High-survival-rate grafting method for grafting pinus sylvestris onto Korean pine in arid region

Abstract

The invention discloses a high-survival-rate grafting method for grafting pinus sylvestris to pinus koraiensis in arid regions, which comprises the following steps: selecting pinus sylvestris stock with the growth period of 4-6 years, flatly cutting below a terminal bud branch point to form a cross section, downwards and obliquely cutting twice from the cross section to form a triangular pyramid-shaped chimeric structure, cutting a semi-lignified pinus koraiensis scion with a terminal bud, and grafting the semi-lignified pinus koraiensis scion with the terminal bud to obtain the pinus sylvestris scion. After main buds are reserved and lateral buds are cut off, two oblique triangular xylem sections are obliquely cut out from the lower portion of spores through two cutters, and a double-slope wedge-shaped joint body is formed; when the rootstock and the scion are cut, the cutting blade is immersed in an aqueous solution containing a plant growth regulator and a bactericide; the double-slope wedge-shaped joint body is inserted into the triangular pyramid-shaped embedding structure, so that the scion medullary layer and the stock medullary layer are overlapped; wrapping the interface with a composite hydrogel film containing a temperature-sensitive polymer and an antibacterial component; shading treatment is carried out after grafting is completed. The method can significantly improve the survival rate of grafted seedlings.

Description

Technical Field

[0001] The present invention relates to the technical field related to grafting technology, and more specifically, to a grafting method with high survival rate for grafting Pinus sylvestris var. mongolica and Pinus koraiensis in arid areas. Background Art

[0002] In the process of cultivating and planting red pine, using Larix gmelinii as the rootstock for red pine grafting can combine the excellent characteristics of Larix gmelinii and Pinus koraiensis, such as the strong stress resistance and wide ubiquity of Larix gmelinii and the high economic value of Pinus koraiensis, thereby achieving efficient cultivation of red pine.

[0003] However, there are still many problems to be solved in the existing technology of grafting Pinus sylvestris var. mongolica to Pinus koraiensis, the most prominent of which is the low grafting survival rate, poor wound healing, and the impact on the yield and wound firmness. During the grafting process, a good callus needs to be formed at the junction of the stock and the scion to achieve effective transmission of nutrients and water. However, the existing grafting method is not fine enough in the treatment of the stock and the scion, which makes it difficult for the cambium and the pith layer of the two to be accurately docked, thereby affecting the formation and development of the callus, and ultimately reducing the success rate of the grafting. The interface is prone to water loss, which is also a major problem in the prior art, especially in arid areas. Moisture retention at the interface is crucial for the formation and growth of callus. However, existing grafting protection measures often fail to provide a stable and suitable humidity environment for the interface. On the one hand, it is difficult to balance the air permeability and moisture retention of traditional wrapping materials (plastic film wrapped in multiple layers). At high temperatures, the water loss at the interface is too fast, resulting in dry interface, affecting the activity and metabolism of cells; at low temperatures, the humidity of the interface cannot be effectively maintained, which is also not conducive to the formation of callus. On the other hand, in the existing grafting operation, improper handling of cutting tools fails to effectively inhibit the growth and reproduction of microorganisms, making the interface susceptible to bacterial infection, further destroying the moisture balance of the interface and aggravating the problem of water loss at the interface.

[0004] Therefore, it is necessary to design a technical solution that can overcome the above-mentioned defects. Summary of the invention

[0005] One object of the present invention is to provide a grafting method with high survival rate for grafting Pinus sylvestris var. mongolica onto Pinus koraiensis in arid areas, which can improve the survival rate of grafted seedlings.

[0006] In order to achieve these purposes and other advantages of the present invention, the present invention provides a high survival rate grafting method for Pinus sylvestris var. mongolica grafted onto Pinus koraiensis in arid areas, comprising: selecting a Pinus sylvestris var. mongolica rootstock with a growth period of 4 to 6 years, flatly cutting below the branch point of the terminal bud to form a cross section, obliquely cutting two knives downward from the cross section to form a triangular pyramid mosaic structure, intercepting a semi-lignified Pinus koraiensis scion with a terminal bud, retaining the main bud and cutting off the side buds, obliquely cutting two oblique triangular xylem sections from below the spore with two knives to form a double pyramid mosaic structure matching the triangular pyramid mosaic structure. Bevel wedge joint body; when cutting the rootstock and scion, immerse the cutting blade in an aqueous solution containing a plant growth regulator and a fungicide, and repeat the dipping treatment after each cutting is completed; insert the double-bevel wedge joint body into the triangular pyramid-shaped mosaic structure so that the pith layer of the scion overlaps the pith layer of the rootstock; use a composite hydrogel film containing a thermosensitive polymer and an antibacterial component to wrap the interface, the hydrogel film remains in a swollen state at low temperatures to maintain the humidity of the interface, and shrinks at high temperatures to increase air permeability; after the grafting is completed, a light-shielding treatment is performed.

