the binding tape on until the scion and rootstock heal. In step one, the specific method for treating the scion in a sealed container is as follows: A porous ceramic granule adsorbent loaded with nano zinc oxide is placed in a sealed container. The adsorbent contains 3 wt% nano zinc oxide and is coated with a 0.8% polydopamine membrane. The mass ratio of adsorbent to scion is 1:

12. During ventilation, a gas stream containing 0.3 ppm of cyclopropene carboxylate is injected into the container. The gas stream is carried by a mixture of nitrogen and carbon dioxide in a volume ratio of 20:1, and the injection time is 4-6 minutes each time ventilation is performed. During ventilation, light regulation was implemented simultaneously, with red light of 660nm intermittently irradiating the wedge-shaped surface of the scion at an intensity of 10-15 μmol / (m²). 2 •s), irradiate for 2-3 minutes each time ventilation is performed; After treatment in a sealed container, immerse the wedge-shaped surface of the scion in a mixture of 0.01% salicylic acid and 0.005% proline for 10-15 seconds, drain, refrigerate at 0-5℃ for 1 hour, and then place it in a compound antibacterial solution. In step one, before soaking in the compound antibacterial solution, the wedge-shaped surface of the scion is first immersed in a pretreatment solution containing 0.5-1 U / mL laccase and 0.05%-0.1% pectin lyase, and treated at 35-38℃ for 8-10 minutes. In step one, the chitosan in the composite antibacterial solution is a cationic nanofiber modified with succinic anhydride, the tea polyphenols are encapsulated by β-cyclodextrin to form a complex with a particle size of <10nm, and 0.005% sodium subtilis lipopeptide is added to the composite antibacterial solution. During immersion in the compound antibacterial solution, apply low-frequency ultrasound at 40-50 kHz, with the sound intensity controlled at 0.5 W / cm². 2 The process was repeated for 3 minutes followed by a 2-minute interval, and repeated 3 times. Finally, the strips were rinsed with deionized water to obtain the pretreated spikelets.

2. The grafting method for Tibetan white pine of the same variety as described in claim 1, characterized in that, In step three, the preparation method of hydrogel containing 0.05%-0.1% nano-silver particles is as follows: sodium carboxymethyl cellulose and sodium alginate are mixed at a mass ratio of 3:2 and dissolved in deionized water to prepare a 2% mixed solution. Nano-silver particles, 0.3% bamboo charcoal fiber and 0.15% polyethylene glycol are added. The mixture is stirred at 60℃ and 200r / min for 2 hours. After cooling to room temperature, 1% calcium chloride solution is added to crosslink and form a hydrogel. The spraying is done in two stages. The first spraying is completed within 10 minutes of grafting, and the second spraying is done 30 minutes after the first spraying. After the second spraying, the graft is irradiated with 365nm ultraviolet light for 10-15 seconds.

3. The grafting method for Tibetan white pine of the same variety as described in claim 2, characterized in that, The nano-silver particles were pretreated by dispersing them in deionized water containing 0.2% polyvinylpyrrolidone and sonicating for 20 min. Then, 0.5% of propyl gallate by mass of the nano-silver particles was added for surface modification. After stirring at 40°C for 1 h, the particles were centrifuged to obtain nano-silver particles with an antioxidant coating on the surface. The bamboo charcoal fiber is pretreated by soaking it in a 3% hydrogen peroxide solution for 30 minutes, then loading 3% by mass of nano zinc oxide particles onto the surface of the bamboo charcoal fiber, and forming a coating layer through dopamine polymerization.

4. The grafting method for Tibetan white pine of the same variety as described in claim 1, characterized in that, In step two, before applying the first layer of composite antibacterial liquid, a layer of extract is applied. The extract includes 2% aloe polysaccharide, 0.01% indolebutyric acid, and 0.3% nano diatomaceous earth by mass. After a 3-minute interval, apply the composite antibacterial solution again. After applying the last layer of composite antibacterial solution, after a 10-minute interval, apply a protective solution. The protective solution includes 0.1% lysozyme, 0.1% houttuynia cordata extract, and 0.003% silver nitrate.

5. The grafting method for Tibetan white pine of the same variety as described in claim 1, characterized in that, In step three, after grafting is completed, the cultivation method is as follows: On the 3rd to 5th day after grafting, spray a healing-promoting solution around the binding band every day. The healing-promoting solution includes 0.3% chitosan oligosaccharide, 0.05% proline and 0.01% zinc sulfate by mass, and adjust the osmotic pressure to 1250 mOsm / kg. From the 6th to the 12th day after grafting, spray the vascular bundle induction solution around the binding tape every day. The vascular bundle induction solution includes 0.8% potassium dihydrogen phosphate, 0.003% sodium naphthaleneacetate and 0.1% nano zinc oxide by mass fraction, and the osmotic pressure is increased to 350-450 mOsm / kg. The spraying frequency is adjusted to once every 2 days. From the 13th day after grafting until the scion and rootstock have healed, spray the plant with a balanced nutrient solution every three days. The balanced nutrient solution includes 0.3% potassium dihydrogen phosphate, 0.1% calcium nitrate, and 0.05% zinc sulfate. At the same time, in the evening, alternately irradiate the grafting site with 450nm blue light and 660nm red light, with a light intensity of 8-12μmol / (m²・s), and each type of light irradiation for 15-20 minutes.

6. The grafting method for Tibetan white pine of the same variety as described in claim 5, characterized in that, Both the healing-promoting solution and the vascular bundle-inducing solution contain 0.03% pH-responsive sustained-release microspheres. The pH-responsive sustained-release microspheres have a three-layer structure: the outer layer is a porous pH-sensitive membrane composed of chitosan-sodium alginate complex; the middle layer is a nanofiber network loaded with γ-aminobutyric acid and silicate; the nanofibers are formed by cross-linking polylactic acid-glycolic acid copolymer and sodium lignosulfonate; the surface of the nanofibers is grafted with 0.1-0.5% catalase; and the core is a sustained-release unit containing nanoscale montmorillonite carrier, which adsorbs zinc ions and borate ions through ion exchange. Furthermore, 5-8% of 2,4-epibrassinolide is intercalated between the montmorillonite layers.

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