Method for grafting pinus massoniana with pinus yunnanensis

Abstract

The application provides a method for grafting Pinus tabulaeformis with Pinus yunnanensis var. biondii, and belongs to the technical field of grafting of needle-leaved trees, and solves the technical problems of slow grafting, high pathogenic infection rate and low survival rate of less than 40% caused by environmental fluctuations. The technical solution points comprise the following steps: cutting a vertical incision of 1.8-2.2 cm deep to the xylem at a position of 2.5-3.5 cm from the surface of the cultivation substrate exposed at the root stem of the stock; immersing the base of the scion into a sodium citrate buffer solution containing polyvinylpyrrolidone, methyl salicylate and calcium chloride after the base is cut into a symmetrical double-wedge shape; coating adsorption glue on both sides of the vertical incision; inserting the scion to align the cambium and fixing the scion with a grafting tape; applying an aqueous ethanol solution containing salicylic acid, trehalose and polysorbate 80 to the grafting interface every 2-3 days; maintaining the day temperature at 23-25 DEG C, the night temperature at 16-18 DEG C and the humidity at 90-95%; girdling the stock when the new shoots grow to 2-3 cm in length, and cutting off the main stem after 7-10 days. The application is used for large-scale propagation of Pinus yunnanensis var. biondii grafted seedlings.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tree grafting. More particularly, the present application relates to a method for grafting Pinus gerardiana Wall. ex D. Don onto Pinus tabulaeformis Carr. BACKGROUND

[0002] Pinus gerardiana Wall. ex D. Don, as an endangered species with high economic value, has a narrow native distribution area. The survival rate of artificial introduction is less than 20% due to low temperature and drought stress. By grafting on Pinus tabulaeformis Carr., the planting range of Pinus gerardiana Wall. ex D. Don can be expanded to the western alpine region with an average annual temperature of ≥-5℃, creating economic benefits for local farmers.

[0003] Grafting Pinus gerardiana Wall. ex D. Don onto Pinus tabulaeformis Carr. is an important way to cultivate resistant seedlings, but the survival rate of this combination has been at a low level for a long time. The cambial cell activity of Pinus tabulaeformis Carr. is quite different from that of Pinus gerardiana Wall. ex D. Don, and it is difficult for them to quickly initiate callus differentiation after contact. After the cut of the stock is exposed, resin secretion will cover the cambium, hindering cell contact. The inherent lignification process of Pinus is relatively fast, and if vascular connection is not established within a short period of time, the cut is easily isolated by the newly formed xylem, leading to healing failure. Traditional methods rely on a single growth regulator, which is difficult to coordinate the cell division cycle of both parents.

[0004] The microenvironment formed by grafting is prone to the growth of Fusarium and Pythium fungi. Once these pathogens invade through the cut, they quickly block the vascular bundle. Conventional protective measures lack sufficient air permeability, which actually exacerbates the proliferation of anaerobic bacteria. Spraying broad-spectrum fungicides can inhibit callus cell division, causing secondary damage. Pinus tabulaeformis Carr. and Pinus gerardiana Wall. ex D. Don have different response thresholds to temperature and humidity: Pinus tabulaeformis Carr. requires higher daytime temperatures for cambium division, while Pinus gerardiana Wall. ex D. Don requires lower nighttime temperatures to reduce respiratory consumption. When grafting outdoors, large diurnal temperature differences can interrupt callus proliferation; low humidity can cause the shoot to lose water faster than the stock can supply, while high humidity can promote the germination of pathogenic spores. Existing technologies cannot balance these three relationships, and the survival rate is often less than 40%.

[0005] Substances that enhance cell activity can accelerate resin secretion, while agents that inhibit resin secretion can damage the cambium. A single agent cannot simultaneously meet the needs of promoting healing and inhibiting bacteria. Activated carbon adsorbs resin but hinders oxygen exchange; air-permeable materials have poor antibacterial effects, and airtight materials lead to hypoxia. Existing grafting tapes cannot balance the isolation of pathogens and the maintenance of microenvironment gas exchange. Greenhouse temperature control consumes too much energy, and outdoor grafting cannot accurately respond to instantaneous changes in temperature and humidity. Intermittent spraying can only maintain local humidity, making it difficult to stabilize the microenvironment of the interface.

[0006] These defects restrict the large-scale application of the technology of grafting Pinus gerardiana Wall. ex D. Don onto Pinus tabulaeformis Carr. in afforestation projects. SUMMARY

[0007] The application provides a method for grafting Pinus tabulaeformis Carr with Pinus yunnanensis Franch, adopts a composite buffer solution to pretreat the scion, coordinates the cell division cycle of the two parent cells, accelerates the initiation of callus, and overcomes the hindrance of resin secretion to the contact of the cambium; designs an interface protection layer with adsorbed resin and air permeability, blocks the path of pathogen invasion, maintains gas exchange in the microenvironment, and avoids the risk of anaerobic bacteria reproduction in the traditional protection method; formulates a combination of temperature and humidity parameters and a periodic nutrition supplement scheme, balances the differentiated needs of Pinus tabulaeformis Carr and Pinus yunnanensis Franch for diurnal temperature difference, and stabilizes the callus proliferation conditions. Finally, the grafting survival rate is improved to the level of scalable application.

[0008] The problems of slow interface healing, easy infection and unstable survival rate are solved.

[0009] The problem of oxidative stress damage of the scion cutting surface is solved.

[0010] The problem of delayed healing caused by insufficient cell activity of the scion is solved.

[0011] The problems of water loss and pathogen invasion of the cutting surface of the stock are solved.

[0012] The problem of slow callus differentiation of the cutting surface of the stock is solved.

[0013] The problem of lack of specific healing factors of Pinus is solved.

[0014] The problem of transitory period of scion transpiration imbalance is solved.

[0015] The problem of poor physiological compatibility of the stock and the scion is solved.

[0016] The problems of resin blocking the cambium and lack of trace elements are solved.

