A grafting method with high survival rate for Pinus sylvestris var. mongolica grafted onto Pinus koraiensis in arid areas

Abstract

The invention discloses a grafting method with a high survival rate of Pinus sylvestris var. mongolica grafted onto Pinus koraiensis in arid areas, comprising: selecting a Pinus sylvestris var. mongolica rootstock with a growth period of 4-6 years, flatly cutting below the terminal bud branch point to form a cross section, obliquely cutting two knives downward from the cross section to form a triangular pyramid-shaped mosaic structure, intercepting a semi-lignified Pinus koraiensis scion with a terminal bud, retaining the main bud and removing the lateral buds, obliquely cutting two triangular xylem sections from below the spore to form a double-bevel wedge-shaped joint body; when cutting the stock and the scion, immersing the cutting blade in an aqueous solution containing a plant growth regulator and a bactericide; inserting the double-bevel wedge-shaped joint body into the triangular pyramid-shaped mosaic structure so that the scion pith layer overlaps the stock pith layer; wrapping the interface with a composite hydrogel film comprising a thermosensitive polymer and an antibacterial component; and performing light shading after the grafting is completed. The present invention 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 particularly to a grafting method with high survival rate for grafting Pinus sylvestris var. mongolica onto 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 red pine, such as the strong stress resistance and wide popularity of Larix gmelinii and the high economic value of red pine, thereby achieving efficient cultivation of red pine.

[0003] However, existing grafting techniques for Pinus sylvestris var. mongolica grafted onto Pinus koraiensis still face numerous challenges, the most prominent of which are low graft survival rates and poor wound healing, which impact fruit yield and wound firmness. During the grafting process, a well-formed callus must form at the junction of the rootstock and scion to ensure efficient transfer of nutrients and water. However, existing grafting methods lack precise handling of the rootstock and scion, making it difficult to accurately align the cambium and pith layers of the two, which in turn affects callus formation and development, ultimately reducing the success rate of the graft. Susceptible water loss at the interface is also a major challenge in existing techniques, particularly in arid regions. Moisture retention at the interface is crucial for callus formation and growth. However, existing graft protection measures often fail to provide a stable and suitable humidity environment at the interface. Traditional wrapping materials (multi-layer plastic film wrapping) struggle to balance breathability and moisture retention. At high temperatures, moisture loss from the interface is rapid, leading to dryness and impacting cell activity and metabolism. At low temperatures, moisture cannot be effectively maintained at the interface, similarly hindering callus formation. On the other hand, improper handling of cutting tools during existing grafting operations fails to effectively inhibit the growth and reproduction of microorganisms, making the interface susceptible to bacterial infection, further disrupting the moisture balance of the interface and exacerbating 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 a 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 objects 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 4-6 years, cutting horizontally below the branch point of the terminal bud to form a cross section, making two oblique cuts downward from the cross section to form a triangular pyramid-shaped mosaic structure, cutting a semi-lignified Pinus koraiensis scion with a terminal bud, retaining the main bud and removing the lateral buds, and then obliquely cutting two triangular xylem sections from below the bud to form a double pyramid-shaped mosaic structure. Beveled wedge-shaped 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-beveled wedge-shaped joint body into the triangular pyramid-shaped mosaic structure so that the pith layer of the scion and the pith layer of the rootstock overlap; 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 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%.

[0008] The cutting blade is dipped for 10-15 seconds each time, and the liquid is left to drain 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 5-15 nm, and the particle spacing is 50-100 nm.

[0009] Furthermore, the preparation method of the composite hydrogel film includes:

[0010] Poly (N-isopropylacrylamide) and 0.1-0.5% by mass of silver nanoparticles are dispersed in deionized water, and 5-15% by mass of chitosan, 0.5-1% dopamine, and 0.1-0.3% quercetin are added, and a uniform prepolymer solution is formed by magnetic stirring.

[0011] The prepolymer solution was coated on the surface of a 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.

[0012] Spray a crosslinker solution containing 0.1-0.5% glutaraldehyde onto the substrate surface and let it stand for 2-4 hours to complete chemical crosslinking to form a composite hydrogel film;

[0013] 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.