[0007] Furthermore, the aqueous solution containing the plant growth regulator and the fungicide comprises 0.05-0.15% indolebutyric acid and 0.3-0.8% carbendazim wettable powder mixed in a volume ratio of 1:1, and nanosilver particles with a particle size of 5-15 nm are added to a final concentration of 0.1-0.3%.

[0008] The cutting blade is dipped for 10-15 seconds each time, and the liquid is left to stand for 5-8 seconds after dipping, so that a uniform liquid film is formed on the surface of the blade; the particle size of the nanosilver particles in the liquid film is 5-15nm, and the particle spacing is 50-100nm.

[0009] Furthermore, the preparation method of the composite hydrogel film comprises: Dispersing poly (N-isopropylacrylamide) and 0.1-0.5% by mass of silver nanoparticles in deionized water, adding 5-15% by mass of chitosan, 0.5-1% by mass of dopamine and 0.1-0.3% by mass of quercetin, and forming a uniform prepolymer solution by magnetic stirring; The prepolymer solution was coated on the surface of the polyethylene substrate, and then frozen at -20°C for 12 hours and thawed at room temperature for 6 hours to form a porous temperature-sensitive substrate. Spraying a crosslinking agent solution containing 0.1-0.5% glutaraldehyde onto the surface of the substrate, and letting it stand for 2-4 hours to complete chemical crosslinking to form a composite hydrogel film; The composite hydrogel film has a swelling ratio of 250-300% at 20°C, and the swelling ratio drops to 60-80% at 37°C.

[0010] Furthermore, the specific operation of the stock incision is: Cut the stem horizontally 3-5 cm below the branch point of the terminal bud to form a 90° incision with a cross-sectional flatness error of ≤0.1 mm; make two oblique cuts downward from the cross section with a cutting length of 3-5 cm to form a triangular pyramid-shaped mosaic structure.

[0011] Furthermore, starting from 2 mm below the spore, two inclined triangles with a length of 3-5 cm are cut along the axis direction of the scion to form a double-bevel wedge-shaped joint body.

[0012] Furthermore, after docking, a pulsed pressure device is used to continuously apply pressure for 5-10 seconds to promote the contact of the medullary parenchyma cells.

[0013] Furthermore, after grafting, a black sunshade net with a shading rate of 75% was covered for 2-3 weeks; The shading is lifted in three stages, with each stage reducing the shading rate by 25%, with an interval of 7 days.

[0014] Furthermore, the step of cutting out two sloped triangles also includes: the angle between the two slopes is 25°-35°, and the cutting blade maintains an inclination angle of 15°-20° with the axis of the scion; The section roughness Ra≤0.8μm, and the intersection line of the two inclined surfaces deviates from the medullary axis by an angle of <1°.

[0015] Furthermore, the specific operation of the pulse pressurizing device is: After the scion and the rootstock are butt-jointed, a pulse pressure of 0.1-0.2 MPa is applied through a pneumatic device, with a pulse frequency of 1-3 Hz, and the pressure is applied continuously for 5-10 seconds; The pressure fluctuation of the device is ≤±5%, the pressure interface is in contact with a silicone pad, the hardness of the silicone pad is Shore A20-30, and the angle deviation between the pressure direction and the scion axis is <3°.

[0016] Furthermore, the post-grafting management also includes: Cover the outside of the scion with a black sunshade net with a shading rate of 75% and continue covering it for 2-3 weeks; From the third week onwards, the shading will be lifted in three stages, with each stage reducing the shading rate by 25%, with an interval of 7 days. The first stage is the third week: the light blocking rate drops to 50%, the light transmission wavelength range is 500-700nm, and the UV blocking rate is ≥85%; The second stage is the fourth week: the light blocking rate drops to 25%, the light transmission wavelength extends to 400-700nm, and the UV blocking rate is ≥70%; The third stage is the fifth week: completely remove the shade nets, and cover them with a temporary net with a 30% shading rate from 10:00 to 14:00 every day within 3 days after the removal of the nets, with a single covering time of ≤2 hours.

[0017] The present invention has at least the following beneficial effects: In the cutting process, the present invention immerses the blade into an aqueous solution containing plant growth regulators, bactericides and nano-silver particles, which can not only promote the growth of stock and scion cells, but also effectively inhibit pathogens, reduce the risk of infection, and lay the foundation for successful grafting. The unique temperature sensitivity of the composite hydrogel film is utilized to make it swell and retain moisture at low temperatures, and shrink and ventilate at high temperatures, providing a stable and suitable humidity and gas environment for the interface, greatly reducing the problem of interface water loss, and is conducive to callus formation and growth. The grafting method of the present invention effectively overcomes the shortcomings of the prior art, significantly improves the survival rate of Korean pine grafting, and has broad application prospects.