[0017] In order to solve the above problems and achieve the purpose and other advantages of the application, a method for grafting Pinus tabulaeformis Carr with Pinus yunnanensis Franch is provided, comprising the following steps:

[0018] At the position of 2.5-3.5 cm from the root stem of the stock to the surface of the cultivation substrate, a vertical incision is made longitudinally to the depth of the xylem, and the length of the vertical incision is 1.8-2.2 cm;

[0019] The base of the scion is cut into a symmetrical double-wedge shape, and the length of the cutting surface is 1.5-2.0 cm;

[0020] The cutting surface of the scion is immersed 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 s, the concentration of the sodium citrate buffer solution is 0.04-0.06 mol / L, and the pH value is 6.0-6.8;

[0021] The vertical cut is coated with adsorbing glue on both sides, and the preparation method of the adsorbing glue is as follows: 40-50 mesh activated carbon powder is mixed with 8-12% hydroxyethyl cellulose solution in a mass ratio of 1:2.5-3.5, and 0.3-0.5% nano silicon dioxide is added, corresponding to the mass of the activated carbon powder, and then stirred uniformly;

[0022] The scion is inserted into the vertical cut of the stock, and the interface is fixed with a grafting tape after the alignment of the layers;

[0023] An ethanol aqueous solution with a volume fraction of 45-55% containing 0.7-0.9% salicylic acid, 1-3% trehalose, and 0.2-0.4% polysorbate 80 in terms of mass fraction is applied to the interface area every 2-3 days, and the application amount is 0.05-0.15 mL / time;

[0024] The day temperature is maintained at 23-25°C, the night temperature is maintained at 16-18°C, and the humidity is maintained at 90-95%;

[0025] When the new shoots of the scion are 2-3 cm long, the stock trunk is girdled 0.8-1.2 cm above the vertical cut to the xylem, and the trunk above the girdling is cut off after 7-10 days;

[0026] After grafting, shading treatment is performed.

[0027] Preferably, in the method for grafting Pinus tabulaeformis onto P. yunnanensis, the scion cut surface is immersed in a sodium citrate buffer solution, then the base of the scion is wrapped with a breathable film for 8-10 min, and then the film is removed before fixing with a grafting tape.

[0028] Preferably, in the method for grafting Pinus tabulaeformis onto P. yunnanensis, the scion cut surface is uniformly sprayed with a 0.02-0.05% naphthaleneacetic acid solution before being inserted into the vertical cut of the stock, and then the subsequent operations are performed after standing for 30-60 s.

[0029] Preferably, in the method for grafting Pinus tabulaeformis onto P. yunnanensis, after cutting off the trunk above the girdling, the cut surface of the stock is immediately coated with a composite protective agent with a thickness of 0.5-1.0 mm, and the surface forms a gel film after standing for 2-4 min;

[0030] The composite protective agent is prepared by mixing the following components in terms of mass percentage: 0.3-0.5% humic acid, 0.1-0.2% chitosan, and 0.01-0.03% nano zinc oxide, with the balance being deionized water.

[0031] The preparation method of the composite protective agent is as follows: humic acid is dissolved in deionized water at 50-60°C, then chitosan is added and stirred until completely dissolved, and finally nano zinc oxide is ultrasonically dispersed for 20-30 min.

[0032] 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.

[0033] 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%.

[0034] Pine polypeptide extract was prepared by the following method:

[0035] Take the phloem tissue from healthy Tibetan white pine trees of the current year, flash freeze it with liquid nitrogen, and then grind it to a particle size ≤0.5mm;

[0036] 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.

[0037] 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.

[0038] The 3-10 kDa fraction was desalted using a Sephadex G-25 column and then lyophilized to obtain a pine polypeptide extract.

[0039] Preferably, in the method for grafting Pinus tabuliformis onto Pinus bungeana, the specific method for shading is as follows:

[0040] 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:

[0041] a) Replace with a shade net with a light transmittance of 50-60% on days 1-3;

[0042] 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.

[0043] Preferably, in the method for grafting Pinus tabulaeformis with Pinus yunnanensis var.

[0044] Select Pinus tabulaeformis seedlings with a diameter of 0.4-0.7 cm and a height of 1-1.5 m as rootstocks;

[0045] Select terminal branches of Pinus yunnanensis var. with a diameter of 0.4-0.7 cm as scions, and cut 8-12 cm sections;

[0046] The grafting tape is a blue polyethylene film with a light transmittance of 60-70% and a thickness of 0.03-0.05 mm.

[0047] Preferably, in the method for grafting Pinus tabulaeformis with Pinus yunnanensis var., before applying the ethanol aqueous solution, use a sterile cotton swab to wipe the interface in one direction along the cambium layer, remove the exuded resin, and stand for 8-10 min after wiping;

[0048] 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, with an application amount of 0.02-0.05 mL per time.

[0049] The present application at least includes the following beneficial effects:

[0050] The present application significantly improves the cell affinity of the rootstock and the scion by pretreating the scion with a composite buffer, effectively solving the problem of asynchronous healing caused by physiological differences between the stock and the scion in traditional grafting. The synergistic effect of the specific components in the buffer not only protects the cell activity of the scion, but also promotes the rapid formation of callus.

[0051] The adsorbing glue designed in the present application has both resin adsorption and air permeability functions, blocking the invasion of pathogenic bacteria while maintaining oxygen exchange in the interface area. This dual-function design overcomes the problem of hypoxia caused by traditional sealing materials, creating an ideal growth environment for callus.

[0052] The present application precisely controls the day-night temperature difference and humidity, meeting the cell division needs of Pinus tabulaeformis and reducing the respiratory consumption of Pinus yunnanensis var. This environmental regulation scheme effectively balances the different requirements of the two plants for growth conditions, significantly improving the success rate of grafting.

[0053] The composite protective agent used after cutting the main stem of the rootstock in the present application protects the cut surface and promotes callus formation through the synergistic effect of multiple active ingredients. This multifunctional protection scheme solves the problem of traditional single sealing materials that cannot balance protection and healing promotion.

[0054] The phased adjustment strategy adopted in the shading treatment, combined with precise atomized water spraying, prevents scion light inhibition and avoids dehydration damage. This dynamic regulation method overcomes the defects of traditional fixed shading methods that cannot adapt to the needs of different healing stages.

[0055] The present application significantly enhances the recognition and lignin synthesis ability of the stock and scion cells by adding Tibetan white bark pine specific active ingredients. This species-specific design solves the problem of lack of targeted active substances in traditional methods.

[0056] The grafting tape designed in the present application ensures the fixing strength while protecting the interface and maintaining the necessary photosynthesis through specific light transmittance and spectral characteristics. This functional design overcomes the shortcomings of traditional grafting tapes with single function.

[0057] The present application effectively solves the problem of vascular connection disorder caused by resin blockage through the synergistic treatment of resin removal and trace element supplementation. This comprehensive treatment scheme makes up for the defects of traditional methods that ignore the metabolic balance of the interface area.

[0058] The standardized operation system established in the present application realizes the stable improvement of grafting survival rate by accurately controlling the parameters of each link. This systematic method provides reliable technical support for large-scale application.

[0059] Other advantages, objectives and features of the present application will be partially embodied by the following description, and partially understood by those skilled in the art through research and practice of the present application. DETAILED DESCRIPTION

[0060] The present application will be further described in detail below so that those skilled in the art can implement it according to the description.