[0014] Furthermore, the specific operation of the stock incision is:

[0015] 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-sectional area, with a cutting length of 3-5 cm, to form a triangular pyramid-shaped mosaic structure.

[0016] Furthermore, starting from 2 mm below the spore, two bevel triangles with a length of 3-5 cm are cut along the axis of the scion to form a double-bevel wedge-shaped joint body.

[0017] Furthermore, after docking, a pulsed pressure device is used to apply pressure for 5-10 seconds to promote contact between the medullary parenchyma cells.

[0018] Furthermore, after grafting, cover with a black sunshade net with a shading rate of 75% for 2-3 weeks;

[0019] The shading is removed in three stages, with each stage reducing the shading rate by 25%, with an interval of 7 days.

[0020] Furthermore, the step of cutting out two bevel triangles also includes: the angle between the two bevels is 25°-35°, and the cutting blade maintains an inclination angle of 15°-20° with the scion axis;

[0021] The section roughness Ra is less than or equal to 0.8 μm, and the intersection line of the two inclined surfaces deviates from the medullary axis by an angle of less than 1°.

[0022] Furthermore, the specific operation of the pulse pressurizing device is:

[0023] After the scion and the rootstock are docked, a pulse pressure of 0.1-0.2 MPa is applied through a pneumatic device with a pulse frequency of 1-3 Hz for 5-10 seconds;

[0024] The pressure fluctuation of the device is ≤±5%, the pressure interface is in contact with a silicone pad with a hardness of Shore A20-30, and the angle deviation between the pressure direction and the scion axis is <3°.

[0025] Furthermore, the post-grafting management also includes:

[0026] Cover the outside of the scion with a black shade net with a shading rate of 75% and continue covering it for 2-3 weeks;

[0027] 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.

[0028] The first stage is the third week: the light blocking rate is reduced to 50%, the light transmission wavelength range is 500-700nm, and the UV blocking rate is ≥85%;

[0029] The second stage is the fourth week: the light blocking rate is reduced to 25%, the light transmission wavelength is extended to 400-700nm, and the UV blocking rate is ≥70%;

[0030] The third stage is the fifth week: completely remove the shade net, and cover it 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 net, with a single covering time of ≤2 hours.

[0031] The present invention has at least the following beneficial effects:

[0032] During the cutting process, the present invention immerses the blade in an aqueous solution containing plant growth regulators, bactericides and nano-silver particles, which not only promotes the growth of stock and scion cells, but also effectively inhibits pathogens, reduces the risk of infection, and lays the foundation for successful grafting. The unique temperature sensitivity of the composite hydrogel membrane is utilized to make it swell and retain moisture at low temperatures, and shrink and breathe 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.

[0033] Other advantages, objectives and features of the present invention will be reflected in part from the following description and will be understood by those skilled in the art through study and practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is a schematic diagram of a longitudinal section of a scion or rootstock according to an embodiment of the present application;

[0035] Figure 2 This is a schematic diagram of a cross section of a scion or rootstock according to an embodiment of the present application;

[0036] Figure 1 and Figure 2 In the figure, 1 represents spore and 2 represents incision. DETAILED DESCRIPTION

[0037] The present invention will be described in further detail below in conjunction with the accompanying drawings so that those skilled in the art can implement the invention with reference to the description.

[0038] It should be understood that terms such as "having," "comprising," and "including" used in the embodiments of this application do not exclude the presence or addition of one or more other elements or combinations thereof. All directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of this application are intended only to explain the relative positional relationships and movement of components in a specific posture. If the specific posture changes, the directional indications will also change accordingly. When an element is referred to as being "fixed to" or "disposed on" another element, it can be directly on the other element or there may be an intervening element. When an element is referred to as being "connected to" another element, it can be directly connected to the other element or indirectly connected to the other element through an intervening element. References to "first," "second," etc. in the embodiments of this application are for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, features designated as "first" or "second" may explicitly or implicitly include at least one of such features.

