Method for grafting Tibetan white bark pine on Chinese pine

Abstract

The invention provides a method for grafting Tibetan white bark pine on Chinese pine, belongs to the technical field of needle-leaved tree species grafting, and solves the technical problem that the survival rate is lower than 40% due to slow joint healing, high pathogen infection rate and environmental fluctuation. According to the technical scheme, the method is characterized by comprising the following steps: cutting 1.8-2.2 cm of a vertical notch reaching xylem at the part, which is 2.5-3.5 cm exposed out of the surface of a culture medium, of a rootstock rhizome part; cutting the scion base into a symmetrical double-sided wedge shape, and immersing the scion base into a sodium citrate buffer solution containing polyvinylpyrrolidone, methyl salicylate and calcium chloride; the two sides of the vertical notch are coated with adsorption glue; inserting a scion alignment cambium and fixing by using a grafting belt; an ethanol water solution containing salicylic acid, trehalose and polysorbate 80 is applied to the interface every 2-3 days; maintaining the day temperature to be 23-25 DEG C, the night temperature to be 16-18 DEG C and the humidity to be 90-95%; when the young sprouts grow to 2-3 cm, the rootstocks are girdled, and trunks are cut off after 7-10 days. The method is used for large-scale breeding of the Tibetan pinus bungeana grafted seedlings.

Description

Technical Field

[0001] This invention relates to the field of tree grafting technology. More specifically, this invention relates to a method for grafting Pinus tabuliformis onto Pinus bungeana. Background Technology

[0002] Tibetan white pine (Pinus gerardiana Wall. ex D. Don) is an endangered species with high economic value. Its native distribution area is narrow, and low temperature and drought stress result in an artificial introduction survival rate of less than 20%. By grafting onto Pinus tabuliformis rootstock, its planting range can be expanded to the high-altitude and cold mountainous areas of western China with an average annual temperature of ≥-5℃, creating economic benefits for local farmers from the pine cone industry.

[0003] Grafting Tibetan white pine onto Chinese pine rootstock is an important method for cultivating stress-resistant seedlings, but the survival rate of this grafting combination has long been low. The cambium cell activity of Chinese pine and Tibetan white pine differs significantly, making it difficult to quickly initiate callus differentiation after contact. After the rootstock cut is exposed, resin secretion covers the cambium, hindering cell contact. The inherently rapid lignification process of pines means that if vascular connections are not established quickly, the cut is easily isolated by newly formed xylem, leading to healing failure. Traditional methods relying on a single growth regulator are insufficient to coordinate the cell division cycles of both parents.

[0004] The microenvironment created by grafting is prone to the growth of Fusarium and Phytophthora, pathogens that quickly block vascular bundles after entering through the incision. Conventional protective measures lack sufficient aeration, which exacerbates the proliferation of anaerobic bacteria. Spraying broad-spectrum fungicides inhibits callus cell division, causing secondary damage. The temperature and humidity response thresholds of Pinus tabuliformis and Pinus bungeana differ: Pinus tabuliformis requires higher daytime temperatures for cambium division, while Pinus bungeana requires lower nighttime temperatures to reduce respiration. During open-air grafting, excessive diurnal temperature fluctuations interrupt callus proliferation; excessively low humidity causes the scion to lose water faster than the rootstock can supply water, while excessively high humidity promotes pathogen spore germination. Current techniques struggle to balance these three factors, resulting in a survival rate often below 40%.

[0005] Substances that enhance cell activity accelerate resin secretion, while resin-inhibiting agents toxically damage the cambium layer. A single agent cannot simultaneously meet the needs of promoting healing and inhibiting bacteria. Activated carbon adsorbs resin but hinders oxygen exchange; breathable materials have poor antibacterial effects, while airtight materials lead to oxygen deficiency. Existing grafting strips cannot simultaneously isolate pathogens and maintain microenvironmental gas exchange. Greenhouse temperature control consumes too much energy, and open-air grafting cannot accurately respond to instantaneous temperature and humidity changes. Intermittent spraying can only maintain local humidity and is insufficient to stabilize the microenvironment in the graft union area.

[0006] These shortcomings have limited the large-scale application of the grafting technique of Pinus tabuliformis onto Pinus bungeana in afforestation projects. Summary of the Invention

[0007] This invention provides a method for grafting Pinus tabuliformis onto Pinus bungeana (Tibetan White Pine). The method employs a composite buffer solution to pretreat the scion, coordinating the cell division cycles of both parents, accelerating callus initiation, and overcoming the barrier of resin secretion to cambium contact. An interface protective layer with both resin adsorption and permeability is designed to block pathogen invasion pathways while maintaining microenvironmental gas exchange, avoiding the risk of anaerobic bacterial growth associated with traditional protective methods. A combination of temperature and humidity parameters and a periodic nutrient supplementation plan are developed to balance the different diurnal temperature requirements of Pinus tabuliformis and Pinus bungeana (Tibetan White Pine), stabilizing callus proliferation conditions. Ultimately, this method aims to improve grafting survival rates to a level suitable for large-scale application.

[0008] This addresses the issues of slow interface healing, susceptibility to infection, and unstable survival rates.

[0009] To address the problem of oxidative stress damage on the cut surface of scions.

[0010] This addresses the problem of delayed healing caused by insufficient scion cell activity.

[0011] To address the issues of water loss and pathogen invasion at the cut surface of rootstock.

[0012] To address the problem of slow callus differentiation on the cut surface of rootstock.

[0013] To address the lack of pine-specific healing factors.

[0014] To address the problem of transpiration imbalance in scions during the shading transition period.

[0015] To address the problem of poor physiological compatibility between rootstock and scion.

[0016] This addresses the issues of resin blockage in the cambium layer and trace element deficiency.

[0017] To address the aforementioned problems and achieve the objectives and other advantages of this invention, a method for grafting Pinus tabuliformis onto Pinus bungeana is provided, comprising the following steps: Make a vertical cut 1.8-2.2 cm deep, reaching the xylem, at a point 2.5-3.5 cm above the surface of the cultivation substrate at the rootstock rhizome. The base of the scion is cut into a symmetrical double-sided wedge shape, with a cut length of 1.5-2.0 cm; Immerse the cut surface of the scion in a sodium citrate buffer solution containing 4-6% polyvinylpyrrolidone, 0.1-0.2% methyl salicylate, and 0.05-0.08% calcium chloride for 60-90 seconds. The concentration of the sodium citrate buffer solution is 0.04-0.06 mol / L, and the pH value is 6.0-6.8. The adsorbent is coated on both sides of the vertical cut. The adsorbent is prepared by mixing activated carbon powder with a particle size of 40-50 mesh and hydroxyethyl cellulose solution with a mass fraction of 8-12% at a mass ratio of 1:2.5-3.5, and then adding nano-silica equivalent to 0.3-0.5% of the activated carbon powder mass, and stirring evenly. Insert the scion into the vertical cut of the rootstock, align the cambium layers, and then fix the interface with grafting tape; Apply an aqueous solution of ethanol containing 0.7-0.9% salicylic acid, 1-3% trehalose, and 0.2-0.4% polysorbate 80 (45-55% by volume) to the interface area every 2-3 days, at a rate of 0.05-0.15 mL per application. Maintain a daytime temperature of 23-25℃, a nighttime temperature of 16-18℃, and a humidity of 90-95%. When the new shoots of the scion are 2-3cm long, make a ring cut on the main trunk of the rootstock 0.8-1.2cm above the vertical cut, reaching the xylem. Cut off the main trunk above the ring cut 7-10 days later. After grafting, the graft should be shaded.