[0061] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0062] The present application provides a method for grafting Pinus tabulaeformis with Tibetan white bark pine, comprising the following steps:

[0063] At the position of 2.5-3.5 cm from the root stem of the stock where the cultivation substrate surface is exposed, a vertical cut is made longitudinally to the depth of the xylem, and the length of the vertical cut is 1.8-2.2 cm;

[0064] The base of the scion is cut into a symmetrical double-wedge shape, and the length of the cut surface is 1.5-2.0 cm;

[0065] The cut surface of the scion is immersed in a sodium citrate buffer solution containing 4-6% by mass of polyvinylpyrrolidone, 0.1-0.2% of methyl salicylate and 0.05-0.08% of 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;

[0066] The vertical cut is coated with adsorbing glue, and the adsorbing glue is prepared by mixing activated carbon powder with a particle size of 40-50 meshes and 8-12% by mass of a hydroxyethyl cellulose solution at a mass ratio of 1:2.5-3.5, and then adding nano silicon dioxide with a mass percentage of 0.3-0.5% of the activated carbon powder and stirring uniformly;

[0067] The scion is inserted into the vertical cut of the stock, and the interface is fixed by using a grafting tape after the alignment of the layers is formed;

[0068] An ethanol aqueous solution with a volume fraction of 45-55% and containing 0.7-0.9% by mass of salicylic acid, 1-3% of trehalose and 0.2-0.4% of polysorbate 80 is applied to the interface area every 2-3 days, and the application amount is 0.05-0.15 mL per time;

[0069] The day temperature is maintained at 23-25℃, the night temperature is maintained at 16-18℃, and the humidity is maintained at 90-95%;

[0070] When the new shoots of the scion are 2-3 cm long, the main stem of the stock is girdled at a depth of the xylem at a position 0.8-1.2 cm above the vertical cut, and the main stem above the girdled position is cut off after 7-10 days;

[0071] After the grafting is completed, shading treatment is performed.

[0072] The traditional method for grafting Pinus tabulaeformis with Pinus yunnanensis var. bretschneideri has significant technical defects. The callus formation of the stock and the scion is not synchronized, resulting in the death of the scion due to insufficient nutrient supply and water loss. The moisture control of the interface area is not good, and the traditional grafting tape or paraffin sealing has poor air permeability to inhibit callus respiration or has too strong air permeability to effectively retain moisture. The supply of nutrients and oxygen is imbalanced, and the local nutrient supply of the interface area is not optimized in the traditional method, the high-concentration ethanol aqueous solution damages the newly formed cells, the low-concentration ethanol aqueous solution has insufficient antibacterial effect, and the xylem healing is delayed due to lack of oxygen.

[0073] The method for grafting Pinus tabulaeformis with Pinus yunnanensis var. bretschneideri provided in the scheme comprises the following steps:

[0074] Selection of the stock and the scion:

[0075] Pinus tabulaeformis seedlings with a diameter of 0.4-0.7 cm and a height of 1-1.5 m are selected as the stock; and the terminal branch of Pinus yunnanensis var. bretschneideri with a diameter of 0.4-0.7 cm is selected as the scion, and a 8-12 cm branch segment is cut off as the scion to ensure that the scion has sufficient meristem activity.

[0076] Stock treatment:

[0077] Make vertical incisions (1.8-2.2 cm long) into the rootstock at a depth of 2.5-3.5 cm below the substrate surface, where the cambium is exposed. This depth prevents contamination of the incision by pathogens in the substrate, and the length of the vertical incision provides sufficient cambium contact surface.

[0078] Scion pretreatment:

[0079] Cut the base of the scion into a symmetrical double-wedge shape (1.5-2.0 cm long), increasing the contact area between the stock and scion. Soak the scion in a sodium citrate buffer solution (0.04-0.06 mol / L, pH 6.4-6.6) containing 4-6% polyvinylpyrrolidone (to promote cell adhesion), 0.1-0.2% methyl salicylate (to inhibit pathogenic bacteria), and 0.05-0.08% calcium chloride (to enhance cell wall stability) for 60-90 seconds. This combination enhances the scion's stress resistance and activates callus differentiation.

[0080] Interface protection:

[0081] Apply an adsorption glue to both sides of the vertical incision, made by mixing 40-50 mesh activated carbon powder (to adsorb exuded resin) with 8-12% hydroxyethyl cellulose solution (as an adhesive) at a ratio of 1:2.5-3.5, and adding 0.3-0.5% nano-silicon dioxide (to enhance air permeability). This forms a physical barrier to block pathogen invasion while maintaining oxygen exchange.

[0082] Graft fixation:

[0083] Insert the scion strictly aligned with the cambium, ensuring effective vascular tissue connection between the stock and scion. Use a blue grafting tape with a light transmittance of 60-70% to fix the graft, which provides moderate shading to reduce transpiration and allows photosynthetic signal substances to penetrate.

[0084] Postoperative management:

[0085] Nutrient supplementation: Apply an ethanol aqueous solution (45-55% by volume) containing 0.7-0.9% salicylic acid (to induce disease resistance), 1-3% trehalose (as an osmoprotectant), and 0.2-0.4% polysorbate 80 (to promote absorption) at 0.05-0.15 mL per time every 2-3 days to maintain cell activity in the interface area.

[0086] Environmental control: Maintain a day temperature of 23-25°C (to promote cell division in Pinus tabulaeformis) combined with a night temperature of 16-18°C (to reduce respiration consumption in Pinus yunnanensis var. beshanzuensis), and a humidity of 90-95% (to prevent water loss in the scion and inhibit oxidation at the incision).

[0087] Physiological intervention: When the new shoots are 2-3 cm long, make a ring cut (deep into the xylem) on the stock to block nutrient backflow. After 7-10 days, cut off the main stem above the ring cut to force the stock to supply nutrients to the grafting interface for healing.

[0088] After grafting, shading treatment is carried out.

[0089] Significance and economic value of grafting Pinus tabulaeformis Carr. with Pinus yunnanensis Franch.

[0090] 1) Break through ecological limitations and expand planting areas

[0091] Pinus yunnanensis Franch. is originally distributed in the Himalayan high-altitude (3000-4000 meters) region and has weak adaptability to cold and arid environments. Pinus tabulaeformis Carr. as a rootstock has strong cold tolerance (can tolerate -30℃ low temperature) and can grow in poor soil. After grafting, Pinus yunnanensis Franch. can be planted in the western high-cold and semi-arid regions (such as Qinghai, western Sichuan, southern Gansu, and other regions with an altitude of 2000-3000 meters) to expand its suitable range.