[0039] 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 this field can implement it. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0040] The embodiments of the present application provide a high survival rate grafting method of Pinus sylvestris var. mongolica onto Pinus koraiensis in arid areas, comprising:

[0041] Select 4-year-old seedlings of Pinus sylvestris or 5-year-old transplanted seedlings, cut them flatly at 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 .

[0042] See also Figure 1 The cutting of the scion starts from 2mm below the spore 1, and the double-bevel triangle 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.

[0043] During the cutting process, the blade should be immersed in a mixed solution containing 0.05-0.15% indolebutyric acid, 0.3-0.8% carbendazim, and 0.1-0.3% nanosilver. Each dip should be for 10-15 seconds, followed by a 5-8 second drain. A double-beveled wedge-shaped joint is inserted into the triangular pyramidal structure, overlapping the pith of the scion and the rootstock, ensuring a minimum overlap of 1.5 cm. The joint is then wrapped with a composite hydrogel membrane (covering 2 cm above and below the incision). Immediately after grafting, a shade net with a 75% shading ratio is applied.

[0044] 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 rootstock cross section can be adaptively adjusted (until the cross section is cut off). Figure 2 The multiple dotted lines in the figure are used to ensure that the pith and cambium of the two overlap. Furthermore, the present embodiment adopts the flat-cut rootstock and double-bevel scion design. Regardless of the diameter difference between the rootstock and the scion, the bevel triangle contact can fit the pith, xylem and cambium tightly, avoiding the scion from being exposed, causing water loss and bacterial infection, which will lead to grafting failure. The cambium contact area 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 to below 8%. The synergistic effect of nanosilver and carbendazim reduces the pathogen infection rate to below 2%, retains the rootstock terminal bud branching point to maintain apical dominance, shortens the callus formation time to 7-10 days, and achieves a survival rate of more than 97%.

[0045] In another embodiment, the blade treatment solution consists of a 1:1 volume ratio of 0.05-0.15% indolebutyric acid (IBA) and 0.3-0.8% carbendazim wettable powder, with 5-15 nm nanosilver particles added to a concentration of 0.1-0.3%. To perform the treatment, the grafting knife is completely immersed in the solution for 10-15 seconds. After removal, it is allowed to stand vertically for 5-8 seconds to form a uniform liquid film 5-10 μm thick on the blade edge. This treatment is repeated before each cut of the stock or scion to ensure that the active ingredient is always adhered to the blade surface.

[0046] Indolebutyric acid promotes callus differentiation through liquid membrane penetration incisions, 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 deep into the xylem vessels, forming sustained and long-term antibacterial protection, avoiding cross-contamination caused by continuous grafting operations, and greatly reducing the infection rate.

[0047] In another embodiment, the hydrogel film is prepared in three steps: first, poly (N-isopropylacrylamide) and 0.1-0.5% silver nanoparticles are dispersed in deionized water. Chitosan (5-15% by weight), dopamine (0.5-1%), and quercetin (0.1-0.3%) are then added and magnetically stirred to form a uniform prepolymer solution. Second, the prepolymer solution is coated on a 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 crosslinker solution containing 0.1-0.5% glutaraldehyde is sprayed onto the substrate surface and allowed to stand for 2-4 hours to complete chemical crosslinking, forming a composite hydrogel film. The hydrogel film has a swelling ratio of 250-300% at 20°C, decreasing 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.

[0048] In this example, quercetin and silver nanoparticles produce a synergistic antimicrobial effect, increasing the inhibition rate against common pathogens (such as Fusarium and Botrytis cinerea) from 85% to 99.5%, and extending the antimicrobial effect to over 30 days. Quercetin's antioxidant properties (free radical scavenging efficiency ≥ 90%) inhibit oxidative stress damage at the interface and reduce the risk of callus necrosis. At 20°C, the swelling ratio of 250-300% locks in moisture, which is particularly beneficial in arid regions. At 37°C, the swelling ratio shrinks to 60-80%, increasing air permeability by three times, preventing interface mold and rot caused by high temperature and humidity.

