Use of natural polysaccharide-based healing agent in tomato grafting

Abstract

The present application relates to the field of agricultural biotechnology, and more particularly to the application of a healing agent based on natural polysaccharide in tomato grafting. The natural polysaccharide is selected from one or more of xanthan gum, pectin, and yellow pectin, and the natural polysaccharide is applied in the preparation of a healing agent for promoting the healing of a tomato grafting wound. The application of the healing agent based on natural polysaccharide in tomato grafting is first compared systematically with the promoting effects of xanthan gum, pectin, yellow pectin, and tea-brown gum on the healing of a tomato grafting wound, and it is found that yellow pectin has the best effect, can significantly accelerate the formation of callus and the regeneration of vascular bundles, and the grafting interface can be basically closed on the 9th day after grafting.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to the application of a healing agent based on natural polysaccharides in tomato grafting. Background Technology

[0002] tomato( Solanum lycopersicum L. Tomatoes are one of the most widely planted and consumed greenhouse vegetables globally. However, due to limited arable land resources and the prevalence of continuous cropping, soil-borne diseases are exacerbated, soil physical and chemical properties deteriorate, and yield and quality are severely affected. Grafting technology can effectively address continuous cropping obstacles, enhance plant resistance, and improve water and fertilizer use efficiency, and has become a key agronomic measure in greenhouse tomato production. Statistics show that the current usage rate of grafted tomato seedlings in greenhouse cultivation in my country exceeds 60%, and in some major producing areas it reaches over 80%.

[0003] However, the mechanical cutting damage to the tomato stem during grafting directly affects the survival rate and subsequent growth of grafted seedlings due to its healing speed and effectiveness. Slow wound healing not only prolongs the recovery period but also makes the exposed wound surface susceptible to pathogen infection, leading to rotting, wilting, and even death of the grafted seedlings. Production data shows that the mortality rate of grafted tomato seedlings due to poor healing is typically 5%–10%, and can reach 15%–20% under conditions of high temperature and humidity or improper management, causing serious economic losses to growers. Therefore, promoting rapid healing of tomato grafting wounds is a key issue in optimizing grafting cultivation techniques.

[0004] Currently, the main measures to promote wound healing in production fall into two categories: environmental control and chemical agents. While environmental control is highly safe, it is easily limited by facility conditions and its effectiveness is unstable. Although chemical healing agents can promote callus formation and inhibit pathogens, they generally pose a risk of pesticide residues, which does not meet the sustainable development requirements of green agriculture to "reduce pesticides and fertilizers."

[0005] Natural polysaccharides are a class of widely available, biocompatible, and biodegradable macromolecular compounds. In the medical field, natural polysaccharides such as xanthan gum and pectin have been successfully used in wound dressings, promoting fibroblast adhesion and proliferation, inhibiting inflammatory responses, and accelerating wound healing in animals by creating a moist environment. However, research on the application of natural polysaccharides in promoting the healing of tomato grafting wounds is still insufficient, and systematic experimental data are lacking to support the differences in effects and mechanisms of action among different polysaccharides.

[0006] There are already some reports in the prior art regarding the application of polysaccharides in plant wound healing. For example, CN106804605B discloses a plant wound healing agent containing sodium alginate oligosaccharides, which can be used for the treatment of pruning, girdling, or grafting wounds in fruit trees and flowers; CN120887749A discloses a nutrient solution for healing grafted fruit and vegetable wounds containing polysaccharide complex enzymes, which utilizes the combination of chitosan and sodium alginate, along with the synergistic effect of cellulase and pectinase, to promote wound healing.

[0007] However, in these technical solutions, polysaccharides are mainly used as auxiliary components or in combination with other active ingredients. There is no systematic research on the direct application of natural polysaccharides such as xanthan gum, pectin, and flavonoid gum as single active ingredients to the healing of grafted wounds in tomatoes, nor is there a systematic comparison of the effects of different natural polysaccharides on the healing of grafted tomatoes.