[0092] 2) Significantly improve economic value

[0093] Economic benefits of pine cones: Pinus yunnanensis Franch. pine cones weigh 15-20g per grain (3 times that of ordinary Pinus tabulaeformis Carr.), pine nuts have an oil content of more than 60%, are rich in unsaturated fatty acids and antioxidant substances, and have a market price of more than 80 yuan / kg. After 5 years of planting grafted seedlings, the yield of pine cones per mu is 300-400 kg, and the economic benefits are much higher than those of ordinary economic forests.

[0094] Ecological industry integration: In the high-cold region, industries such as pine cone picking and ecological tourism can be developed.

[0095] 3) Ecological restoration and carbon sink gain

[0096] Grafted seedlings inherit the stress resistance of Pinus tabulaeformis Carr. and can grow in arid and saline-alkali mountainous areas, which can be used for ecological restoration of degraded mountainous areas.

[0097] The carbon sink capacity of Pinus yunnanensis Franch. is 1.8 times that of Pinus tabulaeformis Carr., and large-scale planting is of great significance for carbon sequestration and emission reduction in high-cold regions.

[0098] 4) Protecting endangered species genetic resources

[0099] Pinus yunnanensis Franch. is listed as an endangered tree species in China, and grafting can achieve large-scale preservation of germplasm resources and avoid ecological damage caused by over-exploitation of the original habitat.

[0100] The large-scale grafting and seedling raising realized by the present application reduces the lower limit of the planting altitude of Pinus yunnanensis Franch. to 2500 meters, providing a new way for the western high-cold regions of Qinghai and Sichuan. The pine cones produced by grafted seedlings have no significant difference in quality from the original species, and the yield per mu can reach more than 24,000 yuan.

[0101] Example 1

[0102] Select 50 strains of Pinus tabulaeformis seedlings with a diameter of 0.5 cm and a height of 1.2 m as rootstocks. Select 50 strains of adult terminal branches of Pinus densata with a diameter of 0.5 cm as scions, and cut 10 cm sections. At the rootstock rhizome, expose the surface of the cultivation substrate by 3 cm, and make a vertical incision that reaches the xylem. The length of the vertical incision is 2 cm. The base of the scion is cut into a symmetrical double-wedge shape, and the length of the cut surface is 1.8 cm. The scion cut surface is 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 s, and the buffer solution has a pH value of 6.5. On both sides of the vertical incision, apply adsorbed glue, which is made of 45-mesh activated carbon powder mixed with 10% hydroxyethyl cellulose solution at a mass ratio of 1:3, and then add 0.4% nanosilicon dioxide equivalent to the mass of the activated carbon powder. Insert the scion into the vertical incision of the rootstock, align the layers, and then use grafting tape to fix the interface. Every 36 h, apply a 50% ethanol aqueous solution containing 0.8% salicylic acid, 2% trehalose, and 0.3% polysorbate 80 to the interface area, with a volume fraction of 0.8% salicylic acid, 2% trehalose, and 0.3% polysorbate 80, and an application amount of 0.1 mL per time. Maintain a day temperature of 24°C, a night temperature of 17°C, and a humidity of 93%. When the scion sprout is 2.5 cm long, ring the trunk of the rootstock 1 cm above the vertical incision to a depth of the xylem, and cut off the trunk 8 days later. After 30 days, the results show that 46 out of 50 grafted seedlings survive, with a survival rate of 92%, the average length of the scion sprout is 6.2 cm, the xylem of the interface callus is complete at a rate of 88%, and there is no mold infection or interface cracking.

[0103] Comparative Example 1

[0104] To address the problem of asynchronous callus of the rootstock and the scion, the methyl salicylate and calcium chloride in the buffer solution are removed, and only polyvinylpyrrolidone is retained. The remaining operations are identical to those in Example 1. After 30 days, the results show that 34 out of 50 grafted seedlings survive, with a survival rate of 68%, the average length of the scion sprout is 4.5 cm, the xylem of the interface callus is complete at a rate of only 60%, and the scion appears to be wilted due to insufficient nutrition. This proves that methyl salicylate and calcium chloride play a key role in coordinating the callus speed of the rootstock and the scion.

[0105] Comparative Example 2

[0106] To address the problem of water control in the interface area, traditional paraffin is used to completely seal the interface area, and the adsorbed glue treatment is removed. The remaining operations are identical to those in Example 1. After 30 days, the results show that 18 out of 50 grafted seedlings survive, with a survival rate of 36%, the average length of the scion sprout is 3.0 cm, and there is a large amount of water-stained lesions at the interface. This shows that paraffin sealing cannot balance the demand for air permeability and moisture retention.

[0107] Comparative Example 3

[0108] For the problem of imbalance between nutrition and oxygen supply, the application of ethanol aqueous solution was cancelled and only distilled water was used instead, and the rest of the operations were the same as in Example 1. After 30 days, the statistics showed that among the 50 grafted seedlings, 29 survived, with a survival rate of 58%, the average length of the new shoots of the scions was 4.2 cm, and the xylem connection was incomplete. It was confirmed that the bacteriostatic function of the ethanol aqueous solution and the nutrition supplement were indispensable for the healing of the graft interface.

[0109] Effects: Example 1 solves the three problems of synchronous healing of stock and scion, microenvironment regulation of the graft interface, and nutrition supply by a composite technical solution. Counterexamples 1-3 respectively verify the irreplaceability of the buffer components, the function of the adsorbing glue, and the ethanol aqueous solution in solving these problems.

[0110] In another scheme, the method for grafting Pinus tabulaeformis Carr. with Pinus yunnanensis Franch. involves immersing the cut surface of the scion in a sodium citrate buffer solution, wrapping the base of the scion with a breathable film for 8-10 min, then removing the film and fixing the grafting tape.

[0111] In the traditional grafting method of Pinus tabulaeformis, the cut surface of the scion is immersed in a buffer solution and then directly fixed with grafting tape without any transitional protection. The existing technology generally ignores the problem of cell dehydration caused by the exposure of the cut surface after buffer treatment and does not consider the short-term moisture retention needs of the scion after immersion. In conventional operations, the scion is immediately fixed after being taken out of the buffer solution, which leads to rapid evaporation of water from the cut surface, a decrease in cell activity, and an impact on the formation of callus.

[0112] In this scheme, after the scion is immersed in the buffer solution, the base of the scion is wrapped with a breathable film for 8-10 min, and then the film is removed for fixation. The existing technology directly fixes the graft interface and cannot solve the problem of moisture retention during the transition period of the scion. The wrapping of the breathable film precisely balances the needs of moisture retention and air permeability by preventing rapid evaporation of water and avoiding hypoxia caused by long-term coverage. The light-transmitting property of the film allows the scion to perform necessary photosynthesis while reducing damage to newly formed cells caused by direct sunlight.