[0018] Other advantages, objectives and features of the present invention will be embodied in part through the following description, and in part will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a schematic diagram of a longitudinal section of a scion or rootstock according to an embodiment of the present application; Figure 2 This is a schematic diagram of a cross section of a scion or rootstock according to an embodiment of the present application; Figure 1 and Figure 2 In the figure, 1 represents spore and 2 represents incision. DETAILED DESCRIPTION

[0020] The present invention is further described in detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0021] It should be understood that the terms such as "having", "including" and "comprising" used in the embodiments of the present application do not exclude the existence or addition of one or more other elements or their combinations. All directional indications (such as up, down, left, right, front, back...) in the embodiments of the present application are only used to explain the relative position relationship, movement, etc. between the components in a certain specific posture. If the specific posture changes, the directional indication will also change accordingly. When an element is referred to as "fixed on" or "set on" another element, it can be directly on the other element or there may be a centering element at the same time. When an element is referred to as "connecting" another element, it can be directly connected to another element or it can be indirectly connected to another element through a centering element. The description of "first", "second", etc. in the embodiments of the present application is only for descriptive purposes, and cannot be understood as indicating or implying its relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" can explicitly or implicitly include at least one of the features.

[0022] It should be noted that the technical solutions between the various embodiments of the present application can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0023] The embodiment of the present application provides a high survival rate grafting method of Pinus sylvestris var. mongolica grafted onto Pinus koraiensis in arid areas, comprising: Select 4-year-old seedlings of Scots pine or 5-year-old transplanted seedlings, cut horizontally 0.5 cm below the branch point of the terminal bud to form a 90° cross section, and use a blade to cut two 3-5 cm long incisions downward from the cross section to form a triangular pyramid mosaic structure. Figure 1 and Figure 2 .

[0024] See also Figure 1 The cutting of the scion starts from 2 mm below the spore 1, and the double-beveled triangular wood section is cut with two blades. Figure 2 The length of the single bevel is 3-5cm, and the angle between the two bevels is 25-35° (preferably 30°), forming a double-bevel wedge-shaped joint; during the cutting process, the angle between the blade and the scion axis is kept at 15-20° to ensure that the cut surface is smooth and free of burrs.

[0025] During the cutting process, the blade needs to be immersed in a mixed solution containing 0.05-0.15% indolebutyric acid, 0.3-0.8% carbendazim and 0.1-0.3% nanosilver, and each time it is dipped for 10-15 seconds, the liquid is drained for 5-8 seconds. Insert the double-bevel wedge-shaped joint body into the triangular pyramid-shaped mosaic structure, so that the pith layer of the scion overlaps with the pith layer of the rootstock, ensuring that the pith layer overlaps by more than 1.5 cm, and wrap the interface with a composite hydrogel film (covering 2 cm above and below the incision), and immediately cover it with a 75% shading rate sunshade net after grafting.

[0026] This embodiment can well adapt to the situation where the scion and the rootstock have different sizes. For example, as the scion size changes, the length of the incision on the cross section of the rootstock can be adaptively adjusted (until the cross section is cut off), see Figure 2 Multiple dotted lines in the figure are used to ensure that the pith and cambium of the two overlap. Further, in this embodiment, the pith and cambium of the two are overlapped by flat-cut stock and double-bevel scion design. Regardless of the diameter difference between the stock and the scion, the bevel triangle contact can fit the pith, xylem and cambium tightly, avoiding the exposure of the scion, water loss, and infection by bacteria, which may cause grafting failure. The contact area of ​​the cambium can be increased by 50%, and the swelling ratio of the hydrogel film at 200-300% at 20°C can be used to control the interface water loss rate below 8%. The synergistic effect of nanosilver and carbendazim reduces the bacterial infection rate to below 2%, retains the branching point of the stock apical bud to maintain the apical dominance, shortens the callus formation time to 7-10 days, and achieves a survival rate of more than 97%.

[0027] In another embodiment, the blade treatment liquid is mixed with 0.05-0.15% indolebutyric acid (IBA) and 0.3-0.8% carbendazim wettable powder in a volume ratio of 1:1, and 5-15nm nanosilver particles are added to 0.1-0.3%. In the specific operation, the grafting knife is completely immersed in the solution for 10-15 seconds, and then taken out and vertically left to stand for 5-8 seconds to form a uniform liquid film of 5-10 μm thickness on the blade edge. This treatment is repeated before each cutting of the rootstock or scion to ensure that the active ingredient is always attached to the blade surface.

[0028] Indolebutyric acid promotes callus differentiation through liquid membrane penetration incision, and the combination of carbendazim and nanosilver produces a synergistic antibacterial effect. The small particle size of nanosilver particles (5-15nm) enables them to penetrate deeply into the xylem vessels, forming continuous and long-term antibacterial protection, avoiding cross-contamination caused by continuous grafting operations, and greatly reducing the infection rate.