[0018] Preferably, in the method of grafting Pinus tabuliformis onto Pinus thunbergii, the cut surface of the scion is soaked in sodium citrate buffer solution, and then the base of the scion is wrapped with a breathable film for 8-10 minutes. After that, the film is removed and the grafting tape is used for fixation.

[0019] Preferably, in the method of grafting Pinus tabuliformis onto Pinus bungeana, before inserting the scion into the vertical cut of the rootstock, the cut surface of the scion is evenly sprayed with a naphthaleneacetic acid solution with a mass fraction of 0.02-0.05%, and left to stand for 30-60 seconds before proceeding with subsequent operations.

[0020] Preferably, in the method of grafting Pinus tabuliformis onto Pinus bungeana, after cutting the main trunk above the girdling point, a composite protective agent is immediately applied to the cut surface of the rootstock with a thickness of 0.5-1.0 mm, and left to stand for 2-4 minutes until a gel film forms on the surface. The composite protective agent is prepared by mixing the following components in the following mass percentages: 0.3-0.5% humic acid, 0.1-0.2% chitosan, 0.01-0.03% nano zinc oxide, and the balance being deionized water; The preparation method of the composite protective agent is as follows: humic acid and deionized water are stirred and dissolved at 50-60℃, then chitosan is added and stirred until completely dissolved, and finally nano zinc oxide is added and ultrasonically dispersed for 20-30 minutes.

[0021] Preferably, in the method for grafting Pinus tabuliformis onto Pinus tabuliformis, after applying a composite protective agent to the cut surface of the rootstock until a gel film forms on the surface, a phosphate buffer containing 0.1-0.3% indolebutyric acid, 0.05-0.1% gibberellin, and 0.01-0.03% vitamin B1 by mass fraction is sprayed at a rate of 0.1-0.2 mL / cm³. 2 The concentration of the phosphate buffer is 0.05-0.1 mol / L, and the pH value is 6.0-6.5.

[0022] Preferably, in the method for grafting Pinus tabuliformis onto Pinus bungeana, the sodium citrate buffer solution further contains 0.002-0.005% by mass of pine polypeptide extract and 0.001-0.003% by mass of copper pinoresinate, wherein the purity of copper pinoresinate is ≥95%. Pine polypeptide extract was prepared by the following method: Phloem tissue from healthy Tibetan white pine trees of the current year was collected, flash-frozen in liquid nitrogen, and then ground to a particle size ≤0.5mm. Add pre-cooled Tris-HCl buffer with a pH of 7.2-7.4 at a mass ratio of 1:8-10. The Tris-HCl buffer contains 0.1 mol / L NaCl and 5 mmol / L EDTA. Perform ultrasonic-assisted extraction at 4°C with an ultrasonic power of 300W. Use a pulse mode with 5 seconds of operation followed by 10 seconds of intermittent operation. The total extraction time is 30 min. Centrifugation was performed at 12,000 rpm for 15 min at 4°C. The supernatant was then fractionated and passed through 10 kDa and 3 kDa ultrafiltration membranes. The 3-10 kDa fraction was desalted using a Sephadex G-25 column and then lyophilized to obtain a pine polypeptide extract.

[0023] Preferably, in the method for grafting Pinus tabuliformis onto Pinus bungeana, the specific method for shading is as follows: Cover the grafted seedlings with a shade net that has a light transmittance of 30-40% for 7-10 days, and then adjust the light exposure in stages: a) Replace with a shade net with a light transmittance of 50-60% on days 1-3; b) From day 4 onwards, completely remove the shade netting. Spray the interface area with atomized water once daily from 10:00 to 14:00, at a rate of 0.1-0.2 mL / cm². 2 Continue until the new shoots of the scion reach a length of 5-6 cm, then stop.

[0024] Preferably, in the method of grafting Pinus tabuliformis onto Pinus bungeana, Select Pinus tabuliformis seedlings with a diameter of 0.4-0.7cm and a height of 1-1.5m as rootstock; Select terminal branches of mature Tibetan white pine trees with a diameter of 0.4-0.7cm as scions, and cut 8-12cm sections of branches; The grafting tape is a blue polyethylene film with a light transmittance of 60-70% and a thickness of 0.03-0.05mm.

[0025] Preferably, in the method of grafting Pinus tabuliformis onto Pinus thunbergii, before applying the ethanol aqueous solution, a sterile degreased cotton swab is used to wipe along the direction of the interface cambium layer in one direction to remove the exuded resin, and the swab is left to stand for 8-10 minutes after wiping. When applying the ethanol aqueous solution, simultaneously apply a trace element solution containing 0.01-0.03% manganese sulfate and 0.005-0.01% zinc sulfate by mass, at a rate of 0.02-0.05 mL per application.

[0026] The present invention has at least the following beneficial effects: This invention significantly improves the cell affinity between rootstock and scion by pretreating the scion with a composite buffer solution, effectively solving the problem of asynchronous healing caused by physiological differences between rootstock and scion in traditional grafting. The synergistic effect of specific components in the buffer solution protects the cell activity of the scion and promotes rapid callus formation.

[0027] The adsorbent designed in this invention combines resin adsorption and air permeability, maintaining oxygen exchange in the interface area while blocking pathogen invasion. This dual-functional design overcomes the hypoxia problem caused by traditional sealing materials, creating an ideal growth environment for callus tissue.

[0028] This invention precisely controls diurnal temperature variation and humidity to meet the cell division needs of Pinus tabuliformis while reducing the respiration consumption of Pinus bungeana. This environmental control scheme effectively balances the different growth requirements of the two plants, significantly improving the grafting success rate.

[0029] This invention utilizes a composite protective agent applied after severing the rootstock trunk. Through the synergistic effect of multiple active ingredients, it both protects the cut surface and promotes healing. This multifunctional protective solution solves the problem that traditional single-component sealing materials cannot simultaneously provide protection and promote healing.

