Method for grafting same variety of pinus bungeana

Abstract

The invention relates to a same-variety grafting method for Tibetan pinus bungeana, and belongs to the technical field of forest vegetative propagation. In order to solve the technical problems that in the high-altitude environment, the grafting survival rate of the white bark pine is low, a connector is prone to being infected by microorganisms, and callus is formed slowly, efficient grafting is achieved through collaborative optimization of scion pretreatment and stock incision treatment. The method comprises the steps that a one-year-old branch with the diameter being 4-6 mm is selected and subjected to temperature and humidity control and composite antibacterial liquid dipping treatment; a sterile healing interface is formed in cooperation with rootstock incision slope cutting and four times of composite antibacterial liquid brushing; a one-year-old branch is cut, at least two or more bud points exist on the branch, two plump bud points are reserved, the branch is cut open from the position between the two plump bud points, and the scion is obtained. According to the method, the grafting survival rate and the interface stress resistance of the pinus bungeana in the alpine region are remarkably improved, the method is suitable for rapid breeding and hereditary characteristic retention of excellent germplasm of the pinus bungeana in Tibet plateau ecological restoration engineering, and more scion resources of the pinus bungeana can be utilized and protected.

Description

Technical Field

[0001] The present invention belongs to the technical field of forest tree cultivation. More specifically, the present invention relates to a grafting method for the same variety of Pinus gerardiana Wall. Background Art

[0002] Pinus gerardiana Wall., as a rare and endangered tree species unique to the southeastern edge of the Qinghai-Tibet Plateau and a key ecological constructive species, has an irreplaceable position in the restoration of alpine fragile ecosystems, biodiversity conservation, and landscape construction. However, the natural regeneration ability of this species is weak, and seed propagation has many limitations such as a long cycle, low germination rate, and poor stress resistance of seedlings. Therefore, grafting propagation technology is regarded as an important way to achieve its rapid asexual propagation, maintain excellent traits, and ex situ conservation of germplasm resources, especially of great significance for genetic improvement and plantation establishment. The grafting propagation of Pinus gerardiana Wall. faces multiple technical bottlenecks in high-altitude areas. In traditional methods, scion preservation often uses normal temperature or simple low-temperature treatment, but the large day-night temperature difference and dry air environment on the plateau easily lead to water imbalance inside the scion, and the activity significantly decreases after storage. During scion pretreatment, conventional disinfection means are difficult to completely remove low-temperature-resistant microorganisms, and these bacteria are prone to invade the cut during the grafting process, causing interface infection. The antibacterial treatment of the rootstock cut mainly relies on single-dose drug application, and the drug efficacy duration is short in a low-temperature environment, unable to effectively inhibit the continuous invasion of microorganisms along the xylem vessels, and the infection risk in the later stage of grafting remains high. Summary of the Invention

[0003] Another object of the present invention is to provide a grafting method for the same variety of Pinus gerardiana Wall. Traditional grafting methods have problems such as insufficient scion pretreatment, poor adaptability of the rootstock cut, and weak later protection in high-altitude areas, resulting in insufficient contact area between the scion and the rootstock cambium, high microorganism infection rate, and long callus formation cycle.

[0004] The number of bud points and cutting positions are not clearly defined in conventional scion preparation, resulting in uneven distribution of scion vitality, unbalanced nutrient supply after grafting, and affecting the uniform differentiation of callus.

[0005] Existing scion storage technologies lack gas component and light regulation, unable to simultaneously inhibit respiratory consumption and activate callus-related genes, resulting in scion metabolic disorders and decreased stress resistance.

[0006] Traditional scion disinfection processes do not thoroughly degrade lignin and pectin, resulting in insufficient softening of the cell wall on the wedge surface, affecting the close fit between the scion and the rootstock and the efficiency of body fluid exchange.

[0007] The penetration depth of conventional antibacterial solutions is insufficient, unable to effectively remove deep-layer microorganisms in the scion, and unreasonable ultrasonic parameter settings are prone to damage the cell structure of the cambium.

[0008] The antibacterial components of the existing graft interface protection materials are prone to loss and have low mechanical strength, and cannot adapt to the drastic changes in temperature and humidity on the plateau, resulting in the failure of water-oxygen balance regulation.

[0009] The stability of the traditional silver nanowire dispersion system is poor, and the adsorption performance of bamboo charcoal fiber is insufficient, resulting in uneven distribution of antibacterial components and low free radical scavenging efficiency.

[0010] Single antibacterial treatment of the rootstock incision is difficult to cover the entire path of microbial invasion, and lacks a sequential release mechanism of wound-healing promoting components, resulting in the contradiction between early infection and late healing.

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

[0012] The existing slow-release technology cannot respond to the pH change of the interface microenvironment, resulting in the mismatch between the release rate of growth regulators and the callus process, causing resource waste or local toxicity.