[0113] In this scheme, the scion is wrapped with a breathable film after being immersed in the buffer solution, mainly to solve the problem of cell dehydration caused by the exposure of the cut surface. The wrapping time of 8-10 min ensures sufficient absorption of the buffer components while avoiding hypoxia. After the film is removed, the graft interface is fixed, ensuring that the scion completes grafting in the best state. This step significantly improves the cell activity of the scion and promotes the rapid formation of callus.

[0114] In another scheme, the method for grafting Pinus tabulaeformis Carr. with Pinus yunnanensis Franch. involves uniformly spraying the cut surface of the scion with a naphthaleneacetic acid solution with a mass fraction of 0.02-0.05% before inserting the scion into the vertical cut of the stock, and then performing the subsequent operations after standing for 30-60 s.

[0115] In the traditional method of Pinus tabulaeformis grafting, the scion is directly inserted into the cut surface of the stock after the treatment of the scion cutting surface, without hormone pretreatment of the scion. The existing technology usually relies on the hormone secretion of the stock itself to induce callus formation, resulting in different speeds of callus formation between the scion and the stock. The conventional operation ignores the key influence of the cell activity of the scion on the initial healing of grafting, and the scion often delays the start of the callus process due to insufficient hormone stimulation, increasing the risk of dehydration.

[0116] In the present scheme, a 0.02-0.05% naphthaleneacetic acid solution is sprayed on the cutting surface before the scion is inserted into the stock, and the solution is left to stand for 30-60s. The existing technology does not have this step, and the scion is only treated with a buffer, with limited hormone effect. As an auxin hormone, naphthaleneacetic acid directly activates cell division of the scion cutting surface, synchronizing the callus formation process with the stock. The standing time ensures sufficient penetration of the hormone but avoids excessive stimulation, avoiding cell dysplasia.

[0117] The spraying of the naphthaleneacetic acid solution in the present scheme aims to start the callus program of the scion in advance, solving the problem of different speeds of callus formation between the scion and the stock. The 30-60s standing balances the speed of hormone absorption and cell response, avoiding tissue damage caused by too long treatment time. This step makes the scion have high-activity callus cells before being inserted into the stock, shortening the healing period after grafting.

[0118] In another scheme, the method of grafting Pinus tabulaeformis with Pinus yunnanensis var. tibetica, after cutting the main stem above the girdling, immediately applies a composite protective agent to the cut surface of the stock, with a thickness of 0.5-1.0mm, and stands for 2-4min until a gel film forms on the surface;

[0119] The composite protective agent is prepared by mixing the following components in mass percentage: humic acid 0.3-0.5%, chitosan 0.1-0.2%, nano zinc oxide 0.01-0.03%, and the balance is deionized water;

[0120] The preparation method of the composite protective agent is: 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 20-30min.

[0121] In the traditional method of Pinus tabulaeformis grafting, after cutting the main stem of the stock, only paraffin or ordinary grafting wax is usually used to seal the cut surface, lacking targeted protection and promotion of healing. In the existing technology, the treatment of the cut surface mainly focuses on physical sealing, ignoring the active regulation of callus formation. The sealing material used in the conventional method has poor air permeability, easily leading to internal hypoxia, and does not have the functions of antibacterial and promoting cell division, affecting the healing speed of the cut surface of the stock and the subsequent growth of the scion.

[0122] The present scheme uses a composite protective agent to smear the cut surface immediately after cutting the main stem of the stock, forming a gel film with air permeability and biological activity. The paraffin sealing in the prior art only provides a physical barrier and cannot solve the problems of hypoxia and pathogen infection. The humic acid and chitosan in the composite protective agent work synergistically to maintain humidity and inhibit pathogens, and the nano zinc oxide further enhances the antibacterial effect. Resting for 2-4 min makes the gel film stable and attached, creating conditions for subsequent spraying of hormone solution.

[0123] The composite protective agent smearing of the present scheme aims to provide multifunctional protection for the cut surface of the stock. Humic acid promotes the division of callus cells, chitosan inhibits the growth of pathogens, and nano zinc oxide enhances the antibacterial performance. The air permeability of the gel film avoids the hypoxia problem of traditional sealing materials. Resting for 2-4 min ensures that the gel film forms a stable structure that can protect the cut surface without interfering with subsequent operations. Compared with traditional methods, this step significantly improves the healing speed of the cut surface of the stock and reduces the risk of infection.

[0124] Example 2

[0125] Select 50 Pinus tabulaeformis seedlings with a diameter of 0.5 cm and a height of 1.2 m as stocks, graft Pinus densata var. Tibetica according to the steps of Example 1. Immediately after cutting the main stem above the ring cut, use a composite protective agent (humic acid 0.4%, chitosan 0.15%, nano zinc oxide 0.02%, and the rest is deionized water) to smear the cut surface, with a thickness of 0.8 mm, and rest for 3 min to form a gel film. After 30 days, the results show that the healing integrity rate of the cut surface of the stock is 95%, and there is no infection or cracking phenomenon, and the average length of the scion shoots is 6.5 cm.

[0126] Comparative Example 4

[0127] Use traditional paraffin to seal the cut surface of the stock, and the rest of the operations are the same as Example 2. After 30 days, the results show that the healing integrity rate of the cut surface of the stock is only 60%, and 15% of the interfaces are necrotic due to hypoxia, and the average length of the scion shoots is 4.0 cm.

[0128] Effect: Example 2 achieves efficient healing of the cut surface of the stock through the multifunctional synergistic effect of the composite protective agent. Comparative Example 4 proves that the traditional sealing method cannot solve the dual needs of air permeability and antibacterial effect, significantly affecting the grafting effect.

[0129] In another scheme, in the method of grafting Pinus densata var. Tibetica onto Pinus tabulaeformis, after smearing the cut surface of the stock with a composite protective agent to form a gel film on the surface, a phosphate buffer solution containing 0.1-0.3% indole-3-butyric acid, 0.05-0.1% gibberellin, and 0.01-0.03% vitamin B1 is sprayed, with a spraying amount of 0.1-0.2 mL / cm 2 , the concentration of the phosphate buffer solution is 0.05-0.1 mol / L, and the pH value is 6.0-6.5.

[0130] The traditional method of Pinus tabulaeformis grafting usually only uses a single protective agent (such as paraffin or ordinary grafting wax) to seal the cut surface after cutting off the main stem of the stock, and lacks active regulation of the callus. In the prior art, the cut surface treatment mainly relies on physical sealing, without combining hormones to promote callus formation, resulting in slow healing of the stock and limited subsequent growth of the scion. The sealing material used in the conventional method has a single function and cannot simultaneously meet the multiple requirements of antibacterial, moisturizing and cell division activation.

