A healing agent and grafting method suitable for grafting Diospyros kaki and chocolate pudding fruit

Abstract

This invention provides a healing agent and grafting method suitable for grafting *Persimmon taiwaniana* and *Chocolate Pudding Fruit*, belonging to the field of plant grafting technology. Addressing the core challenge of cross-ploid and cross-phenological distant grafting of hexaploid temperate *Persimmon taiwaniana* and diploid tropical *Chocolate Pudding Fruit*, this invention pioneers a two-component time-sequential nano-healing system and a two-layer dynamic nutrient balance grafting technology. The healing agent consists of a targeted enzymatic hydrolysis and unblocking solution and a main healing inducer. Active ingredients are targetedly delivered via KH550-modified nano-silica, and a three-dimensional scaffold constructed using nanocellulose guides the orderly growth of callus tissue. The grafting method resolves nutrient conflicts in cross-phenological grafting through functional partitioning of a lower carbon sink energy storage layer and an upper directional energy supply layer. Using this invention, the winter grafting survival rate increases from 2% to 88%, the graft union rot rate is reduced to 2% under high temperature and humidity conditions, and the wind resistance of the graft union is increased by more than four times. This invention has extremely high industrial value.

Description

Technical Field

[0001] This invention belongs to the field of plant grafting technology, and in particular relates to a healing agent and grafting method suitable for grafting persimmon and chocolate pudding fruit. Background Technology

[0002] Taiqiu persimmon is a superior sweet persimmon variety bred by the Fruit Tree Experiment Station of the Ministry of Agriculture, Forestry and Fisheries of Japan. It is a temperate hexaploid persimmon variety, renowned for its excellent fruit quality: the fruit is well-shaped, large, and weighs 200-500g per fruit; the flesh is delicate, seedless, and extremely sweet, with a soluble solids content of 15%-20%. The flesh is tender, crisp, and refreshingly sweet, juicy, and flavorful, enjoying high popularity and reputation in the market. The core value of Taiqiu persimmon lies in its completely sweet nature—it can be eaten directly without astringency removal, overturning the traditional understanding that persimmons must be softened or astringent removed before consumption. Taiqiu persimmon is rich in vitamins B, E, and C, potassium, calcium, magnesium, zinc, selenium, various amino acids, and beta-carotene, among other nutrients. Its vitamin C content is twenty times that of apples, pears, and peaches, making it a hot research topic in fruit tree breeding and cultivation.

[0003] Currently, China has conducted systematic research on the introduction, domestication, cultivation techniques, and variety improvement of Taiqiu persimmon for many years, and has cultivated rootstock varieties suitable for Taiqiu persimmon such as Yalin Persimmon Rootstock No. 6 and Small-fruited Sweet Persimmon. Although some progress has been made in variety selection and basic cultivation management, the explosive market growth of Taiqiu persimmon has not yet been achieved. The core bottleneck lies in the dual problems of Taiqiu persimmon's own resistance defects and the lack of suitable rootstocks: on the one hand, as a temperate hexaploid variety, Taiqiu persimmon has weak resistance to heat, humidity, and waterlogging, slow growth, and weak growth, and cannot adapt to the high temperature and high humidity climate of tropical and subtropical regions. The planting area is limited to a few temperate regions, making it difficult to achieve large-scale planting nationwide. On the other hand, traditional persimmon rootstocks are mostly local conventional varieties, which have poor grafting compatibility with Taiqiu persimmon. During distant grafting, problems such as oxidative browning at the grafting interface, abnormal callus deposition, vascular bundle blockage, and high incidence of cysts are prone to occur, resulting in low grafting survival rate. Moreover, the growth contradiction caused by the difference in phenological stages of rootstock and scion in winter makes the winter grafting survival rate extremely low, making it impossible to achieve large-scale seedling cultivation throughout the year. The rootstock compatibility problem and the low grafting survival rate have led to insufficient seedling supply, which have become the core factors restricting the development of the Taiqiu persimmon industry.

[0004] Distant grafting is a key technique for addressing the resistance deficiencies of Taiqiu persimmon and expanding its planting range. By grafting Taiqiu persimmon onto tropical persimmon rootstocks with excellent resistance, the superior fruit quality of the scion can be combined with the heat and humidity resistance of the rootstock. Black SAPOTE, a diploid tropical persimmon variety, possesses extremely strong resistance to heat, humidity, waterlogging, and other adverse conditions. It adapts exceptionally well to the climate and soil conditions of tropical and subtropical regions, exhibiting vigorous growth and high yield. However, the cross-phenological and cross-ploidy distant grafting of Taiqiu persimmon (hexaploid) and Black SAPOTE (diploid) presents unprecedented technical challenges due to the large genetic distance between the rootstock and scion and the significant phenological differences. Genetic distance leads to compatibility barriers: There are significant differences in cell volume and vascular bundle diameter between hexaploid and diploid plants, and the nutrient transport rate of rootstock and scion is mismatched, which can easily lead to nutrient accumulation and cyst formation at the grafting interface; Nutrient conflicts caused by phenological differences: Persimmon is a temperate deciduous tree species that enters dormancy in winter, while chocolate pudding fruit is a tropical evergreen tree species that has vigorous physiological metabolism in winter. Excessive nutrients and strong root pressure on the rootstock can cause the scion to "drown" or disrupt dormancy. Oxidative browning at the grafting site and blockage of vascular bundles: A large amount of reactive oxygen species will be generated at the distant grafting cut, which will cause oxidative browning of phenolic substances. At the same time, abnormal deposition of callose will block the vascular bundles and block the exchange of nutrients between the rootstock and the scion.

[0005] Currently, there are no successful reports, either domestically or internationally, on grafting Taiqiu persimmon and chocolate pudding fruit. Existing conventional grafting healing agents and grafting methods are designed for grafting fruit trees of the same ploidy and phenology, and cannot solve the special problems of the aforementioned transploid and transphenological distant grafting: conventional healing agents have limited functions and lack the enzymatic hydrolysis, targeted antioxidant, and vascular bundle directional induction functions specifically for transploid grafting; conventional grafting methods often use a single layer of nutrient branches or leave no nutrient branches, which cannot solve the nutrient supply and demand contradiction in winter transphenological grafting; existing technologies do not consider the targeted delivery and three-dimensional scaffolding role of nanomaterials in fruit tree grafting healing, resulting in low utilization rate of active ingredients in healing agents and disordered callus growth.

[0006] Therefore, there is an urgent need to develop a special healing agent and grafting method for transploid and transphenological distant grafting of persimmon and chocolate pudding fruit, in order to break through the core technical bottleneck of industrial development. Summary of the Invention

[0007] The purpose of this invention is to address the shortcomings of existing technologies by providing a healing agent and grafting method suitable for grafting Taiqiu persimmon and Chocolate Pudding fruit. This invention pioneers a two-component time-sequential nano-healing system and a two-layer dynamic nutrient balance grafting technology, systematically solving the core challenges of cross-ploid and cross-phenological distant grafting of Taiqiu persimmon and Chocolate Pudding fruit from three dimensions: molecular, cellular, and physiological levels. It achieves a breakthrough increase in winter grafting survival rate from 2% to 88%, providing core technical support for the large-scale cultivation and promotion of Taiqiu persimmon in tropical and subtropical regions.

[0008] 1. Two-component time-sequential nano-healing system

[0009] The healing agent of this invention consists of a targeted enzymatic hydrolysis and unblocking solution and a primary healing inducer. It is precisely designed to meet the physiological needs of different stages of graft healing, achieving full-cycle regulation from "early enzymatic hydrolysis and unblocking to later targeted healing promotion." The two are not simply used sequentially, but rather have a close synergistic effect: the targeted enzymatic hydrolysis and unblocking solution clears obstacles for the subsequent action of the primary healing inducer, while the primary healing inducer consolidates and amplifies the effect of the targeted enzymatic hydrolysis and unblocking solution.

[0010] The targeted enzymatic hydrolysis and unblocking solution contains the following components by weight percentage: N-acetylcysteine ​​0.1%~0.8%, cell wall degrading enzyme system 0.1%~0.8%, nanocarrier 0.2%~1.0%, plant extract rich in flavonoids and vitamin C 3%~8%, and the balance being water, pH 5.0~6.5; The primary healing inducer comprises the following components by weight percentage: 0.01%~0.08% compound plant growth regulator, 0.05%~0.3% biopolysaccharide, 0.2%~2.0% nanocellulose, 0.05%~0.2% bactericide, 0.01%~0.05% calcium source, 3%~8% plant extract rich in flavonoids and vitamin C, 0.001%~0.005% zeatin, and the balance being water.

[0011] Preferably, the cell wall degrading enzyme system includes β-1,3-glucanase and pectin lyase, and the mass ratio of β-1,3-glucanase to pectin lyase is 1:1 to 4:1; The nanocarrier is KH550 surface-modified nano-silica with a particle size of 50~80 nm and a zeta potential of -35mV.

[0012] Preferably, the compound plant growth regulator is composed of NAA, IBA and EBR, and the mass ratio of NAA, IBA and EBR is 2~5:1:1; The biopolysaccharide is chitosan oligosaccharide and sodium alginate; the mass ratio of chitosan oligosaccharide to sodium alginate is 3~5:3.