[0029] In another embodiment, the preparation of the hydrogel film is divided into three steps: first, poly N-isopropylacrylamide and 0.1-0.5% silver nanoparticles by mass are dispersed in deionized water, chitosan accounting for 5-15% of the total mass of the system, 0.5-1% dopamine and 0.1-0.3% quercetin are added, and a uniform prepolymer solution is formed by magnetic stirring; secondly, the prepolymer solution is coated on the surface of the polyethylene substrate, frozen at -20°C for 12 hours and then thawed at room temperature for 6 hours to form a porous temperature-sensitive substrate; finally, a crosslinking agent solution containing 0.1-0.5% glutaraldehyde is sprayed on the surface of the substrate, and the chemical crosslinking is completed for 2-4 hours to form a composite hydrogel film; the swelling ratio of the hydrogel film is 250-300% at 20°C, and the swelling ratio is reduced to 60-80% at 37°C. . The swelling ratio of the finished membrane is 200-300% at 20°C and drops to 50-80% at 37°C. The silver particles are evenly distributed in the pores through electrostatic adsorption.

[0030] In this embodiment, quercetin and silver nanoparticles produce a synergistic antibacterial effect, and the antibacterial rate of common pathogens (such as Fusarium and Botrytis cinerea) is increased from 85% to 99.5%, and the antibacterial effect is extended to more than 30 days. The antioxidant properties of quercetin (free radical scavenging efficiency ≥ 90%) can inhibit interface oxidative stress damage and reduce the risk of callus necrosis. The swelling ratio of 250-300% at 20°C can lock in moisture, which is particularly beneficial for arid areas. At 37°C, it shrinks to a swelling ratio of 60-80%, and the air permeability is increased by 3 times, avoiding interface mildew caused by high temperature and high humidity environments.

[0031] Thermosensitive materials maintain high moisture content below 25°C, providing the interface with a relative humidity of more than 95%. After shrinking above 30°C, the porosity increases to 40%, preventing tissue decay caused by high temperature and humidity, keeping the interface moist and breathable, and helping to improve the survival rate. Chitosan enhances the adhesion between the membrane and plant tissue, reducing the thermal resistance of the membrane-tissue interface by 40%, and the sustained-release concentration of silver nanoparticles is maintained in the safe antibacterial range of 0.1-0.5ppm, which greatly reduces the infection rate and improves the survival rate of grafted seedlings.

[0032] In another embodiment, a blade is used to cut the stem at a 90° angle to ensure that the cross-sectional flatness error is ≤0.1 mm, and the stem is cut 3-5 cm below the branch point of the apical bud to form a 90° incision with a cross-sectional flatness error of ≤0.1 mm; two oblique cuts are made downward from the cross-section, with a cutting length of 3-5 cm to form a triangular pyramid-shaped mosaic structure, retaining 7-10 healthy needles below the incision to maintain the photosynthetic capacity of the rootstock.

[0033] In another embodiment, see Figure 1 and Figure 2 Scion cutting starts from 2mm below the spore, and a double bevel is cut along the scion axis using a razor blade. The length of a single bevel is 3-5cm, and the angle between the two bevels is 25-35° (preferably 30°), forming a double-bevel wedge-shaped joint. During the cutting process, keep the angle between the blade and the scion axis at 15-20° to ensure that the cut surface is smooth and burr-free. The pith is the central part of the stem, composed of loose thin-walled cells that store nutrients and have active cell division, making it easy to induce callus. When grafting, the pith of the scion and the pith of the rootstock are connected obliquely to reduce the rate of incorrect connection, and through the full fusion of the thin-walled cells of the two, new vascular bundles are gradually differentiated.

[0034] In another embodiment, after docking is completed, a pneumatic pressure device is used to apply a pressure of 0.1-0.2 MPa at the interface for 5-10 seconds. The pressure device uses a silicone pad to contact the interface to ensure that the pressure is evenly distributed and avoid local stress concentration. During the pressurization process, the fit between the scion and the stock is observed to ensure that the pith layer is completely aligned without misalignment.

[0035] In another embodiment, shading management is divided into three stages: covering with 75% shading rate black shading net in the first 21 days after grafting, replacing with 50% shading net on the 22nd to 28th day, and using 25% shading net on the 29th to 35th day.

[0036] The following is a description of specific embodiments.

[0037] Embodiment 1: 1. Material preparation Rootstock: Select 4-year-old Larix gmelinii (top bud diameter about 1.0 cm), seedling height 70-80 cm, and retain 7-10 healthy needles at the base.

[0038] Scion: Collect semi-lignified Korean pine branches (0.8-1.2cm in thickness) of the current year, cut off 8-10cm segments with terminal buds, and remove all side buds.

[0039] Blade treatment fluid: 0.1% indolebutyric acid (IBA) + 0.5% carbendazim wettable powder (volume ratio 1:1) + 10nm nanosilver particles (final concentration 0.2%) were mixed and placed in a 50mL sterile bottle.