[0049] The thermosensitive material maintains a high moisture content below 25°C, providing a relative humidity of over 95% at the interface. After shrinking above 30°C, its porosity increases to 40%, preventing tissue decay caused by high temperature and humidity, keeping the interface moist and breathable, and helping to improve survival rates. Chitosan enhances the adhesion between the membrane and plant tissue, reducing the thermal resistance at the membrane-tissue interface by 40%. The sustained-release silver nanoparticle concentration is maintained within the safe antibacterial range of 0.1-0.5ppm, significantly reducing infection rates and improving the survival rate of grafted seedlings.

[0050] 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 terminal 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.

[0051] In another embodiment, see Figure 1 and Figure 2The scion cutting starts at 2mm below the bud. Use a razor blade to cut a double bevel along the scion axis. The length of each 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, maintain a 15-20° angle between the blade and the scion axis to ensure a smooth and burr-free cut surface.

[0052] The pith, the central part of the stem, is composed of loose, thin-walled cells that store nutrients and undergo active cell division, making it easy to induce callus. During grafting, the pith of the scion and the pith of the rootstock are butted at an angle to minimize misalignment. This allows the parenchyma cells of both grafts to fuse fully, gradually differentiating into new vascular bundles.

[0053] In another embodiment, after docking is completed, a pneumatic pressure device is used to apply 0.1-0.2 MPa of pressure 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 rootstock is observed to ensure that the pith layer is completely aligned and there is no misalignment.

[0054] In another embodiment, shading management is divided into three stages: covering with 75% shading black shading net for 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.

[0055] The following is an explanation with specific examples.

[0056] Example 1:

[0057] 1. Material Preparation

[0058] Rootstock: Select 4-year-old seedlings of Pinus sylvestris var. mongolica (with a top bud diameter of about 1.0 cm), with a seedling height of 70-80 cm and 7-10 healthy needles at the base.

[0059] Scion: Collect semi-lignified Korean pine branches of the current year (0.8-1.2 cm in thickness), cut off 8-10 cm segments with terminal buds, and remove all side buds.

[0060] Blade treatment fluid:

[0061] 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.

[0062] Composite hydrogel film:

[0063] Preparation of prepolymer solution: (1) Disperse poly (N-isopropylacrylamide) and 0.3% silver nanoparticles in deionized water, add 10% chitosan, 0.5% dopamine and 0.2% quercetin, and form a uniform prepolymer solution by magnetic stirring;

[0064] (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;

[0065] (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 to form a composite hydrogel film with a thickness of 0.2 mm (swelling ratio 250% at 20°C, swelling ratio 60% at 37°C).

[0066] 2. Grafting operation steps

[0067] 1. Rootstock treatment

[0068] At 4 cm below the branch 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.

[0069] 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.

[0070] 2. Scion cutting

[0071] 2 mm below the scion spore (6 mm from the top), cut a double-bevel triangle (single bevel length 3.5 cm, angle 30°) at an angle of 15° toward the base to form a double-bevel wedge-shaped junction.

[0072] Each time you cut the scion, soak the blade in liquid again to ensure that there are no burrs on the cut surface.

[0073] 3. Docking and fixing

[0074] 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 rootstock, with the pith layer overlapping by 1-2 cm. Use a pneumatic pressure device (0.15 MPa, for 8 seconds) to make the interface fit tightly.

[0075] 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.

[0076] 4. Shading management

[0077] After grafting, build a 1.5-meter-high sunshade covered with 75% black shade netting, maintaining a light intensity of 1000-1500 lux. On the 22nd day, replace the 50% shade netting (light intensity 2500 lux), on the 29th day, replace the 25% shade netting (light intensity 4500 lux), and on the 36th day, remove the netting completely.

[0078] Comparative Example 1:

[0079] No blade treatment liquid was used, and the remaining parameters and processes were the same as those in Example 1.

[0080] Comparative Example 2:

[0081] Ordinary plastic film (polyethylene) replaced the temperature-sensitive hydrogel film, and the remaining parameters and processes were the same as those in Example 1.

[0082] test:

[0083] 1. Survival rate calculation method:

[0084] 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 normal in color (not 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 dies, or the plant falls over due to severe rot at the interface. 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, the experiment is repeated 3 times, and the average value is taken. 3. Calculation formula Survival rate = number of surviving plants / total number of plants.