[0013] Preferably, the plant extract rich in flavonoids and vitamin C includes finger lemon extract, wherein the finger lemon extract contains quercetin ≥ 0.8% and vitamin C ≥ 500 mg / 100g.

[0014] 2. Double-layer dynamic nutrient balance grafting technology

[0015] This invention creatively proposes a two-layer dynamic nutrient balance grafting technology. Through precise pruning and functional differentiation of branches of the chocolate pudding fruit rootstock, it solves the core contradictions of cross-phenological grafting from three dimensions: carbon source allocation, nutrient transport, and hormone balance. This technology is not simply about "preserving more branches," but rather a precise functional zoning design based on the phenological differences and physiological characteristics of the persimmon and chocolate pudding fruit.

[0016] (1) Lower carbon sink energy storage layer

[0017] Retain 2-3 strong branches with a diameter of 3-10cm at the base of the rootstock, cut them to a length of 50-120cm, and retain 8-15 healthy leaves on each branch.

[0018] Function: As a "carbon sink organ," it converts excess photosynthetic carbon from the rootstock into starch and stores it in the root system, preventing carbon from flowing upwards and disrupting the scion's dormancy; in winter, it consumes excess nutrients from the rootstock, maintaining a metabolic balance between the rootstock and scion. This is key to resolving the phenological conflict between tropical evergreen rootstocks and temperate deciduous scions.

[0019] (2) Upper directional energy supply layer

[0020] 5-8cm below the grafting point on the rootstock, retain 1-2 healthy twigs with a diameter of 0.2-0.8cm, cut to a length of 20-40cm, and retain 2-5 functional leaves on each twig.

[0021] Function: As a "nutrient transport hub", its slender branch characteristics make its vascular bundle vessel diameter highly compatible with the vessel diameter of the Taiqiu persimmon scion, realizing slow-rate, directional transport of nutrients, only meeting the basic needs of callus formation and vascular bundle differentiation, matching the low metabolic level of the dormant scion; at the same time, it autonomously synthesizes auxin and cytokinin, and transports them directionally to the grafting interface, providing continuous hormone signals for callus formation.

[0022] (3) Take the targeted enzymatic hydrolysis and unblocking solution, first apply it to the cambium layer of the rootstock incision, then apply it to the scion incision, let it stand for 10~15s and then insert it; after the scion is inserted, apply the main healing inducer to the rootstock-scion junction and the surrounding 0.5cm area, and seal it with grafting film. (4) 40-50 days after grafting, when the scion buds are 4-6 cm long and the leaves have unfolded, remove the grafting film.

[0023] Preferably, the scion of Taiqiu persimmon is a one-year-old branch of Taiqiu persimmon, cut to a length of 5-9cm, retaining 2-3 plump buds.

[0024] Preferably, after the grafted seedling matures, auxiliary branches of the lower carbon sink energy storage layer are selected for secondary grafting. The number of secondary grafting scions shall not exceed two, and the secondary grafting scions and the main scions shall be triangularly distributed on the rootstock. The distance between the secondary grafting interface and the original main grafting interface shall not be less than 15cm.

[0025] Preferably, the thickness of the primary healing inducer is 0.1~0.2 mm, and the amount applied is 0.2~0.4 ml / plant.

[0026] Preferably, apply 0.6~0.8 ml of the targeted enzymatic hydrolysis solution per plant.

[0027] Preferably, during winter grafting, apply 0.2~0.42ml / plant of targeted enzymatic hydrolysis and unblocking solution to the graft union.

[0028] Compared with existing technologies, the present invention has the following beneficial effects: The special two-component sequential healing agent for grafting Taiqiu persimmon and chocolate pudding fruit provided by the present invention includes a targeted enzymatic hydrolysis unblocking solution and a main healing inducer; by using the special two-component sequential healing agent provided by the present invention, combined with a scientific application method and innovative double-layer dynamic nutrient balance technology, the existing technical bottlenecks have been successfully overcome, and the survival rate of cross-ploid and cross-phenological distant grafting of Taiqiu persimmon and chocolate pudding fruit has been greatly improved. This has solved the core technical bottleneck that restricts the explosive growth of the Taiqiu persimmon industry, and provided exclusive core technical support for the large-scale planting and promotion of Taiqiu persimmon in tropical and subtropical regions. It has extremely high creativity, practicality and industrial value. Attached Figure Description

[0029] Figure 1 This is a schematic diagram of the double-layer dynamic nutrient balance technology for chocolate pudding fruit rootstock.

[0030] Figure 2 This is a schematic diagram illustrating the scientific application method of the targeted enzymatic hydrolysis and unblocking solution and the main healing inducer of this invention.

[0031] Figure 3 A schematic diagram illustrating the application of grafting inducer to grafted seedlings of *Persimmon × Chocolate Pudding Fruit*.

[0032] Figure 4 This is a photograph of the grafted specimen from Example 1.

[0033] Figure 5 This is a photograph of the grafted specimen from Example 2.

[0034] Figure 6 This is a photograph of the grafted specimen from Example 3.

[0035] Figure 7 This is a photograph of the grafted specimen from Example 4.

[0036] Figure 8 This is a photograph of the grafted specimen from Example 5.

[0037] Figure 9 This is a photograph of the grafted specimen from Example 6.

[0038] Figure 10 This is a picture of the grafted specimens from the control group.

[0039] Figures 1-3 The labeling is as follows: 1 - Rootstock trunk (diameter 1.0~1.8cm), 2 - Lower auxiliary branches (diameter 3~5cm, length 50~80cm), 3 - Upper auxiliary branches (diameter 0.2~1cm, length 20~40cm), 4 - Grafting point (50~60cm from the ground), 5 - Rootstock incision (oblique cut); 6 - Scion (Taiqiu persimmon, length 5~9cm), 7 - Scion incision (oblique cut), 8 - Main healing inducer coating layer (thickness 0.1~0.2mm), 9 - Targeted enzymatic hydrolysis and unblocking solution coating layer. Detailed Implementation

[0040] This invention provides a special two-component sequential healing agent suitable for grafting persimmon and chocolate pudding fruit, comprising a targeted enzymatic hydrolysis unblocking solution and a main healing inducer; The targeted enzymatic hydrolysis and unblocking solution contains the following components by weight percentage: N-acetylcysteine ​​0.1%~0.8%, cell wall degrading enzyme system 0.1%~0.8%, nanocarrier 0.2%~1.0%, plant extract rich in flavonoids and vitamin C 3%~8%, and the balance being water, pH 5.0~6.5; the functions of the targeted enzymatic hydrolysis and unblocking solution are: scavenging reactive oxygen species, reducing browning, assisting in the degradation of callosity, improving adhesion, and slow-release components.

[0041] In this invention, the preferred concentration of N-acetylcysteine ​​in the targeted enzymatic hydrolysis solution is 0.18~0.22% (corresponding to 5.4~6.6 mmol / L), and more preferably 0.20%. The mechanism of action of N-acetylcysteine ​​is to remove reactive oxygen species (ROS) generated at the grafting incision site, inhibit the activity of polyphenol oxidase (PPO) and peroxidase (POD), and reduce the oxidative polymerization of phenolic substances. Its function is to solve the problem of oxidative browning at the grafting interface of distant grafting from the root and protect the integrity of the incision cells.

[0042] The cell wall degrading enzyme system is preferably a combination of β-1,3-glucanase and pectin lyase, with the mass ratio of β-1,3-glucanase to pectin lyase preferably being 1:1 to 4:1, more preferably 2:1. The preferred concentration of β-1,3-glucanase is 0.1-0.2%, and the preferred concentration of pectin lyase is 0.05-0.1%. The optimal ratio is 0.15% β-1,3-glucanase and 0.075% pectin lyase. The mechanism of action is that β-1,3-glucanase specifically degrades the key substrate for callosity synthesis, while pectin lyase degrades the pectin components of the cell wall. Its function is to efficiently deconstruct callosity, solving the core problem of vascular bundle blockage and the inability of nutrient exchange between the rootstock and scion, effectively addressing the vascular bundle obstruction problem in the grafting of *Persimmon au au* and *Chocolate Pudding Fruit*.

[0043] In this invention, the nanocarrier is preferably KH550-modified nano-silica with a particle size of 50-80 nm and a Zeta potential of -35 mV. The preferred concentration in the targeted enzymatic hydrolysis solution is 0.4%. The mass concentration of KH550 in the nanocarrier is 2%-5%, the reaction temperature is 60-80℃, and the stirring time is 2-3 h. This modification introduces hydrophilic amino (-NH2) and hydroxyl (-OH) groups: the amino (-NH2) forms strong hydrogen bonds with the carbonyl group (C=O) of pectin in the extracellular matrix of persimmon cambium cells, and the hydroxyl (-OH) forms weak hydrogen bonds with the carboxyl group (-COOH) of proteins, jointly achieving wound anchoring. This anchoring effect keeps the wound moist, guides the orderly arrangement of callus cells, and slowly releases the loaded plant growth regulators, continuously promoting healing. In this invention, the 0.4% concentration represents the optimal balance between the adsorption of active ingredients and the targeted delivery efficiency, avoiding component aggregation or insufficient adsorption, and is suitable for the thin cambium and slow nutrient transport characteristics of persimmon trees.