[0008] Therefore, a healing agent based on natural polysaccharides was designed for application in tomato grafting, providing a technical solution to the aforementioned technical problems. Summary of the Invention

[0009] Therefore, it is necessary to address the aforementioned technical issues by providing a healing agent based on natural polysaccharides for tomato grafting. This would solve the problem that existing polysaccharides are mainly used as auxiliary components or in combination with other active ingredients, and there is no evidence of using natural polysaccharides such as xanthan gum, pectin, and flavonoid gum as single active ingredients directly for healing tomato grafting wounds.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: The application of natural polysaccharides in the preparation of healing agents that promote the healing of grafted tomato wounds, wherein the natural polysaccharides are selected from one or more of xanthan gum, pectin, and flavocarpus.

[0011] Preferably, the natural polysaccharide is xanthan gum.

[0012] Preferably, the mass-volume ratio of natural polysaccharides in the healing agent is 1% to 5% (w / v).

[0013] Preferably, the mass-volume ratio of natural polysaccharides in the healing agent is 2%~3% (w / v).

[0014] The preferred method for preparing the yellow pectin healing agent includes the following steps: Mix xanthan gum and pectin at a mass ratio of 0.5~1.5:1; Add deionized water to prepare a gel-like consistency. The total mass of xanthan gum and pectin to the volume ratio of deionized water is 1.5~2.5g:40mL.

[0015] Preferably, the mass ratio of xanthan gum to pectin is 0.8:1, and the total mass to deionized water volume ratio is 1.8g:40mL.

[0016] A method to promote the healing of grafted tomato wounds, the steps are as follows: (1) Take tomato rootstock and scion, and graft them using the oblique cutting method: make an oblique cut at 45° 2-3 cm above the base of the rootstock stem, cut a matching oblique surface at the corresponding part of the scion, and align the two together. (2) Apply the natural polysaccharide healing agent evenly to the surface of the grafting interface and the surrounding 2 mm area to ensure complete coverage; (3) Use grafting clips to fix the grafting interface so that the cut surfaces fit tightly together.

[0017] A natural polysaccharide healing agent for tomato grafting, the natural polysaccharide healing agent comprising natural polysaccharides and water, wherein the natural polysaccharides are selected from one or more of xanthan gum, pectin, and tartrate gum.

[0018] The application of the natural polysaccharide healing agent for tomato grafting as described in claim 8 in promoting the survival of tomato grafted seedlings.

[0019] It is clear without a doubt that the technical solutions described above by this invention can solve the technical problems that this invention aims to address.

[0020] Meanwhile, through the above technical solutions, the present invention has at least the following beneficial effects: 1. The application of the natural polysaccharide-based healing agent provided by this invention in tomato grafting is the first systematic comparison of the promoting effects of four natural polysaccharides, xanthan gum, pectin, flavocarb gum and tannin, on the healing of tomato grafting wounds. It was found that flavocarb gum had the best effect, which could significantly accelerate the formation of callus tissue and the regeneration of vascular bundles. The grafting interface could be basically closed on the 9th day after grafting.

[0021] 2. The natural polysaccharide healing agent of this invention can significantly reduce the malondialdehyde (MDA) content in tomato leaves after grafting, increase the peroxidase (POD) activity and soluble sugar content, effectively alleviate oxidative stress caused by grafting, and enhance the plant's antioxidant capacity and osmotic regulation capacity.

[0022] 3. The natural polysaccharide healing agent of this invention can significantly promote the growth of plant height and stem diameter of grafted tomato plants, increase chlorophyll content and survival rate of grafted seedlings, and has a positive impact on the later growth of plants.

[0023] 4. The natural polysaccharides used in this invention are widely available, biocompatible, biodegradable, and have no risk of chemical residues. They meet the requirements of sustainable development in green agriculture and can replace traditional chemical healing agents. Attached Figure Description