[0030] The present invention employs a phased adjustment strategy in shading treatment, combined with precise atomized water spraying, to prevent both photoinhibition of the scion and dehydration damage. This dynamic control method overcomes the shortcomings of traditional fixed shading methods, which cannot adapt to the needs of different healing stages.

[0031] This invention significantly enhances the ability of rootstock and scion cells to recognize and synthesize lignin by adding specific active ingredients from Tibetan white pine. This species-specific design solves the problem of traditional methods lacking targeted active substances.

[0032] The grafting tape designed in this invention, while ensuring fixed strength, protects the graft union and maintains necessary photosynthesis through specific light transmittance and spectral characteristics. This functional design overcomes the shortcomings of traditional grafting tapes with only one function.

[0033] This invention effectively solves the problem of vascular connectivity obstruction caused by resin blockage through synergistic treatment of resin removal and trace element supplementation. This comprehensive treatment scheme overcomes the shortcomings of traditional methods that neglect the metabolic balance of the interface area.

[0034] The standardized operating system established in this invention achieves a stable increase in grafting survival rate through precise control of parameters at each stage. This systematic approach provides a reliable technical guarantee for large-scale application.

[0035] 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. Detailed Implementation

[0036] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0037] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0038] This invention provides a method for grafting Pinus tabuliformis onto Pinus bungeana, comprising the following steps: Make a vertical cut 1.8-2.2 cm deep, reaching the xylem, at a point 2.5-3.5 cm above the surface of the cultivation substrate at the rootstock rhizome. The base of the scion is cut into a symmetrical double-sided wedge shape, with a cut length of 1.5-2.0 cm; Immerse the cut surface of the scion in a sodium citrate buffer solution containing 4-6% polyvinylpyrrolidone, 0.1-0.2% methyl salicylate, and 0.05-0.08% calcium chloride for 60-90 seconds. The concentration of the sodium citrate buffer solution is 0.04-0.06 mol / L, and the pH value is 6.0-6.8. The adsorbent is coated on both sides of the vertical cut. The adsorbent is prepared by mixing activated carbon powder with a particle size of 40-50 mesh and hydroxyethyl cellulose solution with a mass fraction of 8-12% at a mass ratio of 1:2.5-3.5, and then adding nano-silica equivalent to 0.3-0.5% of the activated carbon powder mass, and stirring evenly. Insert the scion into the vertical cut of the rootstock, align the cambium layers, and then fix the interface with grafting tape; Apply an aqueous solution of ethanol containing 0.7-0.9% salicylic acid, 1-3% trehalose, and 0.2-0.4% polysorbate 80 (45-55% by volume) to the interface area every 2-3 days, at a rate of 0.05-0.15 mL per application. Maintain a daytime temperature of 23-25℃, a nighttime temperature of 16-18℃, and a humidity of 90-95%. When the new shoots of the scion are 2-3cm long, make a ring cut on the main trunk of the rootstock 0.8-1.2cm above the vertical cut, reaching the xylem. Cut off the main trunk above the ring cut 7-10 days later. After grafting, the graft should be shaded.

[0039] Traditional methods for grafting Tibetan white pine onto Chinese pine have significant technical flaws. The callus formation on the rootstock and scion is asynchronous, leading to the scion's death due to insufficient nutrient supply and dehydration. Moisture control at the graft union is poor; traditional grafting tape or paraffin sealing provides poor aeration, inhibiting callus respiration, or excessive aeration fails to effectively retain moisture. Nutrient and oxygen supply are imbalanced; traditional methods do not optimize local nutrient supply to the graft union, high-concentration ethanol solutions damage new cells, and low-concentration solutions are insufficient for antibacterial effects, delaying xylem healing due to oxygen deficiency.

[0040] The method for grafting Pinus tabuliformis onto Pinus bungeana provided in this solution includes the following steps: Rootstock and scion selection: Select Pinus tabuliformis seedlings with a diameter of 0.4-0.7cm and a height of 1-1.5m as rootstock; select terminal branches of mature Pinus bungeana with a diameter of 0.4-0.7cm, cut 8-12cm branches as scions, and ensure that the scions have sufficient meristematic activity.

[0041] Rootstock treatment: Make a vertical cut (1.8-2.2 cm long) at the base of the rhizome, 2.5-3.5 cm above the substrate surface, reaching the xylem. This height prevents substrate pathogens from contaminating the cut, and the length of the vertical cut provides sufficient cambium contact surface.

[0042] Scion pretreatment: Cut the base of the scion into a symmetrical double-sided wedge shape (cut surface length 1.5-2.0 cm) to increase the contact area between the rootstock and scion. Immerse in a sodium citrate buffer solution (0.04-0.06 mol / L, pH 6.4-6.6) containing 4-6% polyvinylpyrrolidone (promotes cell adhesion), 0.1-0.2% methyl salicylate (inhibits pathogens), and 0.05-0.08% calcium chloride (enhances cell wall stability) for 60-90 seconds to synergistically improve the scion's stress resistance and activate callus differentiation.

[0043] Interface protection: The vertical cut is coated with an adsorbent adhesive made of 40-50 mesh activated carbon powder (adsorbent and exudative resin) and 8-12% hydroxyethyl cellulose solution (adhesive) at a ratio of 1:2.5-3.5, with 0.3-0.5% nano silica (to enhance air permeability) added, forming a physical barrier to block pathogen invasion while maintaining oxygen exchange.

[0044] Grafting fixation: The scion is inserted and the cambium layers are strictly aligned to ensure effective connection of the vascular tissues of the rootstock and scion; the blue grafting tape with a light transmittance of 60-70% provides moderate shading to reduce transpiration while allowing photosynthetic signal substances to penetrate.

[0045] Postoperative management: Nutritional supplementation: Apply 0.05-0.15 mL of ethanol aqueous solution with a volume fraction of 45-55% every 2-3 days, containing 0.7-0.9% salicylic acid (to induce disease resistance), 1-3% trehalose (permeability protectant), and 0.2-0.4% polysorbate 80 (to promote absorption), to maintain cell viability in the interface area. Environmental control: Daytime temperature 23-25℃ (promotes cell division in Pinus tabuliformis) combined with nighttime temperature 16-18℃ (reduces respiration consumption in Pinus bungeana), humidity 90-95% (prevents water loss from scions and inhibits oxidation of the cut). Physiological intervention: When the new shoots are 2-3cm long, girdle the rootstock (deep into the xylem) to block the nutrient return. After 7-10 days, cut off the main stem above the girdled area to force the rootstock to concentrate nutrients to the joint for healing.

[0046] After grafting, the graft should be shaded.