[0044] In this invention, the plant extract rich in flavonoids and vitamin C is preferably finger lemon extract; finger lemon extract is a clear extract obtained by crushing, pressing, filtering and sterilizing fresh finger lemon fruit; the extract contains quercetin flavonoids ≥1.2mg / g, organic acids ≥0.8mg / g, and a total content ≥0.8% (HPLC detection), and vitamin C ≥500mg / 100g; the addition ratio of finger lemon extract in the targeted enzymatic hydrolysis solution is preferably 4%~6%; more preferably 5%; the finger lemon extract can be replaced by Rutaceae plant extract with the same flavonoid and vitamin C content, or an artificially compounded flavonoid + vitamin C mixed solution. The mechanism of action involves the formation of a complex between quercetin and the sulfhydryl groups of NAC, which synergistically reduces H2O2 with vitamin C, while simultaneously disrupting the cell membrane of pathogens. Its functions include auxiliary antibacterial and antioxidant effects, making it suitable for tropical and subtropical high-temperature and high-humidity environments. The flavonoid components exhibit an inhibition rate of over 68% against common grafting pathogens of persimmon trees, and the synergistic effect of vitamin C and NAC can reduce the browning rate by 25%–30%. This extract can be replaced with extracts from Rutaceae plants with equivalent flavonoid and vitamin C content, or artificially formulated flavonoid + vitamin C mixed solutions, ensuring the feasibility of the technology.

[0045] In this invention, the targeted enzymatic hydrolysis solution contains a protein enzyme system that is sensitive to pH, metal ions and storage environment. It is recommended to prepare and use it immediately, or to prepare it as a freeze-dried powder and then reconstitute it before use. When preparing the solution, adjust the pH to 5.5~6.0 and let it stand at room temperature for 10 minutes before use to avoid enzyme inactivation and to adapt it to the acid and alkali tolerance range of persimmon tree cut cells.

[0046] In this invention, the primary healing inducer comprises the following components by weight percentage: 0.01%~0.08% compound plant growth regulator, 0.05%~0.3% biopolysaccharide, 0.2%~2.0% nanocellulose, 0.05%~0.2% bactericide, 0.01%~0.05% calcium source, 3%~8% plant extract rich in flavonoids and vitamin C, 0.001%~0.005% zeatin, and the balance being water. The functions of the primary healing inducer are: promoting callus differentiation, guiding vascular bundle connectivity, antibacterial activity, preventing cyst formation, and water-retaining slow-release.

[0047] In this invention, the composite plant growth regulator is preferably 0.02%~0.07%; it is composed of naphthaleneacetic acid (NAA), indolebutyric acid (IBA), and 24-epibrassinolide (EBR), and the mass ratio of NAA, IBA, and EBR is preferably 2~5:1:1, more preferably 3:1:1. Mechanism of action: NAA promotes cell division and elongation, IBA induces vascular bundle differentiation, and EBR regulates the expression of genes related to callus formation; its function is to accelerate callus differentiation and vascular bundle connection, solving the problem of slow healing. This formulation can shorten callus formation time by more than 30%, increase vascular bundle connectivity efficiency by more than 25%, and achieve a callus induction rate of more than 96%, thus meeting the grafting compatibility improvement requirements of *Persimmon aubergine* and *Chocolate Pudding Fruit*.

[0048] In this invention, the biopolysaccharide is chitosan oligosaccharide and sodium alginate; the preferred mass percentage of chitosan oligosaccharide is 0.04%, and the preferred mass percentage of sodium alginate is 0.03%. The mechanism of chitosan oligosaccharide is to induce the rootstock and scion to produce PR protein, alleviate the genetic rejection between Taiqiu persimmon and chocolate pudding fruit, and destroy the cell membrane of pathogens; the mechanism of sodium alginate is to absorb water to form a breathable and water-retaining gel film, thereby achieving the slow release of active ingredients; the synergy of the two can improve the grafting survival rate, reduce the incidence of cysts, reduce the rate of water loss from the incision, and prolong the release period of active ingredients, thus meeting the grafting healing needs of persimmon trees in tropical and subtropical high-humidity environments.

[0049] In this invention, the concentration of the nanocellulose added to the main healing inducer is preferably 0.3% to 1.0%, more preferably 0.5%. The mechanism is to enhance adhesion by relying on the fibrous network structure and bind to polysaccharides on the surface of persimmon cells; the function is to improve the adhesion of the healing agent, reduce detachment, and solve the problems of smooth grafting cuts and easy detachment of the healing agent in persimmon trees.

[0050] In this invention, the calcium source is calcium nitrate, and its concentration in the main healing inducer is preferably 0.012%~0.02%, more preferably 0.015%. The mechanism is to supplement calcium ions, stabilize the cell membrane structure of persimmon trees, and promote the synthesis of pectin calcium in the cell wall. The function is to reduce the cell necrosis rate, improve the bonding strength between Taiqiu persimmon and chocolate pudding fruit rootstock and scion, and reduce cyst formation. The fungicide is preferably difenoconazole, and its concentration in the main healing inducer is preferably 0.06%~0.09%, more preferably 0.08%. Difenoconazole is a low-toxicity fungicide specifically designed for persimmon planting and grafting. Its mechanism of action is to inhibit the biosynthesis of ergosterol in the cell membrane of pathogens and disrupt the cell wall structure of pathogens. Its function is to specifically inhibit common pathogens such as Fusarium, Anthracnose, and Phytophthora persimmon in the grafting of Taiqiu persimmon and Chocolate Pudding persimmon. It is suitable for the antibacterial needs of tropical and subtropical high temperature and humidity environments. A concentration of 0.08% can reduce the cut rot rate to below 2.5% and has no phytotoxicity to persimmon cells. Its antibacterial effect and safety are far superior to those of broad-spectrum fungicides such as carbendazim.

[0051] In this invention, the plant extract rich in flavonoids and vitamin C is preferably finger lemon extract, which contains quercetin ≥0.8% (HPLC detection) and vitamin C ≥500mg / 100g. The preferred addition ratio of finger lemon extract to the targeted enzymatic hydrolysis solution is 4%~6%; more preferably 5.5%. The finger lemon extract can be replaced by Rutaceae plant extract with the same flavonoid and vitamin C content, or an artificially formulated flavonoid + vitamin C mixture. The mechanism is to synergistically enhance the antioxidant and antibacterial effects of NAC and difenoconazole, further reducing the infection rate of persimmon grafting cuts.

[0052] In this invention, the zeatin content is preferably 0.002% to 0.004%, more preferably 0.003%.

[0053] In this invention, the total amount of the targeted enzymatic hydrolysis and unblocking solution is 1000g (which can be scaled proportionally), and the optimal mass ratio of each component is: NAC 0.20%, β-1,3-glucanase 0.15%, pectin lyase 0.075%, hydrophilic nano-silica aqueous dispersion 0.4%, finger lemon extract 5%, and the remainder is sterile water; the total amount of the main healing inducer is 1000g (which can be scaled proportionally), and the optimal mass ratio of each component is: compound plant growth regulator 0.025%, chitosan oligosaccharide 0.04%, sodium alginate 0.03%, nanocellulose 0.5%, difenoconazole 0.08%, calcium nitrate 0.015%, finger lemon extract 5.5%, and the remainder is sterile water.

[0054] In this invention, the principles for applying the targeted enzymatic hydrolysis and unblocking solution are: first the rootstock, then the scion; thin and even application; focusing on the cambium layer; and precise quantity control. A sterile brush (0.5-1cm bristle length) is used for application, sterilized with 75% alcohol for 30 minutes before application. The cambium layer area inside the rootstock cut is applied first, followed by the surface of the scion cut, avoiding contact with buds and petiole bases. The application thickness is 0.10-0.2mm, with 0.4mL applied per plant, avoiding excessive accumulation that could lead to oxygen deficiency and rotting at the graft union, or insufficient application affecting the effect. After application, allow to stand for 10-15 seconds to allow initial absorption of the components before inserting the scion, ensuring cambium alignment. The principles for applying the main healing inducer are: after scion insertion, apply evenly to the rootstock-scion junction and a 0.5cm radius around it; in low-temperature winter conditions, supplement with 0.2-0.4mL per plant to ensure enzymatic hydrolysis and antioxidant effects, adapting to the phenological characteristics of the chocolate pudding fruit, which exhibits vigorous growth in summer and continuous growth in winter. Operating environment: The entire process is carried out in a sterile environment to avoid cut infection, which is suitable for the characteristics of persimmon grafting cuts that are susceptible to pathogen infection.