[0040] Composite hydrogel membrane: Preparation of prepolymer solution: (1) Disperse poly (N-isopropylacrylamide) and 0.3% by mass of silver nanoparticles in deionized water, add 10% by mass of chitosan, 0.5% dopamine and 0.2% quercetin, and form a uniform prepolymer solution by magnetic stirring; (2) Freeze-thaw: The prepolymer solution is coated on the surface of the polyethylene substrate, frozen at -20°C for 12 hours and then thawed at room temperature for 6 hours to form a porous temperature-sensitive substrate; (3) Chemical crosslinking: Spray a crosslinker solution containing 0.1-0.5% glutaraldehyde onto the substrate surface and let it stand for 2-4 hours to complete the chemical crosslinking and form a composite hydrogel film with a thickness of 0.2 mm (20°C swelling ratio 250%, 37°C swelling ratio 60%).

[0041] 2. Grafting operation steps 1. Stock treatment At 4 cm below the branching point of the terminal bud, the stem was cut flatly (flatness error ≤ 0.1 mm) using a scalpel soaked in the treatment solution to form a 90° cross section.

[0042] Two oblique cuts were made downward from the cross section, with a cutting length of 3.5 cm to form a triangular pyramid-shaped mosaic structure.

[0043] 2. Scion cutting 2mm below the scion spore (6mm from the top), cut a double-bevel triangle (single bevel length 3.5cm, angle 30°) at an angle of 15° toward the base to form a double-bevel wedge-shaped joint.

[0044] Each time you cut the scion, soak the blade in liquid again to ensure that there are no burrs on the cut surface.

[0045] 3. Docking and fixing Insert the double-bevel wedge-shaped joint into the triangular pyramid-shaped mosaic structure so that the pith layer of the scion overlaps with the pith layer of the stock, with the pith layer overlapping by 1-2 cm. Use a pneumatic pressurizing device (0.15 MPa, for 8 seconds) to make the interface fit tightly.

[0046] Immediately wrap the interface with hydrogel film (covering 2 cm above and below the incision), with the edge of the film extending 1 cm beyond the incision, and press lightly with your fingers until it is completely adhered.

[0047] 4. Shading management After grafting, build a 1.5m high sunshade, cover with 75% black sunshade net, and maintain light intensity at 1000-1500 lux. Replace 50% sunshade net (light intensity 2500 lux) on the 22nd day, replace 25% sunshade net (light intensity 4500 lux) on the 29th day, and completely remove the net on the 36th day.

[0048] Comparative Example 1: No blade treatment liquid was used, and the remaining parameters and processes were the same as those in Example 1.

[0049] Comparative Example 2: Ordinary plastic film (polyethylene) replaced the thermosensitive hydrogel film, and other parameters and processes were the same as those in Example 1.

[0050] test: 1. Survival rate calculation method: Definition: The proportion of plants whose scions remain vigorous and produce continuous growth (new shoot elongation ≥ 2 cm) after grafting. 1. Observation standard survival: The terminal bud of the scion germinates, the length of the new shoot is ≥ 2 cm, and the leaves are stretched and the color is normal (non-yellowing). There is no rot at the interface between the rootstock and the scion, and the base of the scion does not loosen when gently pressed with fingers. Failure to survive: The scion withers, the terminal bud necrose, or the interface is severely rotten, causing the plant to fall. 2. Statistical time and sample size Statistical time: 60 days after grafting (the callus tissue is fully mature and the growth trend is stable). Sample size: 200 plants per group, repeat the experiment 3 times, and take the average value. 3. Calculation formula Survival rate = number of surviving plants / total number of plants.

[0051] 2. Methods for determining infection rate Definition: The proportion of plants infected with pathogens (such as mold and bacterial rot) at the grafting interface. 1. Infection criteria Mild infection: White / gray mildew appears on the surface of the interface, with an area of ​​less than 1cm², and has not invaded the wood. Severe infection: The interface rots and turns black, and brown spots can be seen in the wood, accompanied by odor. All infection levels are included in the statistics. 2. Observation time points Regular inspection: Check once on the 7th, 14th, 21st and 30th days after grafting, and record the number of infected plants. Final statistics: The 30-day data shall prevail. 3. Calculation formula Infection rate = number of infected plants / total number of plants. 3. Methods for determining wound healing time Definition: The time from the completion of grafting to the formation of callus visible to the naked eye. 1. Observation method Daily observation: Starting from the 5th day after grafting, observe the interface with a 10x magnifying glass at 9:00 every day and record the date when the callus first appears. Callus standard: Milky white or light yellow spongy tissue is formed at the interface, covering ≥50% of the cut surface. 2. Data processing Single plant record: The callus time is recorded independently for each plant, accurate to 0.1 day (such as 7.2 days = 7 days and 5 hours). Average calculation: Take the median of 30 plants in each group (avoid the influence of extreme values).

[0052] The survival rate, infection rate and healing time data of Comparative Example 1, Comparative Example 2 and Example 1 are shown in the table below.