[0047] The significance and economic value of grafting Tibetan white pine onto Chinese pine: 1) Overcoming ecological limitations and expanding planting areas Tibetan white pine is native to the high-altitude (3000-4000 meters) regions of the Himalayas and has poor adaptability to cold and arid environments. Pinus tabuliformis, as rootstock, possesses extremely strong cold resistance (tolerating temperatures as low as -30℃) and tolerance to poor soil. Grafting allows Tibetan white pine to be promoted and planted in the cold and semi-arid regions of western China (such as Qinghai, western Sichuan, and Longnan, at altitudes of 2000-3000 meters), expanding its suitable habitat.

[0048] 2) Significantly enhance economic value Economic benefits of pine cones: Tibetan white pine cones weigh 15-20g each (3 times that of common pine), with an oil content of over 60%, rich in unsaturated fatty acids and antioxidants, fetching a market price of over 80 yuan / kg. Grafted seedlings reach full production after 5 years, yielding 300-400 kg of pine cones per mu (approximately 0.16 acres), resulting in economic benefits far exceeding those of ordinary economic forests.

[0049] Ecological industry integration: Promoting planting in high-altitude and cold regions can develop industries such as pine cone picking and ecotourism.

[0050] 3) Ecological restoration and carbon sequestration gains Grafted seedlings inherit the stress resistance of Pinus tabuliformis and can grow in arid and saline-alkali mountain areas, making them suitable for ecological restoration of degraded mountain areas.

[0051] The carbon sequestration capacity of Tibetan white pine is 1.8 times that of Chinese pine, and large-scale planting is of great significance for carbon sequestration and emission reduction in high-altitude and cold regions.

[0052] 4) Protect the genetic resources of endangered species Tibetan white pine is listed as an endangered tree species in China. Grafting and propagation can achieve large-scale preservation of germplasm resources and avoid ecological damage caused by excessive seed collection in the native area.

[0053] This invention enables large-scale grafting and seedling cultivation, lowering the minimum planting altitude for Tibetan white pine to 2500 meters, providing a new income source for high-altitude and cold regions in western China such as Qinghai and Sichuan. The pine cones produced from grafted seedlings are of comparable quality to those of the native species, with a yield value of over 24,000 yuan per mu.

[0054] Example 1 Fifty seedlings of Pinus tabuliformis with a diameter of 0.5 cm and a height of 1.2 m were selected as rootstocks. Fifty terminal branches from mature Pinus bungeana trees (0.5 cm in diameter) were selected as scions, with 10 cm sections cut off. A 2 cm long vertical cut was made at the rootstock, extending 3 cm above the surface of the growing medium, reaching the xylem. The base of the scion was shaped into a symmetrical double-sided wedge, with a cut surface length of 1.8 cm. The cut surfaces of the scions were immersed in a 0.05 mol / L sodium citrate buffer solution containing 5% polyvinylpyrrolidone, 0.15% methyl salicylate, and 0.06% calcium chloride for 75 seconds (pH 6.5). An absorbent adhesive was coated on both sides of the vertical cut. The adhesive was made by mixing 45-mesh activated carbon powder and a 10% hydroxyethyl cellulose solution at a mass ratio of 1:3, and then adding 0.4% nano-silica (equivalent to the mass of the activated carbon powder). Insert the scion into the vertical incision of the rootstock, align the cambium layers, and secure the graft union with grafting tape. Every 36 hours, apply 0.1 mL of a 50% ethanol solution containing 0.8% salicylic acid, 2% trehalose, and 0.3% polysorbate 80 to the graft union area. Maintain a daytime temperature of 24℃, a nighttime temperature of 17℃, and a humidity of 93%. When the scion shoot reaches 2.5 cm in length, make a girdling cut on the rootstock trunk 1 cm above the vertical incision, reaching the xylem. Cut off the trunk above the girdling cut after 8 days. After 30 days, the results showed that 46 out of 50 grafted seedlings survived, a survival rate of 92%. The average length of the scion shoot was 6.2 cm, and the callus tissue at the graft union had an 88% lignification rate. No fungal infection or graft union cracking was observed.

[0055] Comparative Example 1 To address the asynchronous callus formation between the rootstock and scion, methyl salicylate and calcium chloride were removed from the buffer solution, retaining only polyvinylpyrrolidone (PVP). All other procedures remained identical to those in Example 1. After 30 days, statistical results showed that 34 out of 50 grafted seedlings survived, a survival rate of 68%. The average length of the new shoots from the scion was 4.5 cm, and the lignification rate of the callus at the graft union was only 60%. Furthermore, the scions showed significant wilting due to insufficient nutrient supply. This demonstrates that methyl salicylate and calcium chloride play a crucial role in coordinating the callus formation rate between the rootstock and scion.

[0056] Comparative Example 2 To address the issue of moisture control in the graft union area, traditional paraffin wax was used to completely seal the area, eliminating the need for absorbent adhesive treatment. All other procedures were the same as in Example 1. After 30 days, statistical results showed that only 18 out of 50 grafted seedlings survived, a survival rate of 36%. The average length of the new shoots from the scions was 3.0 cm, and numerous water-soaked lesions appeared at the graft union. This indicates that paraffin wax sealing cannot balance the needs for breathability and moisture retention.

[0057] Comparative Example 3 To address the imbalance between nutrient and oxygen supply, the application of the ethanol-water solution was discontinued, and distilled water was used instead. The remaining procedures were the same as in Example 1. After 30 days, the results showed that 29 out of 50 grafted seedlings survived, a survival rate of 58%. The average length of the new shoots from the scions was 4.2 cm, and the xylem connection was incomplete. This confirms that the antibacterial function and nutrient supplementation of the ethanol-water solution are indispensable for graft union healing.

[0058] Results: Example 1 solved three major challenges—synchronous healing of rootstock and scion, regulation of the interface microenvironment, and nutrient supply—through a composite technical solution. Comparative Examples 1-3 respectively verified the irreplaceable role of buffer components, adsorbent gel functions, and ethanol-water solutions in solving these problems.

[0059] In another embodiment, the method of grafting Pinus tabuliformis onto Pinus thunbergii involves soaking the cut surface of the scion in sodium citrate buffer solution, wrapping the base of the scion with a breathable film for 8-10 minutes, removing the film, and then fixing it with a grafting tape.

[0060] In traditional Pinus tabuliformis grafting methods, the scion is directly fixed with the grafting tape after the cut surface is dipped in buffer solution, without any transitional protection for the treated scion. Current techniques typically overlook the cell dehydration caused by exposure of the scion cut surface after buffer treatment, and do not consider the short-term moisture retention requirements of the scion after dipping. In routine operations, the scion is fixed immediately after being removed from the buffer solution, leading to rapid evaporation of moisture from the cut surface, decreased cell activity, and impaired callus formation.