[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 This is a schematic diagram of microscopic paraffin sections treated with different methods over 3 days according to the present invention; Figure 2 This is a schematic diagram of microscopic paraffin sections treated with different methods over 6 days according to the present invention; Figure 3 This is a schematic diagram of microscopic paraffin sections treated with different methods over 9 days according to the present invention; Figure 4 This is a schematic diagram illustrating the change in malondialdehyde (MDA) content according to the present invention; Figure 5 This is a schematic diagram illustrating the changes in peroxidase (POD) activity according to the present invention; Figure 6 This is a schematic diagram illustrating the change in soluble sugar content according to the present invention; Figure 7 This is a schematic diagram illustrating the changes in superoxide dismutase (SOD) according to the present invention; Figure 8 This is a schematic diagram illustrating the changes in catalase (CAT) activity according to the present invention; Figure 9 This is a schematic diagram illustrating the changes in tomato seedling height according to the present invention; Figure 10 This is a schematic diagram illustrating the variation in stem thickness of tomato seedlings according to the present invention; Figure 11 This is a schematic diagram illustrating the effects of different treatments of the present invention on the chlorophyll content of tomato leaves; Figure 12 This is a schematic diagram illustrating the effect of different treatments on the survival rate of tomatoes according to the present invention. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0028] It should be noted that, unless otherwise specified, the embodiments and features and technical solutions in the present invention can be combined with each other.

[0029] It should be noted that similar labels and letters in the following figures indicate similar items. Therefore, once an item is defined in one figure, it does not need to be further defined and explained in subsequent figures.

[0030] Reference Figures 1-12Application of healing agents based on natural polysaccharides in tomato grafting.

[0031] Example 1: Preparation of a natural polysaccharide healing agent This embodiment provides four methods for preparing natural polysaccharide healing agents: (1) Xanthan Gum Healing Agent: Weigh 1g of xanthan gum powder, add 20mL of deionized water, stir well to prepare a gel, and use immediately after preparation. The final concentration is 5% (w / v).

[0032] (2) Pectin healing agent: Weigh 1g of pectin powder, add 20mL of deionized water, stir well to prepare a gel, and use immediately after preparation. The final concentration is 5% (w / v).

[0033] (3) Xanthan gum healing agent: Weigh 0.8g xanthan gum powder and 1g pectin powder, add 40mL deionized water, stir evenly to prepare a gel, and use immediately after preparation. The total mass-volume ratio is 4.5% (w / v).

[0034] The preferred method for preparing the yellow pectin healing agent includes the following steps: Xanthan gum and pectin are mixed at a mass ratio of 0.5 to 1.5:1. Preferably, the mass ratio of xanthan gum to pectin is 0.8:1, and the total mass to the volume ratio of deionized water is 1.8 g: 40 mL. Add deionized water to prepare a gel-like consistency. The total mass of xanthan gum and pectin to the volume ratio of deionized water is 1.5~2.5g:40mL.

[0035] (4) Tea brown gum healing agent: Commercially available plant and tree wound healing agent, for direct use.

[0036] In this embodiment, the mass-volume ratio of natural polysaccharides in the healing agent is 1%~5% (w / v), preferably 2%~3% (w / v).

[0037] Example 2: Tomato grafting and healing agent treatment experiment 1. Experimental Materials The tomato variety used in the test was the 'Weilong' series from Shouguang Jinpeng Seed Industry Co., Ltd., with Tomato No. 1 as the scion and the 'Weilong' variety as the rootstock. Seedlings were cultivated using commercial seedling substrate, and grafting was performed uniformly when the plants had 5-6 true leaves and suitable stem diameter.

[0038] Four types of natural polysaccharide healing agents were tested: xanthan gum, pectin, yellow pectin (prepared according to the method in Example 1), and tea brown gum (commercially available).

[0039] Main reagents and uses: The determination of malondialdehyde (MDA) content adopts the thiobarbituric acid (TBA) method, and the required reagents include trichloroacetic acid (TCA), TBA, etc.; the determination of peroxidase (POD) activity adopts the guaiacol method, and the reagents include guaiacol, hydrogen peroxide, etc.; the determination of soluble sugar content adopts the anthrone colorimetric method, and the reagents include anthrone, concentrated sulfuric acid, anhydrous ethanol, etc.

[0040] Other materials and tools: grafting clips (silicone material, 3 mm inner diameter), scissors, scalpel blades, centrifuge tubes, UV spectrophotometer, constant temperature water bath, centrifuge, labels, etc.