[0053] It can be seen that compared with Comparative Examples 1 and 2, Example 1 has significant advantages in survival rate (97.8%), infection rate (1.9%) and healing time (7.0 days). The possible reason is that 0.1% indolebutyric acid (IBA) in the blade treatment liquid accelerates callus differentiation, 0.2% nanosilver (10nm) and 0.5% carbendazim form a broad-spectrum antibacterial system, which significantly reduces the infection rate compared with traditional disinfection, and as the blade achieves instant disinfection of the cut surface, the composite hydrogel film reduces water loss during the moisturizing period while avoiding high temperature suffocation of the seedlings through the thermosensitive swelling properties of poly N-isopropylacrylamide (swelling ratio of 250% at 20°C / shrinkage to 60% at 37°C), and cooperates with chitosan to enhance membrane-tissue adhesion and improve healing efficiency. These technologies work together to significantly improve the survival rate of grafted seedlings.

[0054] According to another embodiment of the present invention, by optimizing the blade dipping treatment process, it is proposed to control the dipping time to 10-15 seconds, and let the drain stand for 5-8 seconds, so that a uniform liquid film with a thickness of 5-10μm is formed on the blade surface. In the prior art, the disinfection treatment of cutting tools mostly adopts a simple immersion or spraying method, which has problems such as uneven adhesion of the liquid and insufficient penetration of the effective ingredients, resulting in unstable antibacterial effect, and excessive drug residues may inhibit callus formation. The present invention ensures that the liquid film on the blade surface fully absorbs the bactericide and growth regulator, and avoids excessive droplet residues causing excessive local concentration by limiting the precise coordination of the dipping time and the drain standing time. Experiments show that when the dipping time is less than 10 seconds, the liquid film coverage is less than 60%, and when it is higher than 15 seconds, the liquid film is too thick (>15μm), resulting in waste of drugs and hindering the respiration of incision cells; when the drain standing time is shorter than 5 seconds, the liquid film is unevenly distributed, and when it is longer than 8 seconds, the liquid film thickness decreases significantly (<3μm), and the antibacterial activity decreases. In addition, nanosilver particles (5-15nm) are evenly dispersed in the liquid film and can penetrate into the micropores of the wood to form a long-lasting antibacterial barrier. This technical feature reduces the pathogen infection rate at the incision to less than 2%, while promoting the directional penetration of indolebutyric acid (IBA), shortening the callus formation time to 7-10 days, which is significantly better than the traditional method (12-14 days).

[0055] According to another embodiment of the present invention, by limiting the coordinated parameters of the stock incision position and depth (cutting the stem flatly at 3-5 cm from the branch point of the apical bud, and making two 3-5 cm cuts obliquely downward from the cross section to form a triangular pyramid-shaped mosaic structure), the problems of insufficient contact area of ​​the cambium layer and high misalignment rate of the pith layer in traditional grafting are innovatively solved. In the prior art, the stock incision position mostly relies on experience-based operations, lacks consideration of the physiological characteristics of the branch point of the apical bud, and easily leads to the incision being too close to damage the apical dominance, or too far to reduce the activity of the cambium layer; the incision depth also often fluctuates due to differences in the stock diameter. When it is too shallow (<3 cm), the pith layer is not fully exposed, and when it is too deep (>5 cm), the mechanical strength of the stock is destroyed, causing the risk of splitting. The present invention has been verified through experiments: when the incision is 3-5cm away from the branching point of the terminal bud, sufficient nutrient transport channels can be retained to maintain apical dominance, while ensuring that the contact area of ​​the cambium is maximized after the scion is inserted (increased by 50%-70%); the precise design of the 3-5cm long bevel cut downward from the cross section makes the overlapping length of the scion and the pith layer of the rootstock ≥1.5cm, promoting the fusion and differentiation of thin-walled cells. Comparative experiments show that when the incision position deviation exceeds ±1cm or the depth deviates from the range of 3-5cm, the callus formation time is extended to 12-15 days, and the survival rate drops below 80%. In addition, the incision flatness error is ≤0.1mm, which further reduces the interface gap and provides a basis for the sealing and fitting of the composite hydrogel membrane. This technical feature significantly improves the interface healing efficiency and makes the grafting survival rate exceed 98%.

[0056] According to another embodiment of the present invention, by limiting the precise parameters of scion cutting (starting from 2mm below the spore, cutting a double-bevel triangle with a length of 3-5cm along the axial direction to form a double-bevel wedge-shaped joint body matching the triangular pyramid-shaped mosaic structure), the problem of high misalignment rate and insufficient contact area between the cambium of the scion and the rootstock in traditional grafting is innovatively solved. In the prior art, the starting point of scion cutting is often randomly selected (such as the base of the spore or a random position), resulting in insufficient exposure of the cambium or deviation in cutting depth; the length of the bevel mostly depends on subjective judgment, and too long (>5cm) is likely to weaken the structural strength of the scion, and too short (<3cm) will result in insufficient contact area, affecting the efficiency of callus fusion. The present invention is verified by experiments: starting from 2mm below the spore, the dense area of ​​the scion vascular bundle can be accurately located, the activity of the apical spore can be retained (the germination rate is increased by 20%), and the cutting depth can be avoided to damage the pith parenchyma cells; the design of the double bevel length of 3-5cm increases the effective contact area of ​​the cambium by 60%-80%, and the angle between the two bevels is controlled at 25°-35° (preferably 30°), forming a stable wedge-shaped structure, which significantly reduces the risk of sliding after the scion is inserted (slip rate <3%). Comparative experiments show that when the cutting starting point deviation is ±1mm or the bevel length deviates from the range of 3-5cm, the interface porosity increases to 15%-20%, the callus formation time is extended to 12-15 days, and the survival rate drops to below 85%. In addition, the blade maintains an inclination angle of 15°-20° with the scion axis during cutting to ensure that the cut surface is smooth and burr-free (roughness Ra≤0.8μm), further reducing microbial attachment and water evaporation. This technical feature enables the grafting survival rate to exceed 98% through parameter synergistic optimization.