[0061] This method involves soaking the scion in buffer solution, then wrapping the base of the scion with a breathable film for 8-10 minutes before removing the film for fixation. Existing techniques that directly fix the graft union cannot address the issue of moisture retention during the scion's transition period. The breathable film wrapping prevents excessive moisture evaporation while also preventing oxygen deficiency caused by prolonged covering, precisely balancing moisture retention and breathability. The film's light transmittance allows the scion to perform necessary photosynthesis while reducing damage to newly formed cells from direct sunlight.

[0062] This method involves soaking the scion in buffer solution and then wrapping it with a breathable film to address cell dehydration caused by exposed cut surfaces. The 8-10 minute wrapping time ensures full absorption of the buffer solution while preventing oxygen deficiency. After removing the film, the graft union is secured, ensuring the scion is in optimal condition for grafting. This step significantly improves scion cell activity and promotes rapid callus formation.

[0063] In another embodiment, in the method of grafting Pinus tabuliformis onto Pinus bungeana, before inserting the scion into the vertical cut of the rootstock, the cut surface of the scion is evenly sprayed with a naphthaleneacetic acid solution with a mass fraction of 0.02-0.05%, and left to stand for 30-60 seconds before proceeding with subsequent operations.

[0064] In traditional Pinus tabuliformis grafting methods, the scion is directly inserted into the rootstock incision after surface preparation, without pre-treatment with hormones. Current techniques typically rely on the rootstock's own hormone secretion to induce callus formation, leading to asynchronous callus rates between the scion and rootstock. Conventional procedures neglect the crucial influence of scion cell activity on early-stage healing, often resulting in delayed callus initiation due to insufficient hormone stimulation, increasing the risk of dehydration.

[0065] This method involves spraying the cut surface of the scion with a 0.02-0.05% (w / w) naphthaleneacetic acid (NAA) solution before inserting the scion into the rootstock and allowing it to stand for 30-60 seconds. Current techniques lack this step; the scion is only treated with a buffer solution, resulting in limited hormonal effects. NAA, as an auxin, directly activates cell division on the scion's cut surface, synchronizing it with the callus process in the rootstock. The standing time ensures sufficient hormone penetration without overstimulation, preventing abnormal cell proliferation.

[0066] The purpose of spraying naphthaleneacetic acid (NAA) solution in this procedure is to initiate the callus process of the scion in advance, resolving the issue of asynchronous healing between the scion and rootstock. A 30-60 second static incubation period balances hormone absorption and cellular response rates, preventing tissue damage due to excessive treatment time. This step ensures that the scion possesses highly active callus cells before insertion into the rootstock, shortening the post-grafting healing cycle.

[0067] In another embodiment, in the method of grafting Pinus tabuliformis onto Pinus bungeana, after cutting the main trunk above the girdling point, immediately apply a composite protective agent to the cut surface of the rootstock with a thickness of 0.5-1.0 mm, and let it stand for 2-4 minutes until a gel film forms on the surface. The composite protective agent is prepared by mixing the following components in the following mass percentages: 0.3-0.5% humic acid, 0.1-0.2% chitosan, 0.01-0.03% nano zinc oxide, and the balance being deionized water; The preparation method of the composite protective agent is as follows: humic acid and deionized water are stirred and dissolved at 50-60℃, then chitosan is added and stirred until completely dissolved, and finally nano zinc oxide is added and ultrasonically dispersed for 20-30 minutes.

[0068] Traditional Pinus tabuliformis grafting methods typically involve sealing the cut surface with paraffin or ordinary grafting wax after severing the rootstock trunk, lacking targeted protection and healing-promoting measures. Current techniques primarily focus on physical sealing of the cut surface, neglecting active regulation of callus formation. The sealing materials used in conventional methods have poor air permeability, easily leading to internal oxygen deficiency, and lack antibacterial and cell division-promoting functions, thus affecting the healing speed of the rootstock cut surface and the subsequent growth of the scion.

[0069] This method involves immediately applying a composite protective agent to the cut surface after severing the rootstock trunk, forming a breathable and bioactive gel film. Existing paraffin sealing techniques only provide a physical barrier and cannot address oxygen deficiency and pathogen infection. The humic acid and chitosan in the composite protective agent work synergistically to maintain humidity while inhibiting pathogens, and nano-zinc oxide further enhances the antibacterial effect. Allowing the mixture to stand for 2-4 minutes allows the gel film to adhere stably, creating conditions for subsequent spraying of the hormone solution.

[0070] This composite protective agent application method aims to provide multifunctional protection for the cut surface of the rootstock. Humic acid promotes callus cell division, chitosan inhibits bacterial growth, and nano-zinc oxide enhances antibacterial properties. The breathability of the gel membrane avoids the oxygen deficiency problem of traditional sealing materials. Allowing it to stand for 2-4 minutes ensures the gel membrane forms a stable structure, protecting the cut surface without interfering with subsequent operations. Compared to traditional methods, this step significantly improves the healing speed of the rootstock cut surface and reduces the risk of infection.

[0071] Example 2 Fifty *Pinus tabuliformis* seedlings with a diameter of 0.5 cm and a height of 1.2 m were selected as rootstocks and grafted with *Pinus bungeana* var. *tibetica* following the steps in Example 1. After cutting the main trunk above the girdled area, a compound protective agent (0.4% humic acid, 0.15% chitosan, 0.02% nano-zinc oxide, with the remainder being deionized water) was immediately applied to the cut surface to a thickness of 0.8 mm and allowed to stand for 3 minutes to form a gel film. Results after 30 days showed that the rootstock cut surface healed completely in 95% of cases, with no infection or cracking observed, and the average length of the new shoots from the scion was 6.5 cm.

[0072] Comparative Example 4 The rootstock cut surface was sealed with traditional paraffin wax, and the remaining operations were the same as in Example 2. Statistical results after 30 days showed that the rootstock cut surface healing integrity rate was only 60%, 15% of the graft unions suffered callus necrosis due to oxygen deficiency, and the average length of the new shoots from the scion was 4.0 cm.

[0073] Results: Example 2 demonstrated that the multifunctional synergistic effect of the composite protectant achieved efficient healing of the rootstock cut surface. Comparative Example 4 proved that traditional sealing methods cannot meet the dual requirements of breathability and antibacterial properties, significantly affecting the grafting effect.

[0074] In another embodiment, the method for grafting Tibetan white pine onto Chinese pine involves applying a composite protective agent to the cut surface of the rootstock until a gel film forms, followed by spraying with a phosphate buffer containing 0.1-0.3% indolebutyric acid, 0.05-0.1% gibberellin, and 0.01-0.03% vitamin B1 at a spraying rate of 0.1-0.2 mL / cm³. 2 The concentration of the phosphate buffer is 0.05-0.1 mol / L, and the pH value is 6.0-6.5.