[0055] This invention provides a grafting method suitable for persimmon and chocolate pudding fruit, comprising the following steps: (1) Select chocolate pudding fruit rootstock with a ground diameter of 1.0~1.8cm; select persimmon scion, cut off the leaves but keep the petiole, and make a slanted cut; (2) Implement a double-layer dynamic nutrient balance technology on chocolate pudding fruit rootstock. Retain 2-3 strong branches with a diameter of 3-10cm at the base of the rootstock as the lower carbon sink energy storage layer, with a length of 50-120cm and 8-15 healthy leaves on each branch. At 5-8cm below the grafting interface of the rootstock, retain 1-2 strong small branches with a diameter of 0.2-1cm as the upper directional energy supply layer, with a length of 20-40cm and 2-5 functional leaves on each branch. (3) Take the targeted enzymatic hydrolysis and unblocking solution, first apply it to the cambium layer of the rootstock incision, then apply it to the scion incision, let it stand for 10~15s and then insert it; after the scion is inserted, apply the main healing inducer to the rootstock-scion junction and the surrounding 0.5cm area, and seal it with grafting film. (4) 40-50 days after grafting, when the scion buds are 4-6 cm long and the leaves have unfolded, remove the grafting film.

[0056] In this invention, it is preferred to select a robust, disease-free, and pest-free chocolate pudding fruit rootstock with a diameter of 1.0-1.8cm, and a robust, disease-free persimmon scion with plump buds. The scion is cut to a length of 5-9cm, retaining 2-3 plump buds, removing the leaves but retaining the petioles, and making a slanted cut. Then, implement a two-layer dynamic nutrient balance pruning technique on the rootstock. Retain 2-3 strong branches with a diameter of 3-10cm at the base of the rootstock as the lower carbon sink energy storage layer, with a length of 50-120cm and 8-15 healthy leaves on each branch. At 5-8cm below the grafting point of the rootstock, retain 1-2 strong small branches with a diameter of 0.2-1cm as the upper directional energy supply layer, with a length of 20-40cm and 2-5 functional leaves on each branch. Pruning tools need to be sterilized with 75% alcohol. No additional disinfection or application of healing agent is required after pruning. The steps for applying the healing agent and sealing the graft union in this invention are as follows: The targeted enzymatic hydrolysis and unblocking solution is prepared fresh and used immediately; using a sterile brush, apply 0.6-0.8 ml / plant of the targeted enzymatic hydrolysis and unblocking solution to the cambium layer of the rootstock cut first, then to the cut of the scion, with a thickness of 0.1-0.2 mm and an application amount of 0.2-0.4 mL / plant; allow to stand for 10-15 seconds before grafting; after the scion is inserted, apply the main healing inducer to the rootstock-scion junction and surrounding area, and seal with grafting film; The film is removed 40-50 days after grafting, when the scion buds have grown to 4-6 cm and the leaves have unfolded.

[0057] In this invention, after the grafted seedling matures, a second grafting is performed on the lower carbon sink energy storage layer. The number of second grafting scions does not exceed two, and the second grafting scions and the main scions are triangularly distributed on the rootstock. The distance between the second grafting interface and the original main grafting interface is not less than 15cm.

[0058] In this invention, during winter grafting, 0.2~0.42ml / plant of targeted enzymatic hydrolysis and unblocking solution is applied to the rootstock-scion junction.

[0059] To address the nutrient supply imbalance problem in cross-phenological grafting of *Persimmon au Autumn* (temperate dormant type) and *Chocolate Pudding Fruit* (tropical evergreen type), this invention innovatively employs a two-layer dynamic nutrient balance grafting technology. Through precise pruning and functional differentiation of *Chocolate Pudding Fruit* rootstock branches, it achieves precise physiological regulation of "root nourishment in the lower layer and nutrient supply in the upper layer." The innovation of this two-layer dynamic nutrient balance grafting technology lies in precisely matching the phenological differences between the *Persimmon au Autumn* scion (temperate dormant type) and the *Chocolate Pudding Fruit* rootstock (tropical evergreen type), achieving precise nutrient regulation through functional zoning. The lower carbon sink storage layer (3-10cm in diameter): As a "carbon sink organ," it converts inorganic nutrients into organic nutrients through photosynthesis. In winter, it can consume excess nutrients from the rootstock, maintaining the metabolic balance between the rootstock and scion, and preventing dormancy disorders in the *Persimmon au Autumn* scion due to nutrient excess in the rootstock. Upper directional energy supply layer (0.2~1cm in diameter): Acting as a "nutrient transport hub," it precisely delivers nutrients to the grafting interface, providing essential active nutrients to the dormant *Persimmon scion* during winter, promoting callus formation and vascular bundle connectivity. This achieves a physiological balance of "root nourishment in the lower layer and nutrient supply in the upper layer," solving the core challenge of cross-phenological grafting. It is also simple to operate, requiring no additional disinfection or application of healing agents, and is well-suited to the vigorous growth characteristics of *Persimmon pudding*. Specific specifications and effects are as follows (the double-layer dynamic nutrient balance technology is suitable for rootstocks older than two years): Lower carbon sink energy storage layer: retain 2-3 strong branches with a diameter of 3-10cm at the base of the rootstock, cut to 40-120cm, and retain 4-10 healthy leaves on each branch; its function is to convert inorganic nutrients into organic nutrients to supply the entire grafting system. In winter, it can consume excess nutrients from the chocolate pudding fruit rootstock, maintain the metabolic balance between rootstock and scion, and prevent excessive nutrients from the rootstock from causing dormancy disorder in the persimmon scion. It is suitable for the characteristics of chocolate pudding fruit, which has continuous photosynthesis and high nutrient accumulation in winter.

[0060] Upper directional energy supply layer: 5-8cm below the grafting point of the rootstock, retain 12 strong twigs with a diameter of 0.2-1cm, cut to 20-50cm, and retain 2-3 functional leaves on each twig; the function is to accurately transport nutrients to the grafting point, deliver the necessary active nutrients and energy to the dormant Taiqiu persimmon scion in winter, promote callus formation and vascular bundle connectivity, and solve the problem of weak nutrient absorption capacity of Taiqiu persimmon during dormancy.

[0061] Technical Results: Through comparative winter grafting experiments, the experimental group using the double-layer dynamic nutrient balance technology of this invention achieved a winter grafting survival rate of 88% for Taiqiu persimmon × chocolate pudding fruit, while the traditional grafting control group without the double-layer dynamic nutrient balance technology had a winter survival rate of only 2%, representing a 4300% increase in survival rate; this technology fully adapts to the growth characteristics of chocolate pudding fruit rootstock.

[0062] Secondary grafting specifications: The double-layer dynamic nutrient balance technology allows for secondary grafting after the grafted seedlings have survived, enabling rapid canopy expansion and increasing early fruiting rate. To avoid concentrated nutrient competition and disruption of the rootstock-scion nutrient balance after secondary grafting, the following specifications must be followed: Only robust lower carbon sink storage layers should be selected for secondary grafting on each Chocolate Pudding Fruit rootstock. The number of secondary grafting scions should not exceed two, and the secondary grafting scions and the main scion should be triangularly distributed on the rootstock. The distance between the secondary grafting interface and the original main grafting interface should not be less than 15cm. The pretreatment of scions, application of healing agent, and sealing of interfaces during secondary grafting should all follow the grafting process specifications of this invention, adapting to the strong nutrient supply capacity of Chocolate Pudding Fruit.

[0063] One-year-old grafted seedlings are small and only suitable for the single-layer auxiliary branch method. At a depth of 5-15 cm below the grafting point, retain two new buds in different directions and remove other excess buds. After 90 days of successful grafting, prune the branches appropriately according to the growth situation to prevent the growth of branches from affecting the growth of the grafted seedling.

[0064] Complete grafting process

[0065] The grafting method of this invention is a cross-phenological and cross-ploidy distant grafting design between temperate hexaploid *Diospyros kaki* scion and diploid tropical chocolate pudding fruit rootstock. It can also be adapted to other cross-phenological distant grafting combinations within the same family. The specific steps are as follows: Selection and pretreatment of rootstock and scion: Select healthy, disease-free seedlings with a diameter of 1.0-1.8cm from the fruit of the chocolate pudding plant as rootstock, and healthy, disease-free one-year-old branches of temperate hexaploid persimmon as scions. Cut the branches to a length of 5-9cm, retain 2-3 plump buds, remove the leaves but retain the petioles, and make a slanted cut to increase the contact area of ​​the cambium.

[0066] Pruning and cultivation of dual-layer dynamic nutrient balance technology: Operate according to the above-mentioned dual-layer dynamic nutrient balance technology pruning specifications, and combine the growth vigor of chocolate pudding fruit to precisely control the thickness, length and number of leaves retained in the auxiliary branches, so as to ensure the carbon sink function of the lower carbon sink energy storage layer and the precise nutrient transport function of the upper directional energy supply layer.

[0067] Special two-component sequential healing agent application and interface sealing: Apply the healing agent scientifically according to the usage specifications. The targeted enzymatic hydrolysis and unblocking solution is prepared and used immediately to ensure enzyme activity. The main healing inducer is precisely controlled in dosage to match the growth characteristics of the persimmon cambium. After the scion is inserted, ensure that the cambium is aligned and tightly wrap the interface with grafting film to prevent rainwater intrusion and moisture loss, providing a moist and sterile environment for graft interface healing, which is suitable for the high humidity and rainy climate characteristics of tropical and subtropical regions.