[0085] 2. Method for determining infection rate

[0086] Definition: The proportion of plants infected with pathogens (such as mold or bacterial rot) at the grafting interface. 1. Infection Criteria: Mild infection: White / gray mold appears on the grafting interface, with an area less than 1 cm² and no penetration into the wood. Severe infection: Blackened rot at the grafting interface, with visible brown lesions in the wood and an odor. All infection levels are included in the statistics. 2. Observation Timepoints: Regular inspections: Inspect once every 7, 14, 21, and 30 days after grafting, and record the number of infected plants. Final statistics: The 30-day data will be used as the basis. 3. Calculation Formula: Infection Rate = Number of Infected Plants / Total Number of Plants.

[0087] 3. Methods for determining wound healing time

[0088] Definition: The time from completion of grafting to visible callus formation. 1. Observation Method: Daily Observation: Starting from the fifth day after grafting, observe the grafting interface using a 10x magnifying glass at 9:00 AM daily and record the date of the first appearance of callus. Callus Criteria: Formation of milky white or light yellow spongy tissue at the grafting interface, covering ≥50% of the cut surface. 2. Data Processing: Individual Plant Recording: Callus formation time is recorded independently for each plant, accurate to the nearest 0.1 day (e.g., 7.2 days = 7 days and 5 hours). Average Calculation: The median of 30 plants in each group is used to avoid the influence of extreme values.

[0089] 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.

[0090]

[0091] 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. In addition, as the blade realizes instant disinfection of the cut surface, the composite hydrogel membrane reduces water loss during the moisturizing period while avoiding high temperature suffocation of seedlings through the thermosensitive swelling properties of poly N-isopropylacrylamide (swelling ratio 250% at 20°C / shrinkage to 60% at 37°C), and cooperates with chitosan to enhance membrane-tissue adhesion, thereby improving healing efficiency. These technologies work together to significantly improve the survival rate of grafted seedlings.

[0092] According to another embodiment of the present invention, by optimizing the blade dipping process, it is proposed to control the dipping time to 10-15 seconds and the standing drain time to 5-8 seconds, so that a uniform liquid film with a thickness of 5-10 μm is formed on the blade surface. Conventional disinfection of cutting tools often involves simple immersion or spraying, which can lead to problems such as uneven liquid adhesion and insufficient penetration of active ingredients, resulting in unstable antibacterial efficacy. Excessive residual liquid can also inhibit callus formation. The present invention precisely coordinates the dipping time with the standing drain time to ensure that the liquid film on the blade surface fully absorbs the bactericide and growth regulator while avoiding excessive residual liquid droplets that could cause localized high concentrations. Experimental results show that when the dipping time is less than 10 seconds, the liquid film coverage is less than 60%, while when it is greater than 15 seconds, the liquid film is too thick (>15 μm), resulting in drug waste and hindering cell respiration at the incision site. When the standing drain time is less than 5 seconds, the liquid film is unevenly distributed, while when it is greater than 8 seconds, the liquid film thickness decreases significantly (<3 μm), reducing antibacterial activity. Furthermore, silver nanoparticles (5-15 nm) are evenly dispersed within the liquid film, penetrating deep into the micropores of the xylem to form a long-lasting antimicrobial barrier. This technical feature reduces the pathogen infection rate at the incision site to below 2%, while also promoting the targeted penetration of indolebutyric acid (IBA), shortening callus formation time to 7-10 days, significantly superior to traditional methods (12-14 days).