[0013] In order to achieve these and other advantages according to the present invention, a grafting method of the same variety of Pinus gerardiana Wall. in Tibet is provided, including the following steps: Step 1: Select a healthy mother tree, cut a 1-year-old branch with a diameter of 4-6 mm and having at least three or more bud points, select two plump bud points on the branch, and cut the branch between the two plump bud points to obtain a scion. The lower part of the scion is obliquely cut to form an inclined surface. The scion is placed in a sealed container at a temperature of 8-12 °C and a humidity of 75-85% for pretreatment for 36-48 h. During the pretreatment, the container is opened for ventilation for 8-10 min every 6 h. After the pretreatment, the scion is immersed in a composite antibacterial solution containing 0.5%-1% chitosan and 0.1%-0.3% tea polyphenols for 10-15 min to obtain a pretreated scion. Before grafting, the pretreated scion is refrigerated at a temperature of -5-0 °C. Step 2: Select a 2-year-old seedling with a ground diameter of 8-12 mm as the rootstock. First, make a horizontal cut to form a top cross-section, make a vertical cut downward from the top cross-section, and make an inclined cut downward on the outside of the rootstock. The vertical cut and the inclined cut form a fitting incision matching the scion. After the cutting is completed, the composite antibacterial solution is applied to the surface of the incision once every 3 minutes, and it is applied four times. Step 3: Embed the pretreated scion into the rootstock incision, and use a binding band to bind it. A hydrogel containing 0.05%-0.1% silver nanowire particles is sprayed on the outer surface of the grafting part after binding. The spraying amount is 0.02-0.03 ml per square centimeter of the grafting surface. The binding band is retained until the scion and the rootstock heal.

[0014] Preferably, in Step 1, the specific method for treating the scion in the sealed container is: Place the porous ceramsite adsorbent loaded with nano-zinc oxide in a closed container. The content of nano-zinc oxide in the adsorbent is 3 wt%, and the surface is coated with 0.8% polydopamine film. The mass ratio of the adsorbent to the scion is 1:12. During ventilation, inject an air stream containing 0.3 ppm ethyl cyclopropenecarboxylate into the container. The air stream is carried by a nitrogen and carbon dioxide mixed gas with a volume ratio of 20:1, and the injection time is 4 - 6 min each time ventilation is carried out. During ventilation, simultaneously perform light regulation. Intermittently irradiate the wedge surface of the scion with red light with a wavelength of 660 nm, the light intensity is 10 - 15 μmol / (m²·s), and irradiate for 2 - 3 min each time ventilation is carried out. After the treatment in the closed container, immerse the wedge surface of the scion in a mixed solution containing 0.01% salicylic acid and 0.005% proline for 10 - 15 s, drain, refrigerate at 0 - 5 °C for 1 h, and then put it into the composite antibacterial solution.

[0015] Preferably, in step one, before soaking in the composite antibacterial solution, first immerse the wedge surface of the scion in a pretreatment solution containing 0.5 - 1 U / mL laccase and 0.05% - 0.1% pectin lyase, and treat at 35 - 38 °C for 8 - 10 min.

[0016] Preferably, in step one, the chitosan in the composite antibacterial solution is a cationic nanofiber modified by succinic anhydride, the tea polyphenols are formed into a complex with a particle size <10 nm through β-cyclodextrin inclusion, and 0.005% subtilin is added to the composite antibacterial solution. Apply 40 - 50 kHz low-frequency ultrasonic waves during soaking in the composite antibacterial solution, control the sound intensity at 0.5 W / cm², continue for 3 min and then intermittently for 2 min, cycle 3 times, and finally rinse with deionized water to obtain the pretreated scion.

[0017] Preferably, in step three, the preparation method of the hydrogel containing 0.05% - 0.1% silver nanoparticles: Mix sodium carboxymethylcellulose and sodium alginate in a mass ratio of 3:2, dissolve in deionized water to prepare a 2% mixed solution, add silver nanoparticles, 0.3% bamboo charcoal fiber and 0.15% polyethylene glycol; stir at 60 °C and 200 r / min for 2 hours, after cooling to room temperature, add 1% calcium chloride solution to crosslink to form a hydrogel. The spraying is carried out in two times. The first time is completed within 10 min after grafting, and the second spraying is carried out 30 min after the first spraying; irradiate with ultraviolet light with a wavelength of 365 nm for 10 - 15 s after the second spraying.

[0018] Preferably, the silver nanoparticles are pretreated. The pretreatment method is as follows: disperse the silver nanoparticles in deionized water containing 0.2% polyvinylpyrrolidone, perform ultrasonic treatment for 20 min, then add propyl gallate accounting for 0.5% of the mass of the silver nanoparticles for surface modification, stir at 40 °C for 1 h, and then perform centrifugal separation to obtain silver nanoparticles with an antioxidant layer coated on the surface; The bamboo charcoal fiber is pretreated. The pretreatment method is as follows: first soak the bamboo charcoal fiber in a 3% hydrogen peroxide solution for 30 min, and then load 3% of the mass of the bamboo charcoal fiber with zinc oxide nanoparticles on the surface of the bamboo charcoal fiber to form a coating layer through dopamine polymerization.

[0019] Preferably, in step two, a layer of extract is applied before the first application of the composite antibacterial liquid. The extract includes 2% aloe polysaccharide, 0.01% indolebutyric acid, and 0.3% nano-diatomite by mass fraction; After an interval of 3 min, apply the composite antibacterial liquid again. After applying the last layer of the composite antibacterial liquid, apply a layer of protective liquid after an interval of 10 min. The protective liquid includes 0.1% lysozyme, 0.1 houttuynin, and 0.003% silver nitrate by mass fraction.