[0075] Traditional Pinus tabuliformis grafting methods typically use only a single protective agent (such as paraffin or ordinary grafting wax) to seal the cut surface after severing the rootstock trunk, lacking active regulation of callus tissue. Current techniques primarily rely on physical sealing of the cut surface, without incorporating hormones to promote callus formation, resulting in slow rootstock healing and limited subsequent growth of the scion. The sealing materials used in conventional methods have limited functionality and cannot simultaneously address the multiple needs of antibacterial activity, moisture retention, and cell division activation.

[0076] This method involves spraying a phosphate buffer solution containing indolebutyric acid, gibberellin, and vitamin B1 after the composite protectant has formed a gel film. Existing technologies lack this step and rely solely on the physical barrier effect of the protectant. The composite hormone solution promotes cell division through indolebutyric acid, coordinates growth rhythms through gibberellin, and enhances stress resistance through vitamin B1, forming a functional complement to the physical barrier of the protectant. The phosphate buffer solution maintains pH stability to ensure hormone activity; the spraying rate is 0.1-0.2 mL / cm³. 2 Precise matching of gel membrane porosity enables uniform penetration.

[0077] This treatment involves spraying a hormone solution to activate the callus process on the rootstock cut surface. Indolebutyric acid accelerates callus cell proliferation, gibberellin balances growth rate, and vitamin B1 reduces oxidative damage. The phosphate buffer (pH 6.0-6.5) is compatible with the protective gel, preventing component conflicts. After spraying, the hormone is released slowly through the gel membrane, providing sustained action for 7-10 days, covering the critical callus healing period. Compared to traditional methods, this step shortens the rootstock cut surface healing time by 30% and increases the growth rate of new shoots from the scion by 15%.

[0078] In another embodiment, the method for grafting Pinus tabuliformis onto Pinus bungeana, wherein the sodium citrate buffer solution further contains 0.002-0.005% by mass of pine polypeptide extract and 0.001-0.003% by mass of copper pinoresinate, wherein the purity of copper pinoresinate is ≥95%; Pine polypeptide extract was prepared by the following method: Phloem tissue from healthy Tibetan white pine trees of the current year was collected, flash-frozen in liquid nitrogen, and then ground to a particle size ≤0.5mm. Add pre-cooled Tris-HCl buffer with a pH of 7.2-7.4 at a mass ratio of 1:8-10. The Tris-HCl buffer contains 0.1 mol / L NaCl and 5 mmol / L EDTA. Perform ultrasonic-assisted extraction at 4°C with an ultrasonic power of 300W. Use a pulse mode with 5 seconds of operation followed by 10 seconds of intermittent operation. The total extraction time is 30 min. Centrifugation was performed at 12,000 rpm for 15 min at 4°C. The supernatant was then fractionated and passed through 10 kDa and 3 kDa ultrafiltration membranes. The 3-10 kDa fraction was desalted using a Sephadex G-25 column and then lyophilized to obtain a pine polypeptide extract.

[0079] In traditional Pinus tabuliformis grafting methods, the buffer solution typically contains only basic components such as polyvinylpyrrolidone and preservatives, lacking active substances specific to the affinity between the scion and rootstock. Current technologies use buffer solutions with limited functionality, primarily serving moisturizing and antiseptic purposes, failing to promote cell recognition and fusion between the scion and rootstock. In routine operations, the buffer solution's pH and ion concentration ranges are relatively wide, resulting in insufficient precision in regulating cell activity and leading to slow or asynchronous callus formation.

[0080] This method involves adding pine polypeptide extract and copper pinoresinate to a sodium citrate buffer solution. Existing techniques do not utilize plant-specific active ingredients, while the pine polypeptide extract contains signaling molecules unique to Tibetan white pine, which can enhance rootstock-scion cell recognition and fusion; copper pinoresinate provides sustained-release copper ions, activating oxidases to promote lignin synthesis. The buffer solution pH is precisely controlled between 6.0 and 6.8, matching the optimal range for pine polypeptide activity and avoiding cell damage. The pine polypeptide extraction employs fractional ultrafiltration and desalting processes to ensure the purity and stability of the active ingredients.

[0081] This regimen incorporates pine peptide extract to leverage its bioactivity and promote rootstock-scion cell fusion. Copper pinoresinate ions participate in oxidase activation, accelerating xylem junction formation. A buffer solution with a pH of 6.0-6.8 maintains cell membrane stability, preventing damage to the scion from excessively high or low pH levels. Ultrasonic-assisted extraction and ultrafiltration fractionation ensure that the molecular weight of the pine peptides is concentrated in the 3-10 kDa range, where peptides exhibit optimal healing-promoting activity.

[0082] Example 3 (containing pine polypeptide and copper pinoresinate) Based on Example 1, 0.003% pine polypeptide extract and 0.002% copper pinoresinate (purity ≥95%) were added to the sodium citrate buffer solution, with all other operations remaining identical. After 30 days, the results showed that 48 out of 50 grafted seedlings survived, a survival rate of 96%. The average length of the new shoots from the scions was 6.8 cm, and the integrity rate of the callus tissue at the graft union was 93%. No fungal infection or cracking at the graft union was observed.

[0083] Results: Example 3, based on Example 1, added pine polypeptide extract and copper pinoresinate, increasing the survival rate from 92% to 96%, the average length of the new shoots from scion to 6.2 cm, and the lignification integrity rate of the callus tissue at the graft union from 88% to 93%. The active ingredients in the pine polypeptide extract significantly enhanced the cell recognition and fusion ability between the rootstock and scion, while copper pinoresinate promoted lignin synthesis efficiency through the slow release of copper ions. The synergistic effect of these two added ingredients further optimized the physiological microenvironment of the graft union area, making the callus process more efficient and stable. Traditional methods did not use these active ingredients, resulting in relatively low callus speed and quality; the technical improvements in Example 3 effectively solved this problem.

[0084] In another embodiment, the specific method for shading in the grafting of Pinus tabuliformis onto Pinus bungeana is as follows: Cover the grafted seedlings with a shade net that has a light transmittance of 30-40% for 7-10 days, and then adjust the light exposure in stages: a) Replace with a shade net with a light transmittance of 50-60% on days 1-3; b) From day 4 onwards, completely remove the shade netting. Spray the interface area with atomized water once daily from 10:00 to 14:00, at a rate of 0.1-0.2 mL / cm². 2 Continue until the new shoots of the scion reach a length of 5-6 cm, then stop.

[0085] Traditional pine grafting methods typically employ single-mode shading after grafting, such as using a shade net with a fixed light transmittance throughout the grafting process or not shading at all, lacking dynamic adjustment of light intensity. Existing techniques have the following problems: a fixed shading rate cannot adapt to the light requirements of grafted seedlings at different healing stages; sudden removal of the shade net easily leads to light stress on the scion; and the lack of combined misting with water spraying to regulate the local microenvironment makes the graft union area prone to dehydration during periods of strong light. In traditional methods, shading and water management are disconnected, causing the scion to stagnate or die from dehydration due to sudden environmental changes.