[0041] Throughout the experiment, the cultivation environment (light, temperature, humidity, water and fertilizer) of each treatment group was kept consistent. Sample collection, sampling and index measurement were carried out in accordance with standard operating procedures to ensure that the data were accurate and reliable.

[0042] 2 Experimental Design The experiment employed a randomized block design, with four treatment groups and one control group. The treatment groups were: xanthan gum treatment group (HY), pectin treatment group (GJ), yellow pectin treatment group (HG), and tea brown gum treatment group (CHA). The control group (CK) was grafted without any healing agent, secured only by grafting clips. Each treatment group was replicated three times, with at least 30 plants per replicate.

[0043] Tomato seedlings with uniform growth and free from pests and diseases were selected and grafted using the oblique grafting method. The specific procedure was as follows: a 45° oblique cut was made 2-3 cm above the base of the rootstock stem, and a matching oblique cut was made on the corresponding part of the scion. The two were then aligned and fitted together. Immediately after cutting, the appropriate healing agent was applied evenly to the surface of the graft union and the surrounding area, ensuring complete coverage. No material was applied after grafting in the control group. The graft union was then secured with grafting clips to ensure a tight seal between the cut surfaces. All treatment groups maintained the same cultivation environment conditions (temperature, humidity, light, water, fertilizer, etc.), differing only in the type of healing agent.

[0044] Subsequent index measurements included: observing callus and vascular bundle regeneration in paraffin sections, measuring malondialdehyde (MDA), peroxidase (POD), soluble sugars in leaves, as well as plant height, stem diameter, and chlorophyll content.

[0045] 3 Experimental Methods 3.1 Tomato Seedling Cultivation After purchasing the tomato seedlings, they were transplanted into seedling pots using a mixture of peat moss, vermiculite, and perlite as the substrate. After transplanting, the seedlings were placed in a greenhouse for management, maintaining substrate moisture and a temperature of 25-27℃. They were watered daily until the seedlings stabilized. Plants that were uniform in growth and free from pests and diseases were selected for grafting.

[0046] 3.2 Stem grafting and callus treatment Tomato seedlings were grafted. Rootstock and scion were selected separately. A 45° angled cut was made approximately 2-3 cm below the cotyledons of the rootstock, and a matching angled cut was made on the scion. The two were then aligned and fitted together. Immediately after grafting, the corresponding healing agent was applied evenly to the graft union and the surrounding 2 mm area, ensuring complete coverage. Four polysaccharide treatment groups were set up: xanthan gum, pectin, yellow pectin, and brown gum; a blank control group (CK) was also included, where grafting was performed without any treatment. Each treatment had three biological replicates. After application, the graft union was immediately fixed with silicone grafting clips to ensure tight contact between the cut surfaces. The treated plants were then placed in a greenhouse for cultivation.

[0047] 4. Sample Collection and Index Determination 4.1 Microstructural Observation On days 0, 3, 6, and 9 after treatment, three seedlings were randomly selected from each treatment group (one seedling per replicate). A stem segment approximately 1-2 cm long was taken from the cut surface and quickly immersed in FAA fixative for 24 h. Then, paraffin sections were prepared according to the standard procedure: sequentially dehydrated with ethanol gradient, cleared with xylene, embedded in paraffin, and cut into continuous sections of 8-10 μm using a rotary microtome. After hematoxylin-eosin staining, the callus and vascular bundle regeneration were observed under an optical microscope and photographed.

[0048] 4.2 Measurement of physiological indicators Leaves from seedlings in each treatment group were collected on days 0, 3, 6, 9, and 12 after treatment. The leaves were shredded, weighed to a fixed weight, packaged, and stored in a -40℃ refrigerator for later use in subsequent index determination.

[0049] Malondialdehyde (MDA) content determination: The thiobarbituric acid (TBA) method was used. Weigh 0.1 g of fresh sample and grind it in a mortar (pre-freezing the mortar for 15 min in an ice bath). Add 1 mL of TCA and grind into a homogenate. Pour the homogenate into a centrifuge tube, then add another 1 mL of TCA and grind again before adding the homogenate to the centrifuge tube. Centrifuge at 1000 r / min and 4℃ for 10 min. Take 1 mL of the supernatant and add 1 mL of TBA solution, mix well, heat in a boiling water bath for 10 min, and after cooling, measure the absorbance at wavelengths of 450 nm, 532 nm, and 600 nm.