[0020] Preferably, in step three, after the grafting is completed, the cultivation method is as follows: On the 3rd - 5th day after grafting, spray a wound healing promoting liquid around the binding band every day. The wound healing promoting liquid includes 0.3% chitosan oligosaccharide, 0.05% proline, and 0.01% zinc sulfate by mass fraction, and the osmotic pressure is adjusted to 1250 mOsm / kg; On the 6th - 12th day after grafting, spray a vascular bundle induction liquid around the binding band every day. The vascular bundle induction liquid includes 0.8% potassium dihydrogen phosphate, 0.003% sodium naphthylacetate, and 0.1% zinc oxide nanoparticles by mass fraction, the osmotic pressure is increased to 350 - 450 mOsm / kg, and the spraying frequency is adjusted to once every two days; From the 13th day after grafting until the scion and rootstock are healed, spray a growth balance nutrient solution on the plant every three days. The growth balance nutrient solution includes 0.3% potassium dihydrogen phosphate, 0.1% calcium nitrate, and 0.05% zinc sulfate. At the same time, alternately irradiate the grafting site with blue light of 450 nm and red light of 660 nm in the evening, the light intensity is 8 - 12 μmol / (m²・s), and each light is irradiated for 15 - 20 min.

[0021] Preferably, 0.03% of pH-responsive sustained-release microspheres are added to both the wound healing promoting liquid and the vascular bundle induction liquid. The pH-responsive sustained-release microspheres include a three-layer structure: an outer layer is a porous pH-sensitive membrane composed of a chitosan-sodium alginate composite, a middle layer is a nanofiber network loaded with γ-aminobutyric acid and silicate, the nanofibers are formed by cross-linking poly(lactic-co-glycolic acid) and sodium lignosulfonate, and the surface of the nanofibers is grafted with 0.1-0.5% by mass of catalase. The inner core is a sustained-release unit containing a nanoscale montmorillonite carrier, which adsorbs zinc ions and borate ions through ion exchange, and 5-8% by mass of 2,4-epibrassinolide is intercalated between the montmorillonite layers.

[0022] The present invention has at least the following beneficial effects: First, through gradient temperature and humidity regulation and gas environment optimization, the present invention significantly improves the activity of scion cells and inhibits the accumulation of metabolic by-products; precisely matches the geometric parameters of the scion wedge surface and the rootstock incision, enhances the contact tightness of the cambium, and lays a foundation for efficient callus formation. Combining enzymatic pretreatment and ultrasonic-assisted penetration technology, deeply softens the cell wall structure of the scion, promotes the directional migration of antibacterial components, and constructs an internally and externally coordinated biocompatible interface; the present invention uses Pinus gerardiana Wall. to graft Pinus gerardiana Wall., and grafting of the same variety can avoid rejection reactions, improve the grafting survival rate, and maintain the inherent excellent shape of Pinus gerardiana Wall.

[0023] Second, the composite antibacterial system of the present invention forms a dynamic protection barrier through triple effects of physical barrier, chemical bacteriostasis, and biological antagonism, effectively inhibiting the invasion path of microorganisms. The synergistic effect of nano-silver hydrogel and functionalized bamboo charcoal fiber constructs a humidity self-adaptive network at the grafting interface, synchronously realizes mechanical strength enhancement and free radical scavenging, and ensures the long-term stability of the healing microenvironment.

[0024] Third, based on the callus differentiation law, the present invention designs a sequential nutrition supply plan, precisely matching the energy and signal requirements at different stages of vascular bundle regeneration. The intelligent response-type sustained-release system triggers the release of active ingredients on demand through environmental perception, and couples with the optical signal regulation technology to optimize the cell polarity distribution, significantly improving the synchrony of the lignification process and the resource utilization efficiency, and finally achieving high-quality callus connection.

[0025] Other advantages, objectives, and features of the present invention will be partially reflected by the following description, and partially will also be understood by those skilled in the art through the research and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of obtaining a scion in one technical solution of the present invention, where the gray line indicates the cutting position; Figure 2In one of the technical solutions of the present invention, the scion is chimeric with the rootstock. Among them, A is the scion and B is the rootstock. Detailed implementation mode

[0027] The following further describes the present invention in detail with reference to embodiments, so that those skilled in the art can implement it according to the text of the specification.

[0028] <Example 1> The implementation steps of the grafting method of the same variety of Pinus gerardiana Wall. in Tibet are as follows: Step 1. Scion pretreatment Select a healthy mother tree, and cut a 1-year-old branch with a diameter of 4-6 mm and at least three or more bud points. Select two plump bud points on the branch (remove the rest of the bud points), and cut the branch between the two plump bud points (as Figure 1 shown), to obtain scions (one branch can obtain two scions); Place the scions in a sealed container at a temperature of 10 °C and a humidity of 80% for 48 hours of pretreatment, and ventilate the container for 10 minutes every 6 hours during this period; After the pretreatment is completed, immerse the scions in a composite antibacterial solution (1% chitosan + 0.3% tea polyphenols + the balance is water) for 15 minutes. When soaking, the bud points cannot be soaked, and the bud points need to be higher than the soaking solution; After soaking, place the scions in a refrigerator at -5 - 0 °C for later use.