[0068] The process synergistically follows these steps: Pre-treatment of the scion and rootstock lays the physiological foundation for grafting, enhancing the activity and stress resistance of the *Persimmon tamarind* scion and the *Chocolate Pudding* rootstock, matching their growth characteristics; a dual-layer dynamic nutrient balance technology achieves precise nutrient regulation and physiological balance, especially addressing winter phenological conflicts and ensuring continuous nutrient supply, adapting to the year-round growth characteristics of *Chocolate Pudding*; scientific application of the main healing inducer combined with standardized application of targeted enzymatic hydrolysis and unblocking solution achieves multiple effects including enzymatic hydrolysis, anti-oxidation, healing promotion, and specialized antibacterial properties, improving healing quality and specifically addressing the core challenges of distant grafting of persimmon trees; interface sealing and post-grafting management provide a stable environment for graft interface healing, adapting to the characteristics of tropical and subtropical climates and ensuring robust growth of the grafted seedlings. These steps work synergistically to form a closed-loop technology, effectively overcoming the core obstacles of cross-phenological and cross-ploidy distant grafting of *Persimmon tamarind* and *Chocolate Pudding*.

[0069] The innovative dual-layer dynamic nutrient balance technology of this invention reveals the nutrient adaptation rules of distant grafting of cross-phenological and transploid fruit trees from three dimensions: carbon source allocation, nutrient transport, and hormone balance.

[0070] 1) The dual-layer dynamic nutrient balance technology of this invention achieves precise allocation and absorption of carbon sources through functional zoning.

[0071] This invention's dual-layer dynamic nutrient balance technology achieves precise allocation and absorption of carbon sources through functional zoning. The lower carbon sink storage layer (3-10cm in diameter) serves as the rootstock's "carbon sink organ," retaining 8-15 healthy leaves to convert excess photosynthetic carbon sources in the rootstock into starch and store it in the rootstock root system, preventing carbon sources from flowing upwards and breaking the scion's dormancy. The upper directional energy supply layer (0.20-1cm in diameter) serves as a "nutrient transport hub," retaining 2-5 functional leaves to directionally transport precisely quantified active carbon sources (soluble sugars) to the grafting interface, meeting only the basic needs for callus formation and vascular bundle differentiation, matching the low metabolic level of the dormant scion.

[0072] 2) Improved the "channel adaptation for nutrient transport in transploid grafting".

[0073] One of the core obstacles to distant grafting of transploid fruit trees is the imbalance in nutrient transport efficiency caused by the difference in cell ploidy between rootstock and scion. There are significant differences in cell volume and vascular bundle diameter between hexaploid persimmon and diploid chocolate pudding fruit, which easily leads to the problem of "mismatch between the nutrient transport rate of the rootstock and the receiving rate of the scion", which in turn causes nutrient accumulation and cyst formation at the grafting point.

[0074] This invention's dual-layer dynamic nutrient balance technology constructs a "gradient adaptation system" for nutrient transport in transploid grafting through precise quantitative design of branch thickness and leaf quantity: the thin branch characteristics (0.2-1cm in diameter) of the upper directional energy supply layer ensure that the diameter of its vascular bundle vessels is highly adapted to the diameter of the vessel in the Taiqiu persimmon scion, achieving slow-rate, directional nutrient transport and avoiding nutrient accumulation at the grafting interface; the thick branch characteristics (3-10cm in diameter) of the lower carbon sink energy storage layer ensure the carbon source storage capacity of the rootstock root system, reserving nutrients for the rapid growth of the scion after breaking dormancy in the later stages of grafting.

[0075] 3) The dual-layer dynamic nutrient balance technology achieves the autonomous synthesis and balance of endogenous hormones by preserving photosynthetic organs. This invention's dual-layer dynamic nutrient balance technology achieves autonomous synthesis and balance of endogenous hormones by preserving photosynthetic organs. The functional leaves of the upper directed energy supply layer can autonomously synthesize auxin (IAA) and cytokinin (CTK), which are then transported to the grafting interface to provide continuous hormonal signals for callus formation and vascular bundle differentiation. The lower carbon sink energy storage layer maintains the hormonal balance between the rootstock and scion by regulating the abscisic acid (ABA) content of the rootstock. In winter, it reduces the amount of ABA synthesized by consuming excess nutrients in the rootstock, avoiding excessively high ABA concentration in the scion that leads to excessive dormancy, while ensuring the basic hormone level at the grafting interface.

[0076] 4) Targeted delivery and scaffold function innovation of nanomaterials in fruit tree grafting healing agents—providing a demonstration for the application of nanotechnology in fruit trees.

[0077] This invention innovatively applies nano-silica (targeting carrier) and nano-cellulose (scaffold material) to the design of a two-component system for fruit tree grafting healing agents. Through surface modification and functional customization of nanomaterials, it achieves targeted delivery, in-situ anchoring, and three-dimensional scaffold support of the active ingredients in the grafting healing agent, constructing a precise action system of "nanomaterials - active ingredients in the healing agent - grafting incision cells". This not only breaks through the technical bottleneck of fruit tree grafting healing agents, but also provides a replicable and scalable technical demonstration for the large-scale application of nano-agricultural technology in fruit tree cultivation, realizing cross-domain technological innovation and application expansion.

[0078] Nanocarriers provide sustained release, enhanced adhesion, and in-situ anchoring—achieving precise targeting of active ingredients in healing agents.

[0079] This invention uses nano-silica (particle size 50~80 nm, Zeta potential -35 mV) modified with KH550 silane coupling agent as a nanocarrier for targeted enzymatic hydrolysis and unblocking solution. By utilizing the surface modification effect and biological targeting of nanomaterials, it solves the core problems of easy loss, dispersion of action sites, and low utilization rate of active ingredients (enzymes, antioxidants) in traditional healing agents, and achieves precise delivery and in-situ anchoring of active ingredients in grafting incision cambium cells.

[0080] The three-dimensional scaffold function of nanocellulose—guiding the orderly growth of callus and the directional differentiation of vascular bundles.

[0081] The key to graft healing in fruit trees lies in the orderly growth of callus tissue and the directional differentiation of vascular bundles. This invention uses nanocellulose (particle size 60~100 nm) as a nanoscaffold material with high specific surface area, high adhesion, and three-dimensional network structure to construct a biomimetic three-dimensional scaffold for the growth of callus tissue at the grafting interface, thus achieving orderly growth of callus tissue and directional differentiation of vascular bundles. Its innovative value is reflected in three aspects: A three-dimensional network structure forms a biomimetic scaffold for callus growth: Nanocellulose has a fibrous microstructure and good film-forming properties. When applied to the grafting incision, it can quickly form an interwoven three-dimensional network film. The pore size (50~100 nm) of this film is highly compatible with the diameter of callus cells, which can provide physical support for the division and growth of callus cells, guide the callus cells to grow in a directional manner along the network structure, avoid cyst formation caused by disordered proliferation, and significantly reduce the incidence of cysts.

[0082] High adhesion enhances the adhesion between the healing agent and the cut—strengthening the close contact between rootstock and scion cells: Nanocellulose can form hydrogen bonds and van der Waals forces with polysaccharide molecules on the surface of persimmon tree cut cells, significantly improving the adhesion of the healing agent and significantly reducing the shedding rate 24 hours after application, ensuring close contact between rootstock and scion cambium cells, and laying the foundation for the directional differentiation of vascular bundles.

[0083] Synergistic active ingredients enable directional induction of vascular bundles: The three-dimensional mesh scaffold of nanocellulose can serve as a directional adsorption carrier for plant growth regulators (NAA:IBA:EBR=3:1:1), fixing the growth regulators that promote vascular bundle differentiation at specific sites on the scaffold, guiding callus cells to differentiate along the scaffold direction to form vascular bundle vessels, shortening the vascular bundle connection time from the traditional 30 days or more to 18-20 days, and improving the connection efficiency.

[0084] This invention combines the three-dimensional scaffold function of nanocellulose with the growth patterns of grafted callus tissue in fruit trees, achieving for the first time the synergistic effect of physical regulation and chemical induction of callus growth. It breaks through the traditional technical approach of "relying solely on chemical substances to regulate callus growth," proposing a new application of nano-agricultural technology: "nano-bionic scaffold guiding the directional growth of plant tissues." The synergistic effect of two nanomaterials—constructing a "nano-regulatory system" for grafted healing in fruit trees—provides a demonstration for the application of nano-agricultural technology in the fruit tree field.

[0085] This invention does not simply involve the superposition of two nanomaterials. Instead, based on the functional sequence of the targeted enzymatic hydrolysis and unblocking solution and the main healing inducer, it achieves the synergistic effect of targeted delivery of nano-silica and the three-dimensional scaffold of nanocellulose, constructing a nano-regulatory system covering the entire grafting healing process from "early enzymatic hydrolysis and unblocking to late-stage healing differentiation": In the early grafting stage (0-3 days), nano-silica precisely delivers enzymes and antioxidants to the cambium cells, achieving callosity deconstruction and ROS clearance, clearing obstacles for callus formation; in the late grafting stage (3-20 days), the three-dimensional scaffold of nanocellulose guides the orderly growth of callus, while simultaneously adsorbing growth regulators in a directional manner, inducing vascular bundle directional differentiation, and achieving efficient affinity between the rootstock and scion. The functional sequence of the two nanomaterials is highly compatible with the physiological process of grafting healing, forming a complete nano-regulatory chain of "targeted delivery - in-situ action - scaffold support - directional differentiation".