[0050] Peroxidase (POD) activity assay: The guaiacol method was used. Weigh 0.1 g of fresh sample, place it in an ice bath (pre-freeze the mortar for 15 min), add 5 mL of POD extraction solution, grind into a paste, pour into a centrifuge tube, and repeat once. Centrifuge at 8400 r / min for 10 min. After adding the reaction reagent, mix immediately and measure the absorbance change at 470 nm wavelength, recording the values ​​at 30 s, 1′30 s, and 2 min.

[0051] Soluble sugar content determination: The anthrone colorimetric method was used. Weigh 0.2 g of sample and grind it in a mortar (pre-freezing the mortar for 15 min in an ice bath). Add 10 ml of distilled water in two batches to form a homogenate. Heat in a boiling water bath for 30 min, cool, and extract the supernatant. Add another 4 ml of distilled water, heat in a boiling water bath for 10 min, cool, and mix the supernatant with the first extract. Take 0.5 mL of the sample solution, add 1.5 mL of distilled water, 0.5 mL of ethyl anthrone acetate, and 5 mL of concentrated sulfuric acid. Heat in a boiling water bath for 10 min, cool, and measure the absorbance at 630 nm.

[0052] 4.3 Growth index determination Three plants were taken from each treatment group on days 3, 6, 9, and 12 after treatment, and the following indicators were measured: Plant height: Measure the vertical distance from the substrate surface to the plant's growing point using a tape measure, in mm.

[0053] Stem diameter: The diameter of the stem is measured 1 cm below the cotyledon node using a digital vernier caliper, in mm.

[0054] 4.4 Chlorophyll content determination On days 3, 6, 9, and 12 after treatment, fully expanded functional leaves were selected from each treatment group, with three leaves measured in each replicate. The relative chlorophyll content (SPAD value) was measured using a portable chlorophyll meter (SPAD-502), and the values ​​were averaged from three different locations on each leaf.

[0055] 4.5 Survival Rate Statistics The survival rate of grafted seedlings in each treatment group was counted on the 12th day after grafting.

[0056] 5. Data Processing All data were first processed using Excel 2016 and then analyzed using SPSS 22.0. Normality and homogeneity of variance tests were performed sequentially. After meeting the criteria, one-way ANOVA was used. Differences between treatments were analyzed using Duncan's multiple range test for multiple comparisons, with a significance level of P < 0.05. Results are expressed as mean ± standard deviation.

[0057] Example 3: Experimental Results: 1. Effects of different treatments on the microstructure of tomato wounds The results of paraffin section observation showed that ( Figures 1-3 In the diagram, sc: scion; st: rootstock; vb: vascular bundle sheath; red arrow indicates the junction; white indicates callus cells. refer to Figure 1Three days after grafting, different polysaccharide healing agents showed significant differences in their regulatory effects on the initial healing process: In the CK control group, the rootstock-scion junction gap was small, the isolation layer was basically degraded, and cells at the cut edge had started active division, resulting in the best initial healing process; In the xanthan gum group, the rootstock-scion junction gap was large, the isolation layer was thick and structurally intact, and the cut cells did not initiate dedifferentiation or healing response, significantly inhibiting the healing process; In the pectin group, the rootstock-scion junction gap was large, the isolation layer was only partially degraded, and cells completed basic activation but did not initiate significant division; In the yellow fruit group, the rootstock-scion junction gap was small, the degree of isolation layer degradation was significant, cells completed dedifferentiation and activation and showed early signs of callus proliferation, with healing ability second only to the control group; In the tea brown group, the rootstock-scion junction gap was the largest, the isolation layer remained intact, and there were no obvious signs of cell activation and division, resulting in a weak initial healing response.