[0029] Step 2. Rootstock treatment Select a 2-year-old seedling with a ground diameter of 8-12 mm as the rootstock. First, make a horizontal cut to form the top cross-section, then make a vertical cut downward from the top cross-section, and make an inclined cut downward on the outside of the rootstock. The vertical cut and the inclined cut form a chimeric incision matching the scion; After cutting, apply the composite antibacterial solution (1% chitosan + 0.3% tea polyphenols + the balance is water) to the incision surface every 3 minutes, and apply it four times in total.

[0030] Step 3. Grafting and protection Embed the pretreated scion into the rootstock incision (as Figure 2 shown), and use a binding band to bind it, and keep the binding band until the scion and the rootstock heal; Spray a hydrogel containing 0.1% silver nanoparticles on the outer surface of the grafting part after binding, and the spraying amount is 0.02 - 0.03 ml per square centimeter of the grafting surface; After grafting is completed (after spraying the hydrogel), put a plastic bag with the opening facing down over the scion, tie the opening of the plastic bag tightly to the rootstock, and then cultivate it under shaded conditions (shading with a sunshade net) for one month before removing the sunshade net. Cut an opening (the diameter of the opening can be specifically 5 - 8 mm) on the 7th day after putting on the plastic bag, cut it once every two days, cut it three times, and remove the plastic bag on the 20th day after bagging.

[0031] <Example 2> The implementation steps of the grafting method of the same variety of Pinus gerardiana Wall. are the same as those of Example 1. The differences from Example 1 are as follows: Step 1. Scion pretreatment Select a healthy mother tree, cut 1-year-old branches with a diameter of 4 - 6 mm and with at least three or more bud points. Select two plump bud points on the branch (remove the remaining bud points), and cut the branch between the two plump bud points (as Figure 1 shown), to obtain scions (two scions can be obtained from one branch); The specific method for treating the scion in a sealed container (temperature 10°C, humidity 80%) for (48 h) is as follows: Place a porous ceramsite adsorbent loaded with nano-zinc oxide in the sealed container. The content of nano-zinc oxide in the adsorbent is 3 wt%, and the surface is coated with 0.8% of polydopamine film. The mass ratio of the adsorbent to the scion is 1:12; During ventilation, inject an air stream containing 0.3 ppm of ethyl cyclopropene carboxylate into the container. The air stream is carried by a nitrogen and carbon dioxide mixed gas with a volume ratio of 20:1, and the injection time is 6 min each time of ventilation; During ventilation, simultaneously perform light regulation, intermittently irradiate the wedge surface of the scion with red light with a wavelength of 660 nm, the light intensity is 15 μmol / (m²·s), and irradiate for 2 - 3 min each time of ventilation; After the treatment in the sealed container is completed, immerse it in a mixed solution containing 0.01% salicylic acid and 0.005% proline (the balance is water) for 10 - 15 s, drain it, and refrigerate it at 0 - 5°C for 1 h; After the pretreatment is completed, immerse the scion in a composite antibacterial solution (1% chitosan + 0.3% tea polyphenols + the balance is water) for 15 min. During soaking, the bud points cannot be soaked, and the bud points need to be higher than the soaking solution; Step 2. Step 3 are the same as those of Example 1.

[0032] <Example 3> The implementation steps of the grafting method of the same variety of Pinus gerardiana Wall. are the same as those of Example 2. The differences from Example 2 are as follows: Step 1. Scion pretreatment Before soaking in the composite antibacterial solution, the scion was first pre-soaked in a pretreatment solution containing 0.5 U / mL laccase and 0.075% pectin lyase, and treated at 35-38 °C for 8-10 min; The chitosan in the composite antibacterial solution is a cationic nanofiber modified by succinic anhydride. Tea polyphenols are formed into a complex with a particle size <10 nm through β-cyclodextrin inclusion, and 0.005% subtilin is added to the composite antibacterial solution; During the soaking with the composite antibacterial solution, low-frequency ultrasonic waves of 40-50 kHz were applied, the sound intensity was controlled at 0.5 W / cm², continued for 3 min and then intermittent for 2 min, cycled 3 times, and finally rinsed with deionized water to obtain the pretreated scion. The bud points should not be soaked during soaking, and the bud points should be higher than the soaking solution; Step 2 and Step 3 are the same as those in Example 2.