[0086] This method employs a phased shading adjustment combined with atomized water spraying, representing a significant improvement over existing technologies. For the initial 7-10 days, a black shade net with 30-40% light transmittance provides high-intensity shading protection. During the transition period (1-3 days), this is replaced with a white shade net with 50-60% light transmittance to gradually enhance light adaptability. After completely removing the shade net, atomized water spraying is applied daily from 10:00 AM to 2:00 PM to mitigate the risk of dehydration under strong light. Existing technologies lack this dynamic adjustment mechanism; excessive shading inhibits photosynthesis, while insufficient shading leads to scion scorching. This method, however, achieves a balance between light intensity and humidity through the synergy of phased shading and precise spraying.

[0087] This shading treatment method achieves precise control through three stages: For the initial 7-10 days, a shade net with 30-40% light transmittance is used to effectively inhibit transpiration of the scion and protect newly formed callus tissue from strong light damage; during the transition period of 1-3 days, the shade net is replaced with one with 50-60% light transmittance to gradually enhance light adaptability and avoid photoinhibition caused by sudden exposure; after completely removing the shade net, atomized water (0.1-0.2 mL / cm³) is sprayed daily from 10:00 to 14:00. 2 This method specifically alleviates the pressure of water evaporation during periods of strong sunlight, continuing until the new shoots reach 5-6 cm in length to ensure a safe transition. By dynamically adjusting the shading rate and combining precise spraying, this method keeps the humidity and light intensity in the interface area within the optimal range, increasing the survival rate by more than 15% compared to traditional fixed shading, and resulting in vigorous new shoot growth without dehydration or scorching.

[0088] In another embodiment, in the method of grafting Pinus tabuliformis onto Pinus bungeana, Pinus tabuliformis seedlings with a diameter of 0.4-0.7 cm and a height of 1-1.5 m are selected as rootstocks; Select terminal branches of mature Tibetan white pine trees with a diameter of 0.4-0.7cm as scions, and cut 8-12cm sections of branches; The grafting tape is a blue polyethylene film with a light transmittance of 60-70% and a thickness of 0.03-0.05mm.

[0089] In traditional Pinus tabuliformis grafting methods, the selection criteria for rootstock and scion are quite broad, usually only considering basic indicators such as diameter and height, lacking precise control over the physiological state of the materials. Existing techniques have the following problems: rootstock diameters that are too large lead to inconsistent cambium thickness, affecting the quality of rootstock-scion contact; scions taken from mature branches have excessive lignification, resulting in insufficient cell division activity; and the selection of grafting strip light transmittance and color is arbitrary, failing to balance fixation strength and photosynthetic requirements.

[0090] This method strictly limits the rootstock to Pinus tabuliformis seedlings with a diameter of 0.4-0.7 cm and a height of 1-1.5 m, ensuring moderate cambium development and ease of operation. The scion is selected from the terminal branches of mature Pinus bungeana trees with a diameter of 0.4-0.7 cm, with 8-12 cm sections cut to ensure apical meristem activity. The grafting tape uses a blue polyethylene film with a light transmittance of 60-70% and a thickness of 0.03-0.05 mm. Existing technologies suffer from poor rootstock-scion diameter matching, excessive lignification of scions from mature trees, and the use of ordinary transparent plastic film for grafting tape, which fails to optimize spectral transmittance.

[0091] Select 0.4-0.7cm diameter seedlings of Pinus tabuliformis as rootstock, with a height of 1-1.5m for easy handling and uniform cambium thickness, improving the contact fit between rootstock and scion. Use terminal branches of mature Pinus bungeana (Tibetan white pine) as scions, with a diameter of 0.4-0.7cm to ensure semi-lignification, and 8-12cm branch segments to retain apical dominance and promote callus formation. A blue polyethylene film with 60-70% light transmittance filters harmful red light wavelengths, and a thickness of 0.03-0.05mm balances fixation and air permeability, while the blue spectrum promotes cambium cell differentiation. This method, through precise control of rootstock and scion specifications and the design of a functional grafting band, solves the callus formation barrier problem caused by differences in the physiological state of materials in traditional grafting.

[0092] In another embodiment, in the method of grafting Pinus tabuliformis onto Pinus thunbergii, before applying the ethanol aqueous solution, a sterile degreased cotton swab is used to wipe along the direction of the interface cambium layer in one direction to remove the exudated resin, and then the swab is left to stand for 8-10 minutes. When applying the ethanol aqueous solution, simultaneously apply a trace element solution containing 0.01-0.03% manganese sulfate and 0.005-0.01% zinc sulfate by mass, at a rate of 0.02-0.05 mL per application.

[0093] Traditional pine grafting methods often involve directly applying an ethanol solution without removing resin exudate during the graft union treatment, leading to the following problems at the graft union: resin accumulation hinders contact between rootstock and scion cells; lack of supplementation of trace elements such as manganese and zinc affects the enzyme activity and lignification process of callus tissue; and the application of the ethanol solution lacks standardized procedures, easily resulting in excessively high local concentrations that damage newly formed cells.

[0094] This protocol adds resin removal and trace element supplementation steps before applying the ethanol-water solution: Using sterile, degreased cotton swabs, the resin is thoroughly removed unidirectionally along the cambium layer without damaging cell structure. The area is left to stand for 8-10 minutes to allow the wound to dry slightly, creating a clean interface for subsequent treatment. Simultaneously, a solution containing manganese sulfate (0.01-0.03%) and zinc sulfate (0.005-0.01%) is applied. Manganese activates phenylalanine ammonia-lyase, promoting lignin synthesis, while zinc enhances superoxide dismutase activity, improving antioxidant capacity and complementing the antibacterial function of the ethanol-water solution. Precise control of the application amount (0.02-0.05 mL / time) ensures that the ethanol-water solution and trace elements evenly cover the interface area without overflow, avoiding excessively high local concentrations that could damage newly formed cells. Existing technologies, lacking synergistic resin removal and trace element treatment, lead to metabolic imbalances in the interface area, delayed lignification, and a significantly increased risk of bacterial growth.

[0095] This solution's resin removal step thoroughly removes resin exudate from the interface area through unidirectional wiping and settling, providing a clean interface for subsequent solution penetration. In the trace element supplementation, manganese sulfate directly promotes the activity of key enzymes in lignin synthesis, while zinc sulfate protects callus tissue from oxidative damage through its antioxidant mechanism. The synergistic effect of these two elements with the ethanol-water solution achieves triple regulation of antibacterial, nutrient, and oxidation control. The precise application design ensures uniform solution distribution without disrupting the microenvironment balance; the application volume of 0.02-0.05 mL is matched to the interface area, meeting requirements while avoiding waste or localized overload. The entire process significantly improves the metabolic efficiency of the interface area, increasing the lignification integrity rate by more than 20%, while reducing the risk of fungal infection to below 5%.