[0131] In the present scheme, after the formation of the gel film of the composite protective agent, a phosphate buffer solution containing indolebutyric acid, gibberellin and vitamin B1 is sprayed. The prior art does not have this step and only relies on the physical isolation effect of the protective agent. The composite hormone solution promotes cell division through indolebutyric acid, coordinates the growth rhythm through gibberellin, and enhances stress resistance through vitamin B1, which is complementary to the physical barrier of the protective agent. The phosphate buffer solution maintains stable pH to ensure the activity of the hormones, and the spraying amount is 0.1-0.2 mL / cm 2 The porosity of the gel film is accurately matched to achieve uniform permeation.

[0132] The spraying of the hormone solution in the present scheme aims to activate the callus program of the cut surface of the stock. Indolebutyric acid accelerates the proliferation of callus cells, gibberellin balances the growth rate, and vitamin B1 reduces oxidative damage. The pH value of the phosphate buffer solution is 6.0-6.5, which is compatible with the protective agent gel and avoids component conflicts. After spraying, the hormones are released slowly through the gel film and have a continuous effect for 7-10 days, covering the key period of callus. Compared with the conventional method, this step shortens the healing time of the cut surface of the stock by 30% and increases the growth rate of the new shoots of the scion by 15%.

[0133] In another scheme, the method for grafting Pinus tabulaeformis with Tibetan white-bark pine, the sodium citrate buffer solution further contains 0.002-0.005% by mass fraction of pine polypeptide extract and 0.001-0.003% of copper abietinate, and the purity of the copper abietinate is ≥95%;

[0134] The pine polypeptide extract is prepared by the following method:

[0135] Healthy Tibetan white-bark pine phloem tissue of the current year is quickly frozen in liquid nitrogen and ground to a particle size of ≤0.5 mm;

[0136] Pre-cooled Tris-HCl buffer solution with a pH value of 7.2-7.4 is added at a mass ratio of 1:8-10, the Tris-HCl buffer solution contains 0.1 mol / L NaCl and 5 mmol / L EDTA, and ultrasonic-assisted extraction is carried out at 4°C, the ultrasonic power is 300 W, the pulse mode is used for 5 s working and 10 s intermittent, and the total extraction time is 30 min;

[0137] Centrifugal separation, centrifugal parameters 12000 rpm, 15 min, 4℃, take supernatant through 10kDa and 3kDa ultrafiltration membrane fractionation intercept in turn;

[0138] The 3-10kDa component is desalted by Sephadex G-25 column, and the pine polypeptide extract is obtained after freeze-drying.

[0139] In the traditional pine grafting method, the buffer usually only contains basic ingredients such as polyvinylpyrrolidone and preservatives, and lacks active substances for the affinity of stock and scion. The buffer used in the prior art has a single function, mainly plays a role in moisturizing and preservative, and cannot promote cell recognition and fusion of the scion and the stock. In the conventional operation, the pH value and ion concentration range of the buffer are wide, and the regulation of cell activity is not accurate enough, resulting in slow or asynchronous formation of callus.

[0140] The present scheme adds pine polypeptide extract and copper abietate in the sodium citrate buffer. The prior art does not use plant-specific active ingredients, while the pine polypeptide extract contains signal molecules unique to Tibetan white-bark pine, which can enhance cell recognition and fusion of the stock and scion; copper abietate provides slow-release copper ions to activate oxidases and promote lignin synthesis. The pH value of the buffer is accurately controlled at 6.0-6.8, which matches the optimal range of the activity of the pine polypeptide and avoids cell damage. The pine polypeptide extract is extracted by fractional ultrafiltration and desalting process to ensure the purity and stability of the active ingredients.

[0141] The addition of pine polypeptide extract in the present scheme aims to promote cell fusion of the stock and scion by utilizing its biological activity. The copper ions of copper abietate participate in the activation of oxidases to accelerate the connection of the xylem. The buffer pH 6.0-6.8 maintains the stability of the cell membrane and avoids damage to the scion caused by excessively high or low pH. Ultrasonic-assisted extraction and ultrafiltration fractionation ensure that the molecular weight of the pine polypeptide is concentrated in the range of 3-10kDa, and the polypeptide in this range has the best promotion of grafting activity.

[0142] Example 3 (containing pine polypeptide and copper abietate)

[0143] On the basis of Example 1, 0.003% pine polypeptide extract and 0.002% copper abietate (purity ≥95%) are additionally added to the sodium citrate buffer, and the remaining operations are exactly the same. After 30 days, the statistical results show that 48 out of 50 grafted seedlings survived, with a survival rate of 96%, the average length of the new shoots of the scions was 6.8 cm, the complete rate of xylem lignification of the callus at the grafting interface was 93%, and there was no phenomenon of mold infection or interface cracking.

[0144] Effect: Example 3 added pine polypeptide extract and copper abietinate to the base of Example 1, and the survival rate increased from 92% to 96%, the average length of the new shoots increased from 6.2 cm to 6.8 cm, and the complete rate of xylemization of the callus at the grafting interface increased from 88% to 93%. The active ingredients in the pine polypeptide extract significantly enhanced the cell recognition and fusion ability of the stock and the scion, and copper abietinate promoted the synthesis efficiency of lignin by releasing copper ions. The synergistic effect of the two added components further optimized the physiological microenvironment of the grafting interface, making the callus process more efficient and stable. The traditional method does not use these active ingredients, resulting in relatively low callus speed and quality, and the technical improvement of Example 3 effectively solves this problem.

[0145] In another aspect, the method for grafting Pinus tabulaeformis Carr. with Pinus yunnanensis Franch. var. tibetica Cheng et L.K.Fu comprises the following steps:

[0146] Covering the grafted seedlings with a shading net having a light transmittance of 30-40% for 7-10 days, and then adjusting the light transmittance in stages:

[0147] a) replacing the shading net with a shading net having a light transmittance of 50-60% for 1-3 days;

[0148] b) removing the shading net completely from the fourth day, and spraying the grafted interface with atomized water once a day from 10:00 to 14:00, with a spraying amount of 0.1-0.2 mL / cm2, until the length of the new shoots reaches 5-6 cm. 2

[0149] The traditional grafting method for Pinus tabulaeformis Carr. usually adopts a single shading treatment after grafting, such as using a shading net with a fixed light transmittance or not shading at all, and lacks dynamic adjustment of light intensity. The prior art has the following problems: a fixed shading rate cannot meet the light requirements of grafted seedlings at different healing stages; sudden removal of the shading net can easily cause light stress of the scion; and without the combination of atomized water spraying to adjust the local microenvironment, the grafted interface is prone to dehydration in strong light periods. In the traditional method, the shading treatment is disconnected from water management, resulting in growth stagnation or dehydration death of the scion due to environmental changes.