[0086] The following describes the invention in further detail with multiple sets of different formulation examples and various control experiments (budding control, branch growth rate control after one year, cyst control, winter survival rate control with and without double-layer dynamic nutrient balance technology, high temperature and high humidity resistance control, grafting interface firmness and vascular bundle connectivity stability control). The scope of protection of the invention is not limited to the following examples.

[0087] Experimental Description: All examples and control experiments used temperate hexaploid *Persimmon tamariscina* scion × diploid tropical chocolate pudding fruit rootstock as the core grafting combination, with 50 plants in each group. The soil fertility, light, and humidity of the experimental plots were consistent (soil pH 5.5-6.0, daytime temperature 28-36℃ in summer and 10-20℃ in winter, humidity 70%-80%). The grafting process of this invention was uniformly followed (except for the control experiment), and the observation indicators and detection methods were consistent.

[0088] Finger lemon extract is a clear extract obtained by crushing, pressing, filtering and sterilizing fresh finger lemon fruits; the extract contains quercetin flavonoids ≥1.2mg / g, organic acids ≥0.8mg / g, and a total content ≥0.8% (HPLC detection), and vitamin C content = 520mg / 100g.

[0089] Example 1

[0090] Targeted enzymatic hydrolysis and unblocking solution (1000g): NAC 0.20% (2.0g), β-1,3-glucanase 0.15% (1.5g), pectin lyase 0.075% (0.75g), nano silica 0.4% (4.0g), finger lemon extract 5% (50g), sterile water 941.75g; among which, the finger lemon extract contains 520mg / 100g of vitamin C; the targeted enzymatic hydrolysis and unblocking solution should be prepared and used immediately, and the pH should be adjusted to 5.8.

[0091] Main healing inducer (1000g): finger lemon extract 5.5% (55g), nanocellulose 0.5% (5.0g), compound plant growth regulator (NAA:IBA:EBR=3:1:1) 0.025% (0.25g), difenoconazole 0.08% (0.8g), calcium nitrate 0.015% (0.15g), chitosan oligosaccharide 0.04% (0.4g), sodium alginate 0.03% (0.3g), zeatin (ZR): 0.003% (0.03g), balance: sterile water; Example 2 (Targeted Enzymatic Hydrolysis and Unblocking Solution without NAC) Targeted enzymatic hydrolysis and unblocking solution (1000g): β-1,3-glucanase 0.15% (1.5g), pectin lyase 0.075% (0.75g), nano silica 0.4% (4.0g), finger lemon extract 5% (50g), sterile water 941.75g; the targeted enzymatic hydrolysis and unblocking solution should be prepared and used immediately, and the pH should be adjusted to 5.8.

[0092] Main healing inducer (1000g): finger lemon extract 5.5% (55g), nanovitamin 0.5% (5.0g), compound plant growth regulator (NAA:IBA:EBR=3:1:1) 0.025% (0.25g), difenoconazole 0.08% (0.8g), calcium nitrate 0.015% (0.15g), chitosan oligosaccharide 0.04% (0.4g), sodium alginate 0.03% (0.3g), zeatin (ZR): 0.003% (0.03g), balance: sterile water; Example 3 (Main healing inducer without NAA) Targeted enzymatic hydrolysis and unblocking solution (1000g): NAC 0.20% (2.0g), β-1,3-glucanase 0.15% (1.5g), pectin lyase 0.075% (0.75g), nano silica 0.4% (4.0g), finger lemon extract 5% (50g), sterile water 941.75g; among which, the finger lemon extract contains 520mg / 100g of vitamin C; the targeted enzymatic hydrolysis and unblocking solution should be prepared and used immediately, and the pH should be adjusted to 5.8.

[0093] Main healing inducer (1000g): finger lemon extract 5.5% (55g), nanocellulose 0.5% (5.0g), compound plant growth regulator (IBA:EBR=1:1) 0.025% (0.25g), difenoconazole 0.08% (0.8g), calcium nitrate 0.015% (0.15g), chitosan oligosaccharide 0.04% (0.4g), sodium alginate 0.03% (0.3g), zeatin (ZR): 0.003% (0.03g), balance: sterile water; Example 4 (Fingerless Lemon Infusion) Targeted enzymatic hydrolysis and unblocking solution (1000g): NAC 0.20% (2.0g), β-1,3-glucanase 0.15% (1.5g), pectin lyase 0.075% (0.75g), nano silica 0.4% (4.0g), and the remainder sterile water. The targeted enzymatic hydrolysis and unblocking solution should be prepared fresh and the pH adjusted to 5.8.

[0094] Main healing inducer (1000g): Nanocellulose 0.5% (5.0g), Compound plant growth regulator (NAA:IBA:EBR=3:1:1) 0.025% (0.25g), Difenoconazole 0.08% (0.8g), Calcium nitrate 0.015% (0.15g), Chitosan oligosaccharide 0.04% (0.4g), Sodium alginate 0.03% (0.3g), Zeatin (ZR): 0.003% (0.03g), with the remainder being sterile water.

[0095] Example 5 (with healing agent, single-layer auxiliary branches)

[0096] Targeted enzymatic hydrolysis and unblocking solution (1000g): NAC 0.20% (2.0g), β-1,3-glucanase 0.15% (1.5g), pectin lyase 0.075% (0.75g), nano silica 0.4% (4.0g), finger lemon extract 5% (50g), sterile water 941.75g; the targeted enzymatic hydrolysis and unblocking solution should be prepared and used immediately, and the pH should be adjusted to 5.8.

[0097] Main healing inducer (1000g): finger lemon extract 5.5% (55g), nanocellulose 0.5% (5.0g), compound plant growth regulator (NAA:IBA:EBR=3:1:1) 0.025% (0.25g), difenoconazole 0.08% (0.8g), calcium nitrate 0.015% (0.15g), chitosan oligosaccharide 0.04% (0.4g), sodium alginate 0.03% (0.3g), zeatin (ZR): 0.003% (0.03g), balance: sterile water.

[0098] Grafting method: The optimal grafting process was adopted, with a single layer of auxiliary branches, and the remaining operations were the same as in Example 1.

[0099] Example 6 (with healing agent, without bilayer dynamic nutrient balance technology)

[0100] Targeted enzymatic hydrolysis and unblocking solution (1000g): NAC 0.20% (2.0g), β-1,3-glucanase 0.15% (1.5g), pectin lyase 0.075% (0.75g), nano silica 0.4% (4.0g), finger lemon extract 5% (50g), sterile water 941.75g; the targeted enzymatic hydrolysis and unblocking solution should be prepared and used immediately, and the pH should be adjusted to 5.8.

[0101] Main healing inducer (1000g): finger lemon extract 5.5% (55g), nanocellulose 0.5% (5.0g), compound plant growth regulator (NAA:IBA:EBR=3:1:1) 0.025% (0.25g), difenoconazole 0.08% (0.8g), calcium nitrate 0.015% (0.15g), chitosan oligosaccharide 0.04% (0.4g), sodium alginate 0.03% (0.3g), zeatin (ZR): 0.003% (0.03g), balance: sterile water.

[0102] Grafting method: Select 1-3 year old rootstock, without double-layer dynamic nutrient balance technology, retain 2 new buds in different directions 5-15cm below the grafting point, and remove excess buds; the rest of the operation is the same as in Example 1.

[0103] Compared to the treatment without healing agents and without bilayer dynamic nutrient balance technology

[0104] Control Experiment 1: Germination Control Experiment

[0105] Experimental Design: Six sets of experimental results and a blank control group were selected. In each set, 50 *Persimmon 'Taiqiu'* grafted onto *Chocolate Pudding Fruit* seedlings were selected. Grafting was carried out from January 15 to 17, 2025 (daily temperature 12-20℃, humidity 50%-60%). Bud emergence was managed in a standardized manner. Bud emergence began 17 days after grafting. The bud emergence rate at 60 days after grafting, the actual survival rate of buds at 120 days, and the average bud emergence time were statistically analyzed to verify the effect of the two-component healing agent and the two-layer dynamic nutrient balance technology on the short-term graft healing effect.