[0096] Although the 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.

Claims

1. A method for grafting Tibetan white pine onto Chinese pine, characterized in that, Includes the following steps: Make a vertical cut 1.8-2.2 cm deep, reaching the xylem, at a point 2.5-3.5 cm above the surface of the cultivation substrate at the rootstock rhizome. The base of the scion is cut into a symmetrical double-sided wedge shape, with a cut length of 1.5-2.0 cm; Immerse the cut surface of the scion in a sodium citrate buffer solution containing 4-6% polyvinylpyrrolidone, 0.1-0.2% methyl salicylate, and 0.05-0.08% calcium chloride for 60-90 seconds. The concentration of the sodium citrate buffer solution is 0.04-0.06 mol / L, and the pH value is 6.0-6.

8. The adsorbent is coated on both sides of the vertical cut. The adsorbent is prepared by mixing activated carbon powder with a particle size of 40-50 mesh and hydroxyethyl cellulose solution with a mass fraction of 8-12% at a mass ratio of 1:2.5-3.5, and then adding nano-silica equivalent to 0.3-0.5% of the activated carbon powder mass, and stirring evenly. Insert the scion into the vertical cut of the rootstock, align the cambium layers, and then fix the interface with grafting tape; Apply an aqueous solution of ethanol containing 0.7-0.9% salicylic acid, 1-3% trehalose, and 0.2-0.4% polysorbate 80 (45-55% by volume) to the interface area every 2-3 days, at a rate of 0.05-0.15 mL per application. Maintain a daytime temperature of 23-25℃, a nighttime temperature of 16-18℃, and a humidity of 90-95%. When the new shoots of the scion are 2-3cm long, make a ring cut on the main trunk of the rootstock 0.8-1.2cm above the vertical cut, reaching the xylem. Cut off the main trunk above the ring cut 7-10 days later. After grafting, the graft should be shaded.

2. The method for grafting Pinus tabuliformis onto Pinus tabuliformis as described in claim 1, characterized in that, After soaking the cut surface of the scion in sodium citrate buffer, wrap the base of the scion with a breathable film for 8-10 minutes, then remove the film and fix it with a grafting tape.

3. The method for grafting Pinus tabuliformis onto Pinus tabuliformis as described in claim 1, characterized in that, Before inserting the scion into the vertical cut of the rootstock, spray the cut surface of the scion evenly with a 0.02-0.05% naphthaleneacetic acid solution, let it stand for 30-60 seconds, and then proceed with the subsequent operations.

4. The method for grafting Tibetan white pine onto Chinese pine as described in claim 1, characterized in that, After cutting the main trunk above the girdling point, immediately apply a composite protective agent to the cut surface of the rootstock, with a thickness of 0.5-1.0 mm, and let it stand for 2-4 minutes until a gel film forms on the surface; The composite protective agent is prepared by mixing the following components in the following mass percentages: 0.3-0.5% humic acid, 0.1-0.2% chitosan, 0.01-0.03% nano zinc oxide, and the balance being deionized water; The preparation method of the composite protective agent is as follows: humic acid and deionized water are stirred and dissolved at 50-60℃, then chitosan is added and stirred until completely dissolved, and finally nano zinc oxide is added and ultrasonically dispersed for 20-30 minutes.

5. The method for grafting Pinus tabuliformis onto Pinus tabuliformis as described in claim 4, characterized in that, After applying a compound protective agent to the cut surface of the rootstock until a gel film forms, spray with a phosphate buffer solution containing 0.1-0.3% indolebutyric acid, 0.05-0.1% gibberellin, and 0.01-0.03% vitamin B1 at a concentration of 0.1-0.2 mL / cm². 2 The concentration of the phosphate buffer is 0.05-0.1 mol / L, and the pH value is 6.0-6.

5.

6. The method for grafting Pinus tabuliformis onto Pinus tabuliformis as described in claim 1, characterized in that, The sodium citrate buffer also contains 0.002-0.005% by mass of pine polypeptide extract and 0.001-0.003% by mass of copper pinoresinate, with a copper pinoresinate purity ≥95%. Pine polypeptide extract was prepared by the following method: Phloem tissue from healthy Tibetan white pine trees of the current year was collected, flash-frozen in liquid nitrogen, and then ground to a particle size ≤0.5mm. Add pre-cooled Tris-HCl buffer with a pH of 7.2-7.4 at a mass ratio of 1:8-10. The Tris-HCl buffer contains 0.1 mol / L NaCl and 5 mmol / L EDTA. Perform ultrasonic-assisted extraction at 4°C with an ultrasonic power of 300W. Use a pulse mode with 5 seconds of operation followed by 10 seconds of intermittent operation. The total extraction time is 30 min. Centrifugation was performed at 12,000 rpm for 15 min at 4°C. The supernatant was then fractionated and passed through 10 kDa and 3 kDa ultrafiltration membranes. The 3-10 kDa fraction was desalted using a Sephadex G-25 column and then lyophilized to obtain a pine polypeptide extract.

7. The method for grafting Pinus tabuliformis onto Pinus tabuliformis as described in claim 1, characterized in that, The specific method for shading is as follows: Cover the grafted seedlings with a shade net that has a light transmittance of 30-40% for 7-10 days, and then adjust the light exposure in stages: a) Replace with a shade net with a light transmittance of 50-60% on days 1-3; b) From day 4 onwards, completely remove the shade netting. Spray the interface area with atomized water once daily from 10:00 to 14:00, at a rate of 0.1-0.2 mL / cm². 2 Continue until the new shoots of the scion reach a length of 5-6 cm, then stop.

8. The method for grafting Pinus tabuliformis onto Pinus tabuliformis as described in claim 1, characterized in that, Select Pinus tabuliformis seedlings with a diameter of 0.4-0.7cm and a height of 1-1.5m as rootstock; Select terminal branches of mature Tibetan white pine trees with a diameter of 0.4-0.7cm as scions, and cut 8-12cm sections of branches; The grafting tape is a blue polyethylene film with a light transmittance of 60-70% and a thickness of 0.03-0.05mm.

9. The method for grafting Pinus tabuliformis onto Pinus tabuliformis as described in claim 1, characterized in that, Before applying the ethanol aqueous solution, use a sterile degreased cotton swab to wipe along the direction of the interface cambium in one direction to remove the exuded resin. After wiping, let it stand for 8-10 minutes. When applying the ethanol aqueous solution, simultaneously apply a trace element solution containing 0.01-0.03% manganese sulfate and 0.005-0.01% zinc sulfate by mass, at a rate of 0.02-0.05 mL per application.