[0093] According to another embodiment of the present invention, by limiting the coordinated parameters of the incision position and depth of the rootstock (cutting the stem flatly 3-5 cm from the branch point of the apical bud, and making two 3-5 cm incisions obliquely downward from the cross section to form a triangular pyramid-shaped mosaic structure), the problems of insufficient cambium contact area and high misalignment rate of the pith layer in traditional grafting are innovatively solved. In the prior art, the incision position of the rootstock mostly relies on experience-based operations, lacking consideration of the physiological characteristics of the branch point of the apical bud, which can easily lead to the incision being too close to damage the apical dominance, or too far to reduce the activity of the cambium; the incision depth also often fluctuates due to differences in the diameter of the rootstock. 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 rootstock is damaged, causing the risk of splitting. The present invention has been verified through experiments: when the incision is 3-5 cm 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 after the scion is inserted is maximized (increased by 50%-70%); the precise design of the 3-5 cm long bevel cut downward from the cross section makes the overlapping length of the scion and the pith layer of the rootstock ≥1.5 cm, promoting the fusion and differentiation of thin-walled cells. Comparative experiments show that when the incision position deviation exceeds ±1 cm or the depth deviates from the range of 3-5 cm, the callus formation time is extended to 12-15 days, and the survival rate drops to below 80%. In addition, the incision flatness error is ≤0.1 mm, 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%.

[0094] 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 that matches the triangular pyramid-shaped mosaic structure), an innovative solution is provided to the problems of high misalignment rate and insufficient contact area between the cambium of the scion and the rootstock in traditional grafting. In the prior art, the starting point for scion cutting is often arbitrarily selected (such as the base of the spore or a random position), resulting in insufficient exposure of the cambium or deviation in the cutting depth; the length of the bevel is mostly dependent on subjective judgment. If it is too long (>5cm), it will easily weaken the structural strength of the scion, and if it is too short (<3cm), the contact area will be insufficient, affecting the efficiency of callus fusion. Experimental validation demonstrates that this invention precisely locates the densely packed scion vascular bundle, preserving apical spore activity (increasing germination rate by 20%) while avoiding excessive cutting depth that could damage the pith parenchyma cells. The design of a double bevel with a length of 3-5 cm 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 that significantly reduces the risk of slippage after scion insertion (slip rate <3%). Comparative experiments show that when the cutting starting point deviates by ±1 mm or the bevel length deviates from the 3-5 cm range, the interface porosity increases to 15%-20%, the callus formation time is extended to 12-15 days, and the survival rate drops below 85%. Furthermore, maintaining a 15-20° inclination angle between the blade and the scion axis during cutting ensures a smooth, burr-free cut surface (roughness Ra ≤ 0.8 μm), further reducing microbial attachment and water evaporation. This technical feature, through coordinated parameter optimization, has achieved a grafting survival rate exceeding 98%.

[0095] According to another embodiment of the present invention, by introducing a pulsed pressurization device and limiting the pressure parameters (duration 5-10 seconds, pulse frequency 1-3Hz, pressure 0.1-0.2MPa), it innovatively solves the problems of uneven contact of pith parenchyma cells and local stress concentration caused by traditional static pressurization. In the prior art, grafting interfaces mostly use fixed pressure clamping or simple binding, which has defects such as uneven pressure distribution and arbitrary pressure application time. This can easily cause scion slippage or pith layer dislocation (dislocation rate >15%), and prolonged pressure application (>15 seconds) may damage the activity of parenchyma cells. The present invention has been verified through experiments: pulsed pressurization (frequency 1-3Hz) can dynamically adjust the direction of pressure, promote micron-level reciprocating displacement (amplitude 10-20μm) between the scion and the rootstock incision, effectively eliminate interfacial gaps (porosity reduced to <2%), and accelerate changes in cell membrane permeability through periodic stress stimulation, promoting the diffusion of growth factors. Limiting the pressure application time to 5-10 seconds (preferably 8 seconds) can balance pressure intensity and cell tolerance, avoiding prolonged pressure-induced cell membrane rupture (survival rate > 98%). Comparative experiments have shown that after pulsed pressure application, the contact area of ​​the pith parenchyma cells increased by 40%-60%, the callus formation time was shortened to 5-7 days, and the survival rate 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 cell fusion efficiency through the coordinated optimization of dynamic pressure mode and parameters.