[0150] The present application adopts shading adjustment in stages combined with atomized water spraying, which has significant improvements compared with the prior art: a black shading net having a light transmittance of 30-40% is used to provide high-intensity shading protection for 7-10 days in the early stage, a white shading net having a light transmittance of 50-60% is used to gradually enhance light adaptability for 1-3 days in the transition period, and atomized water is sprayed from 10:00 to 14:00 every day after the shading net is completely removed to alleviate the risk of dehydration in strong light. The prior art lacks such a dynamic adjustment mechanism, and excessive shading inhibits photosynthesis or insufficient shading causes scion burns, while the present application achieves a balance between light intensity and humidity through the synergy of phased shading and precise spraying.

[0151] ​The shading treatment method of the scheme realizes accurate regulation through three stages: in the initial stage of 7-10 days, the shading net with a light transmittance of 30-40% is used to effectively inhibit the transpiration of the scion and protect the newly formed callus from damage by strong light; in the transition period of 1-3 days, the shading net with a light transmittance of 50-60% is replaced to gradually enhance the light adaptation and avoid photoinhibition caused by sudden exposure; after the shading net is completely removed, 0.1-0.2 mL / cm 2 of atomized water is sprayed from 10:00 to 14:00 every day to specifically alleviate the water evaporation pressure during the strong light period, which continues until the new shoots grow to 5-6 cm to ensure a safe transition. This method can keep the humidity and light intensity of the grafting interface within the optimal range by dynamically adjusting the shading rate and precise spraying, and the survival rate is increased by more than 15% compared with the traditional fixed shading, and the new shoots grow healthily without dehydration or burning.

[0152] In another scheme, in the method for grafting Pinus tabulaeformis with Pinus yunnanensis var. tibetica, the Pinus tabulaeformis seedlings with a diameter of 0.4-0.7 cm and a height of 1-1.5 m are selected as the rootstocks;

[0153] The terminal branches of Pinus yunnanensis var. tibetica with a diameter of 0.4-0.7 cm are selected as the scions, and 8-12 cm branch segments are cut off;

[0154] The grafting tape is a blue polyethylene film with a light transmittance of 60-70% and a thickness of 0.03-0.05 mm.

[0155] In the traditional grafting method of Pinus tabulaeformis, the selection criteria of the rootstock and the scion are relatively wide, usually only considering basic indicators such as diameter and height, and lacking precise control of the physiological state of the material. The existing technology has the following problems: the diameter range of the rootstock is too large, resulting in inconsistent cambium thickness, affecting the contact quality of the rootstock and the scion; the scion is taken from the adult tree branches, and the degree of lignification is too high, and the cell division activity is insufficient; the light transmittance and color of the grafting tape are selected randomly, and the light intensity and photosynthetic demand cannot be considered.

[0156] In the present scheme, the rootstock is strictly limited to Pinus tabulaeformis seedlings with a diameter of 0.4-0.7 cm and a height of 1-1.5 m, ensuring that the cambium develops moderately and is easy to operate; the scion is selected from the terminal branches of Pinus yunnanensis var. tibetica with a diameter of 0.4-0.7 cm, and 8-12 cm branch segments are cut off to ensure the activity of the apical meristem; the grafting tape is a blue polyethylene film with a light transmittance of 60-70% and a thickness of 0.03-0.05 mm. In the existing technology, the diameter matching of the rootstock and the scion is poor, the degree of lignification of the adult tree scion is too high, and the grafting tape is usually ordinary transparent plastic film, which cannot optimize the spectral transmittance.

[0157] Select the diameter of 0.4-0.7 cm of Pinus tabulaeformis seedling as rootstock, height 1-1.5 m for easy operation and uniform cambium thickness, improve the contact of stock and scion; Select adult Pinus yunnanensis terminal branch as scion, diameter 0.4-0.7 cm to ensure semi-lignified state, 8-12 cm branch to retain apical dominance to promote callus; Blue polyethylene film transmittance 60-70% to filter harmful red light band, thickness 0.03-0.05 mm to balance the fixing force and air permeability, blue spectrum to promote cambium cell differentiation. This method solves the problem of callus obstacle caused by physiological state difference of materials in traditional grafting through precise control of stock and scion specifications and functional grafting tape design.

[0158] In another aspect, in the method of grafting Pinus tabulaeformis with Pinus yunnanensis, before applying the ethanol aqueous solution, a sterile cotton swab is used to wipe in one direction along the interface cambium direction to remove exuded resin, and after wiping, it is left to stand for 8-10 min.

[0159] When applying the ethanol aqueous solution, a trace element solution containing 0.01-0.03% manganese sulfate and 0.005-0.01% zinc sulfate is applied at the same time, and the application amount is 0.02-0.05 mL / time.

[0160] In the traditional Pinus tabulaeformis grafting method, during the interface treatment process, the ethanol aqueous solution is usually applied directly without removing the resin exudate, which causes the following problems in the interface area: resin accumulation hinders the contact of stock and scion cells; manganese, zinc and other trace elements are not supplemented, affecting the enzyme activity and lignification process of callus; the application of ethanol aqueous solution lacks standardized operation, which easily causes local concentration to be too high to damage the newly formed cells.

[0161] This aspect increases the steps of resin removal and trace element supplementation before applying the ethanol aqueous solution: a sterile cotton swab is used to wipe in one direction along the cambium direction to completely remove the resin without damaging the cell structure, and it is left to stand for 8-10 min to make the wound slightly dry to create a clean interface for subsequent treatment; a solution containing manganese sulfate (0.01-0.03%) and zinc sulfate (0.005-0.01%) is applied at the same time, manganese activates phenylalanine ammonia lyase to promote lignin synthesis, zinc enhances superoxide dismutase activity to improve antioxidant capacity, which complements the antibacterial function of the ethanol aqueous solution; by precisely controlling the application amount (0.02-0.05 mL / time), it is ensured that the ethanol aqueous solution and trace elements uniformly cover the interface area without overflow, avoiding local concentration to be too high to damage the newly formed cells. Due to the lack of resin removal and trace element cooperative treatment in the prior art, the metabolic imbalance, lignification delay and risk of pathogen growth in the interface area are significantly increased.

[0162] The resin removal step of the scheme thoroughly removes the resin exudate in the interface zone by one-way wiping and static treatment, providing a clean interface for subsequent solution infiltration; in the trace element supplement, manganese sulfate directly promotes the activity of key enzymes in lignin synthesis, zinc sulfate protects the callus from oxidative damage through an antioxidant mechanism, and the synergistic effect of the two with ethanol aqueous solution realizes triple regulation of bacteria inhibition, nutrition, and oxidation; precise application design ensures uniform distribution of the solution and does not destroy the microenvironment balance, and an application amount of 0.02-0.05 mL matches the area of the interface zone, which meets the needs and avoids waste or local overload. The whole process significantly improves the metabolic efficiency of the interface zone, increases the lignification integrity rate by more than 20%, and reduces the risk of mold infection to less than 5%.