[0033] <Example 4> The implementation steps of the grafting method of the same variety of Pinus gerardiana Wall. are the same as those in Example 3. The difference from Example 3 is: Step 3. Grafting and protection Embed the pretreated scion into the incision of the rootstock and tie it with a binding band; Spray a hydrogel containing 0.1% silver nanoparticles on the outer surface of the grafting part after tying. The preparation method of the hydrogel containing 0.1% silver nanoparticles: Mix sodium carboxymethylcellulose and sodium alginate according to a mass ratio of 3:2, dissolve in deionized water to prepare a 2% mixed solution, add silver nanoparticles, 0.3% bamboo charcoal fiber and 0.15% polyethylene glycol; Stir at 60 °C and 200 r / min for 2 hours, cool to room temperature, and then add 1% calcium chloride solution to crosslink to form a hydrogel; The spraying is carried out in two times. The first time is completed within 10 min after grafting, and the second spraying is carried out 30 min after the first spraying; After the second spraying, irradiate with ultraviolet light with a wavelength of 365 nm for 10-15 s; The silver nanoparticles are pretreated. The pretreatment method is: Disperse the silver nanoparticles in deionized water containing 0.2% polyvinylpyrrolidone, ultrasonically treat for 20 min, then add 0.5% propyl gallate based on the mass of the silver nanoparticles for surface modification, stir at 40 °C for 1 h and then centrifuge to obtain silver nanoparticles with an antioxidant layer on the surface; The bamboo charcoal fiber is pretreated. The pretreatment method is: First soak the bamboo charcoal fiber in 3% hydrogen peroxide solution for 30 min, then load 3% silver oxide nanoparticles based on its mass on the surface of the bamboo charcoal fiber, and form a coating layer through dopamine polymerization; After grafting is completed (after spraying the hydrogel), put a plastic bag with the opening facing down over the scion, tie the mouth of the plastic bag tightly to the rootstock, and then cultivate it under shaded conditions (shading with a sunshade net) for one month before removing the sunshade net. Cut an opening (the diameter of the opening can be specifically 5 - 8 mm) on the 7th day after the plastic bag is put on, cut it once every two days, and cut it three times. Remove the plastic bag on the 20th day after bagging; After grafting is completed, the cultivation method (the process from grafting completion to the healing of the scion and rootstock) is as follows: On the 3rd - 5th day after grafting, spray a healing promotion liquid around the binding tape every day (temporarily untie the connection between the mouth of the plastic bag and the rootstock, roll up the plastic bag upwards to expose the grafted part, and tie the mouth of the plastic bag tightly to the rootstock after spraying). The healing promotion liquid includes 0.3% chitosan oligosaccharide, 0.05% proline, and 0.01% zinc sulfate by mass fraction, and the osmotic pressure is adjusted to 1250 mOsm / kg; On the 6th - 12th day after grafting, spray a vascular bundle induction liquid around the binding tape every day (the plastic bag treatment method is the same as that on the 3rd - 5th day). The vascular bundle induction liquid includes 0.8% potassium dihydrogen phosphate, 0.003% sodium naphthylacetate, and 0.1% nano - zinc oxide by mass fraction, the osmotic pressure is increased to 350 mOsm / kg, and the spraying frequency is adjusted to once every 2 days; From the 13th day after grafting until the scion and rootstock heal, spray a growth - balancing nutrient solution on the plant every three days (at this time, the grafted part is already exposed). The growth - balancing nutrient solution includes 0.3% potassium dihydrogen phosphate, 0.1% calcium nitrate, and 0.05% zinc sulfate. At the same time, irradiate the grafting part alternately with blue light of wavelength 450 nm and red light of wavelength 660 nm in the evening, the light intensity is 12 μmol / (m²・s), and each light is irradiated for 15 - 20 min.

[0034] Both the healing promotion liquid and the vascular bundle induction liquid are added with 0.03% pH - responsive sustained - release microspheres. The pH - responsive sustained - release microspheres include 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, the nanofibers are formed by cross - linking poly(lactic - co - glycolic acid) and sodium lignosulfonate, and 0.1 - 0.5% catalase by mass fraction is grafted on the surface of the nanofibers. The inner core is a sustained - release unit containing a nanoscale montmorillonite carrier, which adsorbs zinc ions and borate ions through ion - exchange, and 5 - 8% 2,4 - epi - brassinolide is intercalated between the montmorillonite layers.

[0035] Step 1 and Step 2 are the same as in Example 3.

[0036] <Example 5> The implementation steps of the grafting method for the same variety of Pinus gerardiana Wall. in Tibet are the same as in Example 4. The difference from Example 4 is: Step 2. Rootstock treatment Select 2-year-old seedling with ground diameter of 8-12 mm as the rootstock, make a transverse cut to form the top cross-section, make a vertical cut downward from the top cross-section, and make an inclined cut downward on the outside of the rootstock. The vertical cut and the inclined cut form a fitting incision matching the scion; After cutting, apply the compound antibacterial liquid (1% chitosan + 0.3% tea polyphenols + the balance is water) on the incision surface every 3 minutes for a total of four times; Apply a layer of extraction liquid before applying the compound antibacterial liquid for the first time. The extraction liquid includes 2% aloe polysaccharide, 0.01% indolebutyric acid and 0.3% nano-diatomite by mass fraction, and the balance is water; Step 1 and Step 3 are the same as those in Example 4.

[0037] <Comparative Example 1> The grafting is carried out by the method of Example 5, wherein the difference is that neither the silver nanoparticles nor the bamboo charcoal fibers are pretreated; The remaining steps are the same as those in Example 5.