[0106] Table 1 Comparison of budding quality and survival rate

[0107] Germination rate, 90-day survival rate, and germination time directly reflect the quality of grafting healing and the efficiency of nutrient exchange between rootstock and scion, and are core indicators for the vigorous growth of grafted seedlings after survival. Examples 1-6, grafted between January 15th and 17th in winter, showed an average germination rate exceeding 80% after 90 days, with all buds surviving within 90 days and an average germination time of less than 25 days, significantly better than the control group. Example 1, in particular, performed best. The core reason lies in the dual-component nano-inducing healing agent's synergistic effects of enzymatic hydrolysis, anti-oxidation, specific antibacterial properties, and healing promotion, rapidly opening vascular pathways and enabling efficient nutrient exchange between rootstock and scion. The dual-layer dynamic nutrient balance technology's "lower layer nourishes roots, upper layer supplies nutrients" function continuously provides sufficient nutrients for bud germination, especially in winter, precisely regulating nutrient distribution to match the nutrient needs of the dormant persimmon scion, preventing delayed germination and weakened growth due to insufficient nutrients. In the fingerless lemon infusion group (Example 4), due to the lack of synergistic antioxidant and antibacterial effects of flavonoids and vitamin C, the browning rate at the grafting interface increased, the nutrient exchange efficiency decreased, the germination time was prolonged, and the germination rate was slightly lower. In Example 6, due to the contradiction between rootstock and scion phenology in winter, the nutrient imbalance was severe. Although a healing agent was used, the double-layer dynamic nutrient balance technology was not employed, and the germination rate dropped to 72%. In the control group, without a healing agent, the vascular bundle connection was slow, the germination time was greatly prolonged, and the buds that did emerge were only pseudo-surviving, and the buds slowly withered and died. The control group, without a healing agent and without the double-layer dynamic nutrient balance technology, had the lowest germination rate and the slowest germination time, which fully verified the core role of the healing agent and the double-layer dynamic nutrient balance technology of this invention in improving germination quality.

[0108] Control Experiment 2: Control Experiment on Branch Growth Rate One Year After Grafting

[0109] Experimental Design: Example 1 (optimal group), Example 5 (with healing agent and single-layer auxiliary branches), Example 6 (with healing agent but without double-layer dynamic nutrient balance technology), and control group (without healing agent and without double-layer dynamic nutrient balance technology) were selected. 30 surviving grafted seedlings were selected from each group. Standardized water and fertilizer management and pest and disease control were uniformly implemented. The average increase in branch diameter (3cm diameter above the grafting point) was recorded within 6 months after grafting. The top was uniformly pinched off at 70 days, and a second pinching off was performed at 120 days. The average crown width was measured at 180 days to verify the effect of the invention's technology on improving the long-term growth vigor of grafted seedlings.

[0110] Table 2. Comparison of growth after grafting one year (unit: cm, %)

[0111] The growth indicators of one-year-old branches of grafted seedlings directly reflect the compatibility between rootstock and scion, nutrient translocation efficiency, and overall plant resistance. In Examples 1-6, the average thickness increase exceeded 30%, and the growth vigor was significantly better than that of the control group. The core mechanism is as follows: The two-component healing agent of this invention not only improves the grafting survival rate, but also accelerates vascular bundle differentiation through compound auxin and strengthens the bond between rootstock and scion through calcium nitrate, achieving efficient and stable nutrient exchange between rootstock and scion; the dual-layer dynamic nutrient balance technology continues to play a role in nutrient regulation and photosynthetic enhancement after the grafted seedlings survive, improving the overall carbon sequestration capacity of the plant and providing sufficient organic nutrients for branch growth; at the same time, the synergistic antibacterial effect of difenoconazole and finger lemon extract reduces the occurrence of diseases during the growing season, further ensuring the healthy growth of the plant. Although Example 5 had a healing agent to ensure graft survival, the lack of post-graft nutrient regulation and photosynthetic enhancement resulted in insufficient nutrient supply to the plants and slow branch growth. In the control group, due to poor vascular bundle connectivity and low efficiency of nutrient exchange between rootstock and scion, the growth of branch thickness and height was significantly limited, the grafting healing quality was poor, the rootstock-scion compatibility was low, and the growth vigor was the weakest. This fully verifies the sustained effect of the technical system of this invention on improving the long-term growth vigor of grafted seedlings.

[0112] Control Experiment 3: Control Experiment on Browning Rate and Purulent Discharge at the Grafting Interface

[0113] Experimental Design: Examples 1 (optimal group), 2 (enzymatic hydrolysis ratio deviation), 4 (without finger lemon extract), 5 (with healing agent, single-layer auxiliary branches), 6 (with healing agent, without double-layer dynamic nutrient balance technology), and a control group (without healing agent, without double-layer dynamic nutrient balance technology) were selected, with 40 plants in each group. 180 days after winter grafting, the browning rate of the grafting interface (browning judgment criteria: brown or dark brown discoloration of the cambium and surrounding tissues of the grafting interface, with an area ≥10%) and the incidence of purulent discharge (purulent discharge judgment criteria: viscous turbid liquid exudation from the grafting interface, a typical characteristic of pathogen infection) were counted to verify the inhibitory effect of the technology of this invention on browning and purulent discharge of the grafting interface of distant grafting.

[0114] Table 3. Browning rate and purulent discharge at the grafting site (unit: %)

[0115] Browning and purulent exudation at the grafting site are among the core obstacles to the distant grafting of *Persimmon 'Taiqiu'* and *Chocolate Pudding Fruit*. Browning is essentially caused by the accumulation of reactive oxygen species at the cut, leading to the oxidation of phenolic substances and cell necrosis. Purulent exudation is a secondary infection caused by pathogens after browning. Both damage the integrity of the cambium and hinder vascular bundle connectivity, potentially causing the grafted seedlings to wither and die in severe cases. Experimental data show that in Example 1 (the optimal group), the browning rate at the grafting site was less than 10% in winter, and the incidence of purulent exudation was 0%, demonstrating a significantly better inhibitory effect than other groups. The core reason for this is: In the two-component nano-inducing healing agent, NAC in the targeted enzymatic hydrolysis and unblocking solution can efficiently scavenge reactive oxygen species and inhibit the activity of polyphenol oxidase and peroxidase, reducing the oxidation of phenolic substances from the root and lowering the browning rate. Flavonoids in the finger lemon extract synergistically enhance the antioxidant effect with vitamin C, while simultaneously destroying the cell membrane of pathogens, inhibiting their reproduction, and preventing purulent discharge. β-1,3-glucanase and pectin lyase, in an optimal 2:1 ratio, decompose callosity, avoiding localized cellular hypoxia and browning caused by abnormal callosity deposition. The main healing inducer, difenoconazole, is a persimmon-specific fungicide that specifically inhibits rot-causing pathogens such as Fusarium and anthracnose, further reducing the risk of infection and purulent discharge. The double-layered auxiliary branches, through precise nutrient regulation of "root nourishment in the lower layer and nutrient supply in the upper layer," achieve a balance between nutrient supply and demand in the rootstock and scion, reducing cellular metabolic disorders caused by nutrient accumulation, and synergistically enhancing the browning and purulent discharge inhibition effects of the healing agent.

[0116] Example 4 (lemon extract without fingers) showed a significant decrease in antioxidant and antibacterial effects due to the lack of flavonoids and vitamin C, and a significant increase in the browning rate and pus discharge rate at the grafting interface. Example 6, although containing a healing agent, lacked nutrient regulation by the double-layer auxiliary branches. The conflict between rootstock and scion phenology led to nutrient accumulation and cell metabolic imbalance, resulting in a browning rate of 32% and a pus discharge rate of 22%. The control group, without any protective or regulatory measures, experienced severe oxidative browning at the cut, with a large number of pathogens invading, resulting in a browning rate exceeding 82% and a pus discharge rate of 38%. The grafting interface completely lost its healing ability. This fully verifies the significant inhibitory effect of the technical system of this invention on browning and pus discharge at the grafting interface of distant grafting, thus overcoming the core grafting obstacle.

[0117] Control Experiment 4: Effectiveness Control Experiment of Two-Layer Dynamic Nutrient Balance Technology

[0118] Experimental Design: Example 1 (optimal group), Example 5 (single-layer auxiliary branches with healing agent), Example 6 (with healing agent but without double-layer dynamic nutrient balance technology), and control group (without healing agent and without double-layer dynamic nutrient balance technology) were selected, with 50 plants in each group. The survival of grafted seedlings was examined during the low-temperature period after the fall of leaves of Taiqiu persimmon in winter (average daily temperature 10~20℃, humidity 70%~75%). Due to the huge difference in phenological period between Taiqiu persimmon and chocolate pudding fruit, a large number of scions died in winter if the double-layer dynamic nutrient balance technology was not adopted.

[0119] Table 4. Comparison of the effectiveness of the two-layer dynamic nutrient balance technology (unit: %, survival rate)

[0120] A survival rate of over 80% is excellent, a survival rate of 60%-79.9% is good, a survival rate of 40%-59.9% is acceptable, and a survival rate of less than 40% is unacceptable.

[0121] Due to the significant difference in phenological periods between *Persimmon auburn* and *Chocolate Pudding Fruit*, *Persimmon auburn* sheds its leaves and enters dormancy in November, while *Chocolate Pudding Fruit*, an evergreen tropical plant, has vigorous physiological metabolism in winter. Seedlings without the dual-layer dynamic nutrient balance technology suffer from high mortality rates during winter. Seedlings with only a single layer of auxiliary branches have a significantly higher winter mortality rate than those using the dual-layer dynamic nutrient balance technology. This is because the *Persimmon auburn* scion is dormant, and the intense root pressure and excess water from the "decapitated" tropical evergreen rootstock can directly "drown" the scion or cause it to wither due to nutrient deficiency after dormancy is broken. The dual-layer dynamic nutrient balance technology acts as a perfect buffer, providing "pressure relief and precise nutrient transfer."