[0096] According to another embodiment of the present invention, a post-grafting management method has been designed that involves a phased, gradient removal of shading (initial shading at 75% for 2-3 weeks, followed by a three-stage reduction of 25% every 7 days). This effectively addresses the issues of light stress and dehydration damage to the graft caused by sudden changes in light intensity during traditional post-grafting shading management. Existing techniques typically employ either long-term coverage with a fixed shading ratio (e.g., shading at 75% until the graft survives) or a one-time removal of shading after grafting. The former can easily lead to excessive graft growth and deterioration of photosynthetic capacity, while the latter can cause chloroplast damage (photoinhibition rate >30%) and a surge in transpiration water loss (>50%) due to the sudden increase in light intensity. Experimental verification of the present invention demonstrates that an initial 75% shading (25% transmittance) can suppress the transpiration rate of the scion to 40%-50% of the normal value, while retaining sufficient photosynthetically active radiation (PAR ≥ 200 μmol·m⁻²·s⁻¹) to maintain basal metabolism. A phased, gradient removal of shading (reducing the shading rate by 25% every 7 days) allows the scion to gradually rebuild its light adaptation mechanism, steadily increasing the chlorophyll a / b ratio from an initial 2.8 to 3.5 (±0.2), and maintaining the maximum quantum efficiency of photosystem II (Fv / Fm) above 0.75 (whereas the control group experienced a sharp drop to 0.55). Comparative experiments showed that the wilting rate of the scion reached 35% when shading was removed all at once, while the wilting rate was less than 5% under the phased treatment. Furthermore, the elongation of the new shoots in the phased treatment group was 40%-60% higher than that in the fixed shading group, and the lignification rate was accelerated by 15%. In addition, the black shade net (absorption rate >90% at wavelengths of 400-700nm) effectively blocks ultraviolet radiation (UV-B transmittance <5%) and reduces membrane lipid peroxidation (MDA content is reduced to 0.8μmol / g FW), further ensuring a scion survival rate exceeding 98%. This technical feature dynamically controls light intensity and acclimatization cycles.

[0097] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. 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 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 horizontally below the branch point of the terminal bud to form a cross section, and then cut two oblique cuts downward from the cross section to form a triangular pyramid mosaic structure. Then, cut a semi-lignified Korean pine scion with a terminal bud, retain the main bud and remove the lateral buds. Then, cut two oblique triangular xylem sections from below the bud with two oblique cuts to form a double-bevel wedge-shaped joint body that matches 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 and the pith layer of the rootstock overlap; The interface is wrapped with a composite hydrogel membrane containing a thermosensitive polymer and an antibacterial component. The hydrogel membrane remains swollen at low temperatures to maintain interface humidity, and shrinks at high temperatures to increase air permeability. After grafting is completed, shading treatment is performed; 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 allowed to stand for 5-8 seconds after dipping to form a uniform liquid film on the blade surface. The particle size of the nanosilver particles in the liquid film is 5-15 nm, and the particle spacing is 50-100 nm. The preparation method of the composite hydrogel film comprises: Poly (N-isopropylacrylamide) and 0.1-0.5% by mass of silver nanoparticles are dispersed in deionized water, and 5-15% by mass of chitosan, 0.5-1% dopamine, and 0.1-0.3% quercetin are added, and a uniform prepolymer solution is formed by magnetic stirring. The prepolymer solution was coated on the surface of a 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. Spray a crosslinker solution containing 0.1-0.5% glutaraldehyde onto the substrate surface and let 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 a swelling ratio of 60-80% at 37°C; 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-sectional area, with a cutting length of 3-5 cm, to form a triangular pyramid-shaped mosaic structure.

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

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

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

5. The grafting method according to claim 2, wherein The step of cutting out two bevel triangles also includes: the angle between the two bevels is 25°-35°, and the cutting blade maintains an inclination angle of 15°-20° with the scion axis; The section roughness Ra is less than or equal to 0.8 μm, and the intersection line of the two inclined surfaces deviates from the medullary axis by an angle of less than 1°.

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

7. The grafting method according to claim 4, wherein Post-graft management also includes: Cover the outside of the scion with a black shade 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 is reduced 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 is reduced to 25%, the light transmission wavelength is extended to 400-700nm, and the UV blocking rate is ≥70%; The third stage is the fifth week: completely remove the shade net, and cover it 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 net, with a single covering time of ≤2 hours.

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