[0038] <Comparative Example 2> The grafting is carried out by the method of Example 5, wherein the difference is that the pH-responsive slow-release microspheres are not added; The remaining steps are the same as those in Example 5.

[0039] <Comparative Example 3> The grafting is carried out by the method of Example 5, wherein the difference is that the chitosan in the compound antibacterial liquid is modified with succinic anhydride, and 0.005% subtilopeptin sodium is not added to the compound antibacterial liquid.

[0040] <Experimental characterization> 1. Sample grouping Grafting is carried out according to the methods of Examples 1-5 and Comparative Examples 1-3, a total of eight groups, and 100 grafted seedlings are prepared in each group.

[0041] 2. Experimental detection 2.1 Callus formation time Method: Take the interface tissue every 5 days, make paraffin sections and stain with safranin-fast green, and count the starting time of dedifferentiation of parenchyma cells under the microscope (based on 50% of the samples showing callus cells). The results are shown in Table 1; 2.2 Statistics of grafting survival rate 60 days after grafting, count the number of surviving grafted samples in each group, and calculate the grafting survival rate. The formula is: grafting survival rate = (number of surviving samples ÷ total number of samples) × 100%. The results are shown in Table 1; 2.3 Measurement of new shoot growth At 90 days after grafting, the lengths of the new shoots of the surviving samples in each group were measured, and the average value was taken as the index of new shoot growth. The results are shown in Table 1; 2.4 Number of survived transplants The survived plants were transplanted to a simulated nursery of the original habitat in Tibet at an altitude of 3200 m (soil pH 6.8 ± 0.2, organic matter content 1.2% ± 0.1%). After transplantation, unified management was carried out (watering once a day, shading rate 50%, weeding regularly). For each group, 50 survived seedlings were selected for transplantation, and the number of survived plants at 90 days after transplantation was recorded. The results are shown in Table 1; Table 1 Experimental data Callus formation time (days) Grafting survival rate New shoot growth (cm) Number of survived transplanted plants 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 By comparing and analyzing the callus formation times in Table 1, the callus formation times of Examples 1-5 were successively shortened. The callus formation time of Example 5 was only 6.1 days, and those of Comparative Examples 1-3 were between 6.4 - 6.9 days. It can be seen that in Examples 2-5, through a closed environment with a low temperature of 8-12 °C and a humidity of 75-85%, the respiration rate of the scion was reduced. The nano-zinc oxide ceramsite adsorbent (containing 3 wt% nano-zinc oxide) could effectively adsorb ethylene, reduce the ethylene content in the sealed box, and delay cell senescence; in Examples 2-5, an air flow containing 0.3 ppm ethyl cyclopropenecarboxylate was injected during ventilation to block ethylene signal transduction; in Examples 2-5, the wedge surface of the scion was irradiated with red light at 10-15 μmol / (m²·s) during ventilation to activate the phytochrome receptor and up-regulate the expression level of key callus genes, thereby increasing the cell dedifferentiation rate; in Examples 3-5, treatment with 0.5-1 U / mL laccase + 0.05% pectin lyase was used to increase the softening degree of the cell wall of the wedge surface. Combined with 40 kHz low-frequency ultrasound, the penetration depth of the antibacterial solution extended from the epidermal layer to the cambium layer, removing contamination and reducing callus initiation obstacles; in Example 5, a 2% aloe polysaccharide and 0.01% indolebutyric acid extract were first applied to the cut of the rootstock. The aloe polysaccharide formed a nano-level moisture-holding film to prevent dehydration of the cut caused by the dry environment in the plateau, increasing the cell survival rate. Indolebutyric acid diffused into the cambium layer of the rootstock, increasing the cell division index.

[0042] Comparatively analyzing the grafting survival rates in Table 1, the survival rates of Examples 1-5 increased from 64% to 96%, and those of Comparative Examples 1-3 were 81%-90%. The sodium carboxymethylcellulose-sodium alginate hydrogel (containing 0.1% nano silver) in Examples 4-5 formed a continuous barrier with a thickness of 0.3-0.5 mm through double-layer spraying (forming a fast film within 10 minutes for the first time and filling gaps 30 minutes later), reducing the microbial penetration rate; succinic anhydride-modified chitosan was positively charged and electrostatically adsorbed with the bacterial cell membrane (negatively charged), and the antibacterial rate was improved compared with ordinary chitosan. β-cyclodextrin inclusion complex of tea polyphenols (particle size <10 nm) slowly released active ingredients, and the antioxidant capacity maintenance time was extended; subtilosin A (Example 5) inserted into the fungal cell membrane to form pores, inhibiting the growth of dominant plateau pathogenic bacteria. In Example 5, the rootstock incision was first coated with 2% aloe polysaccharide, and its molecular structure competitively bound to the surface receptor of the pathogenic bacteria, reducing the colonization opportunity; the subsequent 0.1% lysozyme protective solution hydrolyzed the peptidoglycan of the bacterial cell wall, forming a double defense line of "biological occupation + enzymatic killing". Example 5 was modified with polyvinylpyrrolidone and propyl gallate, and an antioxidant layer was formed on the surface of nano silver. The bamboo charcoal fiber in Example 5 had enhanced free radical adsorption ability after being coated with dopamine polymerization, inhibiting soil pathogenic bacteria.