[0163] Although the embodiments of the present application have been disclosed as above, they are not limited only to the uses listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily made by those skilled in the art, and therefore the present application is not limited to specific details, without departing from the general concept defined by the claims and equivalent scope.

Claims

1. A method for grafting Pinus tabulaeformis onto Pinus yunnanensis var. bretschneideri, characterized in that, The method comprises the following steps: a vertical incision is made on the rootstock at a position 2.5-3.5 cm from the surface of the cultivation substrate, the vertical incision reaches the xylem and has a length of 1.8-2.2 cm; the base of the scion is cut into a symmetrical double-wedge shape, and the length of the cut surface is 1.5-2.0 cm; the cut surface of the scion is immersed 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 s, the concentration of the sodium citrate buffer solution is 0.04-0.06 mol / L, and the pH value is 6.0-6.8; absorbing glue is applied on both sides of the vertical incision, and the absorbing glue is prepared by mixing activated carbon powder with a particle size of 40-50 mesh and a hydroxyethyl cellulose solution with a mass fraction of 8-12 % at a mass ratio of 1:2.5-3.5, and then adding nano silicon dioxide with a mass fraction of 0.3-0.5 % of the activated carbon powder and uniformly stirring; the scion is inserted into the vertical incision of the rootstock, and the grafting tape is used to fix the grafting interface after the scion is aligned with the cambium; a 45-55 % ethanol aqueous solution containing 0.7-0.9 % salicylic acid, 1-3 % trehalose and 0.2-0.4 % polysorbate 80 is applied to the grafting interface every 2-3 days, and the application amount is 0.05-0.15 mL / time; the day temperature is maintained at 23-25 ℃, the night temperature is maintained at 16-18 ℃, and the humidity is maintained at 90-95 %; when the new shoots of the scion are 2-3 cm long, the main stem of the rootstock is girdled at a position 0.8-1.2 cm above the vertical incision and reaches the xylem, and the main stem above the girdling position is cut off after 7-10 days; after grafting, shading treatment is performed.

2. The method of growing Pinus oiliva x P. yunmensis as claimed in claim 1, wherein, after the cut surface of the scion is immersed in the sodium citrate buffer solution, the base of the scion is wrapped with a breathable film for 8-10 min, and then the film is removed before the grafting tape is fixed.

3. The method of growing Pinus sylvestris grafted with Pinus yunnanensis as claimed in claim 1, wherein, before the scion is inserted into the vertical incision of the rootstock, a naphthaleneacetic acid solution with a mass fraction of 0.02-0.05 % is uniformly sprayed on the cut surface of the scion, and then the subsequent operations are performed after the scion is left to stand for 30-60 s.

4. The method of growing Pinus sylvestris grafted with Pinus yunnanensis as claimed in claim 1, wherein, after the main stem above the girdling position is cut off, a composite protective agent is immediately applied to the cut surface of the rootstock, the application thickness is 0.5-1.0 mm, and the surface is left to stand for 2-4 min until a gel film is formed on the surface; the composite protective agent is prepared by mixing the following components at a mass percentage: humic acid 0.3-0.5 %, chitosan 0.1-0.2 %, nano zinc oxide 0.01-0.03 %, and deionized water as the balance; the composite protective agent is prepared by stirring and dissolving humic acid and deionized water at 50-60 ℃, then adding chitosan and continuously stirring until the chitosan is completely dissolved, and finally adding nano zinc oxide and ultrasonic dispersing for 20-30 min.

5. The method of growing Pinus sylvestris grafted with Pinus yunnanensis as claimed in claim 4, wherein, After the cutting surface of the stock is coated with the composite protective agent to form a gel film on the surface, a phosphate buffer containing 0.1-0.3% indole butyric acid, 0.05-0.1% gibberellin and 0.01-0.03% vitamin B1 is sprayed on the gel film, with the spraying amount being 0.1-0.2 mL / cm 2 , the concentration of the phosphate buffer being 0.05-0.1 mol / L, and the pH value being 6.0-6.

5.

6. The method of growing Pinus oiliva x P. yunnanensis as claimed in claim 1, wherein, the sodium citrate buffer solution further contains 0.002-0.005 % pine polypeptide extract and 0.001-0.003 % copper turpentine acid, and the purity of the copper turpentine acid is ≥95 %; the pine polypeptide extract is prepared by the following method: healthy one-year-old phloem tissues of Pinus roxburghii are rapidly frozen in liquid nitrogen and ground to a particle size of ≤0.5 mm; The pre-cooled Tris-HCl buffer with pH 7.2-7.4, containing 0.1 mol / L NaCl and 5 mmol / L EDTA, was added in a mass ratio of 1:8-10, and the ultrasonic-assisted extraction was carried out at 4 ℃, with an ultrasonic power of 300 W, in a pulse mode of 5 s working and 10 s intermittent, for a total extraction time of 30 min; Centrifugal separation was carried out at 12000 rpm for 15 min at 4 ℃, and the supernatant was sequentially subjected to fractionation by 10 kDa and 3 kDa ultrafiltration membranes; The 3-10 kDa component was desalted by a Sephadex G-25 column, and the pine polypeptide extract was obtained after freeze-drying.

7. The method of growing Pinus oiliva x P. yunnanensis as claimed in claim 1, wherein, The specific method of shading treatment is as follows: Cover the grafted seedlings with a shading net with a light transmittance of 30-40% for 7-10 days, and then adjust the light transmittance in stages: a) Change to a shading net with a light transmittance of 50-60% for 1-3 days; b) On the 4th day, the shading net was completely removed, and the interface area was sprayed with atomized water once a day from 10:00 to 14:00, with a spraying amount of 0.1-0.2 mL / cm 2 , until the length of the new shoots reached 5-6 cm.

8. The method of growing Pinus oiliva x P. yunmensis as claimed in claim 1, wherein, Select Pinus tabulaeformis seedlings with a diameter of 0.4-0.7 cm and a height of 1-1.5 m as rootstocks; Select terminal branches of P. densata adult trees with a diameter of 0.4-0.7 cm as scions, and cut 8-12 cm long branches; The grafting tape is a blue polyethylene film with a light transmittance of 60-70% and a thickness of 0.03-0.05 mm.

9. The method of growing Pinus oiliva x P. yunmensis as claimed in claim 1, wherein, Before applying the ethanol aqueous solution, use a sterile cotton swab to wipe the interface in one direction to remove the exuded resin, and then stand for 8-10 min 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, with an application amount of 0.02-0.05 mL / time.