[0057] According to another embodiment of the present invention, by introducing a pulse pressurization device and limiting the pressurization parameters (lasting 5-10 seconds, pulse frequency 1-3Hz, pressure 0.1-0.2MPa), the problems of uneven contact of pith thin-walled cells and local stress concentration caused by traditional static pressurization are innovatively solved. In the prior art, the grafting interface mostly adopts fixed pressure clamping or simple bundling, which has defects such as uneven pressure distribution and large randomness of pressurization time, which can easily cause the scion to slide or the pith layer to be dislocated (dislocation rate>15%), and excessive pressure (>15 seconds) may damage the activity of thin-walled cells. The present invention has been verified through experiments: pulse pressurization (frequency 1-3Hz) can dynamically adjust the direction of pressure, promote micron-level reciprocating displacement (amplitude 10-20μm) between the scion and the stock incision, effectively eliminate the interface gap (porosity reduced to <2%), and accelerate the change of cell membrane permeability through periodic stress stimulation, and promote the diffusion of growth factors. Limiting the pressure application time to 5-10 seconds (preferably 8 seconds) can balance the pressure intensity and cell tolerance, and avoid cell membrane rupture caused by long-term pressure application (survival rate > 98%). Comparative experiments show that after pulsed pressure application, the contact area of ​​the medullary thin-walled cells increases by 40%-60%, the callus formation time is shortened to 5-7 days, and the survival rate is increased to more than 98%. In addition, the design of combining the silicone pad with the pneumatic device (pressure fluctuation ≤±5%) ensures uniform pressure application (local stress difference <0.01MPa), further reducing the risk of interface damage. This technical feature significantly improves the efficiency of cell fusion through the coordinated optimization of dynamic pressure mode and parameters.

[0058] According to another embodiment of the present invention, a post-grafting management method is designed to remove shading in stages (the initial shading rate is 75% and maintained for 2-3 weeks, and the shading rate is reduced by 25% every 7 days in three stages), which effectively solves the problem of light stress and dehydration damage to the scion caused by sudden changes in light in traditional post-grafting shading management. In the prior art, long-term coverage with a fixed shading rate (such as continuous shading at 75% until the scion survives) or one-time removal of shading is often used after grafting. The former is likely to cause the scion to grow too long and the photosynthetic capacity to deteriorate, while the latter causes chloroplast damage (light inhibition rate > 30%) and a surge in transpiration water loss rate (> 50%) due to a sudden increase in light. The present invention has been verified through experiments: the initial 75% shading rate (transmittance 25%) can inhibit the transpiration rate of the scion to 40%-50% of the normal value, while retaining sufficient photosynthetically active radiation (PAR≥200μmol·m⁻²·s⁻¹) to maintain basal metabolism; the shading is removed in stages (reducing the shading rate by 25% every 7 days) so that the scion gradually rebuilds the light adaptation mechanism, the chlorophyll a / b ratio is steadily increased from the initial 2.8 to 3.5 (±0.2), and the maximum quantum efficiency of the photosystem II (Fv / Fm) is maintained above 0.75 (the control group drops sharply to 0.55). Comparative tests show that the wilting rate of the scion reaches 35% when the shading is removed once, while the wilting rate is less than 5% under staged treatment; and the elongation of the new shoots of the scion in the staged group is increased by 40%-60% compared with the fixed shading group, and the lignification speed is accelerated by 15%. In addition, the black sunshade net (absorption rate of wavelength 400-700nm> 90%) can effectively block ultraviolet radiation (UV-B transmittance <5%), reduce membrane lipid peroxidation (MDA content reduced to 0.8μmol / g FW), and further ensure that the survival rate of the scion exceeds 98%. This technical feature dynamically regulates light intensity and adaptation cycle.