[0043] Comparatively analyzing the new shoot growth in Table 1, the new shoot growth of Examples 1-5 increased progressively, reaching 13.8 cm in Example 5, a 72.5% increase compared with the basic group, and those of Comparative Examples 1-3 were 9.8-10.3 cm. Examples 4-5 were sprayed with a 1250 mOsm / kg hypertonic healing solution (containing 0.3% chitosan oligosaccharide + 0.05% proline). The hypertonic osmotic pressure induced the scion to synthesize osmotic adjustment proteins, and the proline content increased. A 350 mOsm / kg hypotonic induction solution (containing 0.8% potassium dihydrogen phosphate + 0.003% sodium naphthylacetate) promoted cell vacuolization. Combined with 450 nm blue light irradiation (enhancing microtubulin polymerization), the nutrient transport efficiency was improved; red light irradiation + 0.1% calcium nitrate nutrient solution promoted the synthesis of calmodulin, and the photosynthetic products transported to the top were improved; the inner montmorillonite carrier slowly released 2,4-epibrassinolide, enhancing the stability of the chloroplast thylakoid membrane; the outer chitosan-sodium alginate membrane dissolved and released γ-aminobutyric acid in the callus environment, promoting cell lateral expansion; Comparatively analyzing the transplanting survival quantity in Table 1, the transplanting survival numbers in Examples 1-5 increased from 32 plants to 48 plants, and those in Comparative Examples 1-3 were 41-44 plants. In Examples 2-5, the scion wedge surfaces were soaked with 0.01% salicylic acid + 0.005% proline to induce the expression of the DREB1A stress-resistant gene, and the dehydration resistance was significantly enhanced; in Example 5, the rootstock incision was painted with 0.01% indolebutyric acid, which was transported to the roots through polar transport to induce the expression of the ARF gene, and the adaptability to the plateau arid soil was significantly enhanced. The hydrogel in Example 5 continuously released Ag within 15 days after transplantation + , inhibiting rhizosphere pathogens and the root incidence rate.

[0044] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the field, additional modifications can be easily achieved. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to the specific details and the embodiments shown and described herein.

Claims

1. A grafting method for the same variety of Pinus gerardiana Wall. var. wangii, characterized in that, It includes the following steps: Step 1: Select healthy mother trees and cut 1-year-old branches with a diameter of 4-6 mm, and the branches should have at least three or more bud points. Select two plump bud points on the branches and cut the branches between the two plump bud points to obtain scions. The lower part of the scion is obliquely cut to form an inclined plane. Place the scion in a sealed container at a temperature of 8-12 °C and a humidity of 75-85% for pretreatment for 36-48 h. During the pretreatment, open the container for ventilation for 8-10 min every 6 h. After the pretreatment, soak the scion in a composite antibacterial solution containing 0.5%-1% chitosan and 0.1%-0.3% tea polyphenols for 10-15 min to obtain a pretreated scion. Before grafting, store the pretreated scion at a temperature of -5-0 °C. Step 2: Select 2-year-old seedlings with a ground diameter of 8-12 mm as rootstocks, make a transverse cut to form a top cross-section, make a vertical cut downward from the top cross-section, and make an inclined cut downward on the outside of the rootstock. The vertical cut and the inclined cut form a chimeric incision matching the scion. After the cutting is completed, apply the composite antibacterial solution to the surface of the incision once every 3 minutes, and apply it four times. Step 3: Embed the pretreated scion into the rootstock incision and tie it with a binding band. Spray a hydrogel containing 0.05%-0.1% silver nanoparticles on the outer surface of the grafting part after tying. The spraying amount is 0.02-0.03 ml per square centimeter of the grafting surface. Keep the binding band until the scion and the rootstock heal.

2. The method for grafting the same variety of Pinus gerardiana Wall. in Tibet as claimed in claim 1, characterized in that, In Step 1, the specific method for treating the scion in a sealed container is as follows: Place a porous ceramic adsorbent loaded with zinc oxide nanoparticles in the sealed container. The content of zinc oxide nanoparticles in the adsorbent is 3 wt%, and the surface is coated with 0.8% of polydopamine film. The mass ratio of the adsorbent to the scion is 1:

12. During ventilation, inject a gas stream containing 0.3 ppm of ethyl cyclopropene carboxylate into the container. The gas stream is carried by a mixed gas of nitrogen and carbon dioxide with a volume ratio of 20:

1. The injection time for each ventilation is 4-6 min. During ventilation, synchronously perform light regulation, and intermittently irradiate the wedge surface of the scion with red light with a wavelength of 660 nm. The light intensity is 10-15 μmol / (m²·s), and irradiate for 2-3 min each time ventilation is carried out. After the treatment in the sealed container, immerse the wedge surface of the scion in a mixed solution containing 0.01% salicylic acid and 0.005% proline for 10-15 s, drain it, store it at 0-5 °C for 1 h, and then put it into the composite antibacterial solution.

3. The method for grafting the same variety of Pinus gerardiana Wall. as claimed in claim 1, wherein In Step 1, before soaking in the composite antibacterial solution, first immerse the wedge surface of the scion in a pretreatment solution containing 0.5-1 U / mL of laccase and 0.05%-0.1% of pectin lyase, and treat it at 35-38 °C for 8-10 min.