[0122] Control Experiment 5: High Temperature and High Humidity Resistance Control Experiment (Verifying Adaptability to Tropical and Subtropical Regions)

[0123] Experimental Design: Example 1 (optimal group), Example 5 (with healing agent, single-layer auxiliary branches), Example 6 (with healing agent, without double-layer dynamic nutrient balance technology), and control group (without healing agent, without double-layer dynamic nutrient balance technology) were selected. Each group had 30 surviving grafted seedlings. They were placed in a high-temperature and high-humidity environment in Zhongshan (daily maximum temperature 35-38℃, humidity 80%-90%). The daily rainfall in Zhongshan's Minzhong Street was 105mm on June 17, 2025; 147mm on July 10, 2025; and 120mm on September 24, 2025. The survival rate of the plants and the rate of rot at the grafting point were statistically analyzed. The growth of the leaves was observed (no wilting or yellowing was considered normal) to verify the resistance of the technology of this invention to high temperature and high humidity environments (the core difficulties of high temperature and high humidity are: easy rot at the grafting point, easy infection of the plants with diseases, easy wilting of the leaves, and the plant's tolerance to waterlogging).

[0124] Table 5. Comparison of high temperature and high humidity resistance (unit: %)

[0125] The core advantage of chocolate pudding fruit rootstock is its adaptability to tropical and subtropical climates. However, *Persimmon 'Taiqiu'* natively has weak resistance to high temperatures and humidity. If the plant's resistance is insufficient after distant grafting, it will lead to poor plant growth and graft union rot, failing to realize the advantages of the rootstock. Experimental data shows that Example 1 (the optimal group) achieved a 92% survival rate and only a 2% graft union rot rate after 270 days in a high-temperature and high-humidity environment, demonstrating significant resistance. The core reason for this is: The two-component nano-inducing healing agent contains difenoconazole, a specialized antibacterial ingredient that specifically inhibits fungi and bacteria that thrive in high-temperature and high-humidity environments, reducing grafting site rot and plant diseases. Chitosan oligosaccharides increase plant cell wall thickness, enhance plant resistance, and reduce leaf wilting and yellowing caused by high temperature and humidity. The nanocarrier controls the slow release of the healing agent components, ensuring sustained antibacterial and stress-resistant effects while preventing rapid component loss due to high temperature and humidity. The dual-layer dynamic nutrient balance technology improves plant ventilation and light penetration, reduces water accumulation in leaves, lowers the probability of disease, and simultaneously regulates nutrient distribution, enhancing root water absorption capacity and alleviating water imbalance caused by high temperature and humidity.

[0126] The control group, which had no healing agent, had a high grafting interface rot rate, a plant survival rate of only 52%, and a high rate of leaf wilting and yellowing, clearly indicating that it was not adapted to the climate conditions of Zhongshan. This fully verifies that the technical system of this invention can significantly improve the high temperature and humidity resistance of grafted seedlings, perfectly matching the climate adaptability advantages of chocolate pudding fruit rootstock, and expanding the planting range of Taiqiu persimmon to tropical and subtropical regions.

[0127] Control Experiment 6: Typhoon Area Adaptability Control Experiment (Verifying Lodging Resistance and Graft Joint Strength)

[0128] Experimental Design: Example 1 (optimal group), Example 5 (with healing agent, single-layer auxiliary branches), Example 6 (with healing agent, without double-layer dynamic nutrient balance technology), and control group (without healing agent, without double-layer dynamic nutrient balance technology) were selected. 50 seedlings from each group were selected and planted in the ground with rootstocks planted in 2024, grafted in January 2025. Plant lodging was observed, and the lodging rate and graft union breakage rate were statistically analyzed. The graft union strength (unit: N) was measured to verify the adaptability of this invention to typhoon-prone areas (the core difficulties in typhoon-prone areas are: easy lodging of plants and weak graft union leading to breakage). The data were based on averages from Typhoon Wipha (11-level gusts) in Zhongshan Minzhong Street on July 21, 2025, Typhoon Tapah (10-level gusts) in Zhongshan Minzhong Street on September 7, 2025, and Typhoon Hagasar (11-12-level gusts) in Zhongshan Minzhong on September 23, 2025. Table 6. Typhoon Region Suitability Comparison Table (Unit: %)

[0129] The dual-layer dynamic nutrient balance technology optimizes plant crown shape, reduces wind resistance, and simultaneously enhances overall plant growth, resulting in a more developed root system and increased resistance to lodging and wind breakage. Without this technology, insufficient nutrient supply to the callus tissue leads to a significant decrease in graft union mechanical strength and vascular bundle connectivity, resulting in a higher rate of grafting drop after typhoons. This fully verifies the unique suitability of this invention's technology system for typhoon-prone areas.

[0130] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A healing agent suitable for the grafting of Diospyros kaki and pudding chocolate, characterized in that, It consists of a two-component time-series system of targeted enzymatic hydrolysis and unblocking solution and main healing inducer. The targeted enzymatic hydrolysis and unblocking solution contains the following components by weight percentage: N-acetylcysteine ​​0.1%~0.8%, cell wall degrading enzyme system 0.1%~0.8%, nanocarrier 0.2%~1.0%, plant extract rich in flavonoids and vitamin C 3%~8%, and the balance being water, pH 5.0~6.5; The primary healing inducer comprises the following components by weight percentage: 0.01%~0.08% compound plant growth regulator, 0.05%~0.3% biopolysaccharide, 0.2%~2.0% nanocellulose, 0.05%~0.2% bactericide, 0.01%~0.05% calcium source, 3%~8% plant extract rich in flavonoids and vitamin C, 0.001%~0.005% zeatin, and the balance being water.

2. The healing agent of claim 1, wherein, The cell wall degrading enzyme system includes β-1,3-glucanase and pectin lyase, and the mass ratio of β-1,3-glucanase to pectin lyase is 1:1 to 4:

1. The nanocarrier is KH550 surface-modified nano-silica with a particle size of 50~80 nm and a Zeta potential of -35 mV.

3. The healing agent of claim 1, wherein, The compound plant growth regulator is composed of NAA, IBA and EBR, and the mass ratio of NAA, IBA and EBR is 2~5:1:1; The biopolysaccharide is chitosan oligosaccharide and sodium alginate; the mass ratio of chitosan oligosaccharide to sodium alginate is 3~5:

3.

4. The healing agent of claim 1, wherein, The plant extract rich in flavonoids and vitamin C includes finger lemon infusion, wherein the finger lemon infusion contains quercetin ≥0.8% and vitamin C ≥500mg / 100g.

5. A method of grafting suitable for Diospyros kaki and chocolate pudding fruit, characterized by, Includes the following steps: (1) Select chocolate pudding fruit rootstock with a diameter of 1.0~1.8cm; select persimmon scion, cut off the leaves but keep the petiole, and make a slanted cut; (2) Implement a double-layer dynamic nutrient balance technology on chocolate pudding fruit rootstock. Retain 2-3 strong branches with a diameter of 3-10cm at the base of the rootstock as the lower carbon sink energy storage layer, with a length of 50-120cm and 8-15 healthy leaves on each branch; retain 1-2 strong small branches with a diameter of 0.2-0.8cm 5-8cm below the grafting interface of the rootstock as the upper directional energy supply layer, with a length of 20-40cm and 2-5 functional leaves on each branch. (3) Take the targeted enzymatic hydrolysis and unblocking solution, first apply it to the cambium layer of the rootstock incision, then apply it to the scion incision, let it stand for 10~15s and then insert it; after the scion is inserted, apply the main healing inducer to the rootstock-scion junction and the surrounding 0.5cm area, and seal it with grafting film. (4) 40-50 days after grafting, when the scion buds are 4-6 cm long and the leaves have unfolded, remove the grafting film.

6. The grafting method according to claim 5, characterized in that, The scion of Taiqiu persimmon is a one-year-old branch of Taiqiu persimmon, cut to a length of 5-9cm, retaining 2-3 plump buds.

7. The grafting method according to claim 5, wherein After the grafted seedling matures, select the lower auxiliary branches for secondary grafting. The number of secondary grafting scions should not exceed 2, and the secondary grafting scions and the main scions should be distributed in a triangular shape on the rootstock. The distance between the secondary grafting interface and the original main grafting interface should not be less than 15cm.

8. The grafting method according to claim 5, characterized in that, The thickness of the primary healing inducer application is 0.1~0.2mm, and the application amount is 0.2~0.4ml / plant.

9. The grafting method according to claim 5, characterized in that, Apply 0.6~0.8ml of the targeted enzymatic hydrolysis solution per plant.

10. The grafting method according to claim 5, characterized in that, When grafting in winter, apply 0.2~0.42ml / plant of targeted enzymatic hydrolysis solution to the graft union.

Images