[0059] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A grafting method with high survival rate for Pinus sylvestris var. mongolica grafted onto Pinus koraiensis in arid areas, characterized in that: include: Select a 4-6-year-old Scots pine rootstock, cut flatly below the branch point of the terminal bud to form a cross section, cut two cuts obliquely downward from the cross section to form a triangular pyramid mosaic structure, cut a semi-lignified Korean pine scion with a terminal bud, retain the main bud and cut off the side buds, and then cut two oblique triangle wood sections from below the spore with two cuts obliquely to form a double-bevel wedge-shaped joint body matching the triangular pyramid mosaic structure; When cutting rootstock and scion, dip the cutting blade into a water solution containing plant growth regulators and fungicides, and repeat the dipping treatment after each cut; Insert the double-bevel wedge-shaped joint body into the triangular pyramid-shaped mosaic structure so that the pith layer of the scion overlaps with the pith layer of the rootstock; The interface is wrapped with a composite hydrogel film containing a thermosensitive polymer and an antibacterial component, wherein the hydrogel film remains swollen at low temperatures to maintain the humidity of the interface and shrinks at high temperatures to increase air permeability; After the grafting is completed, shading should be carried out.

2. The grafting method according to claim 1, characterized in that The aqueous solution containing the plant growth regulator and the fungicide comprises a mixture of indolebutyric acid with a mass concentration of 0.05-0.15% and carbendazim wettable powder with a mass concentration of 0.3-0.8% in a volume ratio of 1:1, and nanosilver particles with a particle size of 5-15 nm are added to a final concentration of 0.1-0.3%; The cutting blade is dipped for 10-15 seconds each time, and the liquid is left to stand for 5-8 seconds after dipping, so that a uniform liquid film is formed on the surface of the blade; the particle size of the nanosilver particles in the liquid film is 5-15nm, and the particle spacing is 50-100nm.

3. The grafting method according to claim 1, characterized in that The preparation method of the composite hydrogel film comprises: Dispersing poly (N-isopropylacrylamide) and 0.1-0.5% by mass of silver nanoparticles in deionized water, adding 5-15% by mass of chitosan, 0.5-1% by mass of dopamine and 0.1-0.3% by mass of quercetin, and forming a uniform prepolymer solution by magnetic stirring; The prepolymer solution was coated on the surface of the polyethylene substrate, and then frozen at -20°C for 12 hours and thawed at room temperature for 6 hours to form a porous temperature-sensitive substrate. Spraying a crosslinking agent solution containing 0.1-0.5% glutaraldehyde onto the surface of the substrate, and letting it stand for 2-4 hours to complete chemical crosslinking to form a composite hydrogel film; The composite hydrogel film has a swelling ratio of 250-300% at 20°C, and the swelling ratio drops to 60-80% at 37°C.

4. The grafting method according to claim 1, characterized in that The specific operation of the stock incision is: Cut the stem horizontally 3-5 cm below the branch point of the terminal bud to form a 90° incision with a cross-sectional flatness error of ≤0.1 mm; make two oblique cuts downward from the cross section with a cutting length of 3-5 cm to form a triangular pyramid-shaped mosaic structure.

5. The grafting method according to claim 1, characterized in that Starting from 2mm below the spore, cut out two inclined triangles with a length of 3-5cm along the axis of the scion to form a double-bevel wedge-shaped joint body.

6. The grafting method according to claim 1, characterized in that After docking, a pulsed pressure device is used to apply pressure for 5-10 seconds to promote contact between the medullary parenchyma cells.

7. The grafting method according to claim 1, characterized in that After grafting, cover with a black shade net with a shading rate of 75% for 2-3 weeks; The shading is lifted in three stages, with each stage reducing the shading rate by 25%, with an interval of 7 days.

8. The grafting method according to claim 5, characterized in that The step of cutting out two sloped triangles also includes: the angle between the two slopes is 25°-35°, and the cutting blade maintains an inclination angle of 15°-20° with the scion axis; The section roughness Ra≤0.8μm, and the intersection line of the two inclined surfaces deviates from the medullary axis by an angle of <1°.

9. The grafting method according to claim 1 or 6, characterized in that: The specific operation of the pulse pressurizing device is as follows: After the scion and the rootstock are butt-jointed, a pulse pressure of 0.1-0.2 MPa is applied through a pneumatic device, with a pulse frequency of 1-3 Hz, and the pressure is applied continuously for 5-10 seconds; The pressure fluctuation of the device is ≤±5%, the pressure interface is in contact with a silicone pad, the hardness of the silicone pad is Shore A 20-30, and the angle deviation between the pressure direction and the scion axis is <3°.

10. The grafting method according to claim 1 or 7, characterized in that: The post-grafting management also includes: Cover the outside of the scion with a black sunshade net with a shading rate of 75% and continue covering it for 2-3 weeks; Starting from the third week, the shading will be lifted in three stages, with each stage reducing the shading rate by 25%, with an interval of 7 days, as follows: The first stage is the third week: the light blocking rate drops to 50%, the light transmission wavelength range is 500-700nm, and the UV blocking rate is ≥85%; The second stage is the fourth week: the light blocking rate drops to 25%, the light transmission wavelength extends to 400-700nm, and the UV blocking rate is ≥70%; The third stage is the fifth week: completely remove the shade nets, and cover them with a temporary net with a 30% shading rate from 10:00 to 14:00 every day within 3 days after the removal of the nets, with a single covering time of ≤2 hours.

Images