4. The method for grafting the same variety of Pinus gerardiana Wall. as described in claim 3, characterized in that, In Step 1, the chitosan in the composite antibacterial solution is a cationic nanofiber modified by succinic anhydride. The tea polyphenols are complexed through β-cyclodextrin inclusion to form a complex with a particle size of <10 nm, and 0.005% of subtilin is added to the composite antibacterial solution. During the soaking with the composite antibacterial solution, apply low-frequency ultrasonic waves of 40 - 50 kHz, control the sound intensity at 0.5 W / cm², continue for 3 min and then intermittently for 2 min, cycle 3 times, and finally rinse with deionized water to obtain the pretreated ear shoots.

5. The method for grafting the same variety of Pinus gerardiana Wall. in Tibet as claimed in claim 1, characterized in that, In step three, the preparation method of the hydrogel containing 0.05% - 0.1% silver nanoparticles: Mix sodium carboxymethylcellulose and sodium alginate at a mass ratio of 3:2, dissolve them in deionized water to prepare a 2% mixed solution, add silver nanoparticles, 0.3% bamboo charcoal fibers and 0.15% polyethylene glycol; stir at 60 °C and 200 r / min for 2 hours, after cooling to room temperature, add 1% calcium chloride solution to crosslink and form a hydrogel; The spraying is carried out in two times. The first time is completed within 10 min after grafting, and the second spraying is carried out 30 min after the first spraying; after the second spraying, irradiate with ultraviolet light of wavelength 365 nm for 10 - 15 s.

6. The method for grafting the same variety of Pinus gerardiana Wall. as claimed in claim 5, characterized in that, The silver nanoparticles are pretreated. The pretreatment method is: Disperse the silver nanoparticles in deionized water containing 0.2% polyvinylpyrrolidone, ultrasonically treat for 20 min, then add 0.5% propyl gallate based on the mass of the silver nanoparticles for surface modification, stir at 40 °C for 1 h and then centrifuge to obtain silver nanoparticles with an antioxidant layer on the surface; The bamboo charcoal fibers are pretreated. The pretreatment method is: First soak the bamboo charcoal fibers in 3% hydrogen peroxide solution for 30 min, and then load 3% zinc oxide nanoparticles based on its mass on the surface of the bamboo charcoal fibers to form a coating layer through dopamine polymerization.

7. The grafting method of the same variety of Pinus gerardiana Wall. as claimed in claim 1, characterized in that, In step two, before the first brushing of the composite antibacterial solution, first brush a layer of extract. The extract includes 2% aloe polysaccharide, 0.01% indolebutyric acid and 0.3% nano-diatomite by mass fraction; After an interval of 3 min, brush the composite antibacterial solution again. After brushing the last layer of the composite antibacterial solution, brush a layer of protective solution after an interval of 10 min. The protective solution includes 0.1% lysozyme, 0.1 houttuynin and 0.003% silver nitrate by mass fraction.

8. The method for grafting the same variety of Pinus gerardiana Wall. in Tibet as claimed in claim 1, characterized in that, In step three, after grafting, the cultivation method is: On the 3rd - 5th day after grafting, spray the wound healing promoting solution around the binding band every day. The wound healing promoting solution includes 0.3% chitosan oligosaccharide, 0.05% proline and 0.01% zinc sulfate by mass fraction, and the osmotic pressure is adjusted to 1250 mOsm / kg; On the 6th - 12th day after grafting, spray the vascular bundle induction solution around the binding band every day. The vascular bundle induction solution includes 0.8% potassium dihydrogen phosphate, 0.003% sodium naphthylacetate and 0.1% zinc oxide nanoparticles by mass fraction, and the osmotic pressure is increased to 350 - 450 mOsm / kg, and the spraying frequency is adjusted to once every 2 days; From the 13th day after grafting until the scion and rootstock are healed, a growth balance nutrient solution is sprayed on the plants every three days. The growth balance nutrient solution includes 0.3% potassium dihydrogen phosphate, 0.1% calcium nitrate, and 0.05% zinc sulfate. At the same time, the grafting site is irradiated alternately with blue light of wavelength 450 nm and red light of wavelength 660 nm in the evening, with a light intensity of 8 - 12 μmol / (m²・s), and each type of light is irradiated for 15 - 20 minutes.

9. The method for grafting the same variety of Pinus gerardiana Wall. in Tibet as claimed in claim 8, characterized in that, A 0.03% pH-responsive slow-release microsphere is added to both the healing promotion liquid and the vascular bundle induction liquid. The pH-responsive slow-release microsphere includes 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, the nanofiber is formed by cross-linking poly(lactic-co-glycolic acid) and sodium lignosulfonate, and the surface of the nanofiber is grafted with 0.1 - 0.5% by mass of catalase. The inner core is a slow-release unit containing a nanoscale montmorillonite carrier, which adsorbs zinc ions and borate ions through ion exchange, and 5 - 8% by mass of 2,4-epibrassinolide is intercalated between the montmorillonite layers.

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