[0058] refer to Figure 2 Six days after grafting, the regulatory effects of different polysaccharide healing agents on the grafting healing process varied significantly: In the CK control group, the rootstock-scion junction gap further narrowed, the isolation layer was mostly degraded, callus tissue continued to proliferate and cell division activity was vigorous, and the rootstock-scion junction became denser; in the xanthan gum group, the rootstock-scion junction gap was still relatively large, the degree of isolation layer degradation was extremely low, only a small number of cells at the edge showed weak signs of activation, and callus formation was severely delayed; in the pectin group, the rootstock-scion junction gap narrowed somewhat, the isolation layer underwent local degradation, callus cell division activity increased, and a small amount of disordered callus tissue began to form; in the yellow fruit group, the rootstock-scion junction gap narrowed significantly, the isolation layer was degraded over a large area, callus tissue proliferated vigorously, cells were arranged in an orderly manner, and vascular bundle primordia had begun to differentiate in some areas, and the healing process was significantly better than the other treatment groups; in the tea brown group, the rootstock-scion junction gap did not improve significantly, the isolation layer remained intact, the degree of callus cell activation was low, only a very small amount of callus tissue was formed, and the healing process remained slow.

[0059] refer to Figure 3 Nine days after grafting, the effects of different polysaccharide healing agents on the grafting healing process remained significantly different: In the CK control group, the rootstock-scion junction had achieved complete adhesion, the isolation layer had completely degraded, the callus tissue was dense and continuously differentiating, and a large number of vascular bundle primordia had formed and constructed a preliminary connection network; in the xanthan gum group, obvious rootstock-scion junction gaps remained, the degree of isolation layer degradation was weak, callus tissue proliferation was slow, there were no signs of vascular bundle primordia differentiation, and the healing process was significantly hindered; in the pectin group, the rootstock-scion junction gaps were healed, the isolation layer underwent large-scale degradation, the callus tissue proliferated in an orderly manner, and the differentiation process of vascular bundle primordia had begun; in the yellow fruit group, the rootstock-scion junction was completely sealed, the isolation layer was completely degraded, all callus tissues had completed differentiation and formed mature connection tissues, the vascular bundle network structure was dense and initially interconnected, and the healing quality was significantly better than other treatment groups; in the tea brown group, although the rootstock-scion junction gaps were reduced, obvious gaps still existed, the isolation layer degradation was incomplete, the degree of callus tissue differentiation was low, only a small number of vascular bundle primordia were observed to form, and the overall healing level was relatively lagging.

[0060] The above results indicate that fructose gum is most effective in promoting callus formation and vascular bundle regeneration. The grafting interface was basically closed on the 9th day after grafting, and the healing quality was significantly better than that of other treatment groups.

[0061] 2. Effects of different treatments on physiological and biochemical indicators of tomato leaves 2.1 Changes in MDA content refer to Figure 4 MDA (malondialdehyde) is the final decomposition product of membrane lipid peroxidation, and its content directly reflects the degree of cell membrane damage. The results of malondialdehyde (MDA) content determination showed significant differences in the effects of different natural polysaccharide treatments on the degree of membrane lipid peroxidation in tomato leaves after grafting. On day 3 after treatment, MDA levels in all treatment groups increased compared to day 0, because grafting stress triggered oxidative stress, but the increase in the four natural polysaccharide treatment groups was significantly lower than that in the control group. After day 6, the MDA content in the xanthan gum, xanthan gum, and pectin treatment groups began to decrease slowly, while the control group continued to increase until reaching its maximum on day 9. Overall, the xanthan gum treatment group maintained a low MDA level at all time points, especially on day 6 after grafting, significantly lower than the control and other polysaccharide treatment groups, indicating the mildest degree of cell membrane damage; the xanthan gum and pectin treatment groups were next, the tea brown gum treatment group had higher MDA content at some points, and the control group had the most severe membrane lipid peroxidation. The above results indicate that xanthan gum treatment can more effectively reduce grafting stress-induced MDA accumulation, alleviate membrane lipid peroxidation damage, thereby enhancing the plant's tolerance to oxidative stress and promoting graft union healing.

[0062] 2.2 Changes in POD activity refer to Figure 5 Based on the dynamic measurement results of peroxidase (POD) activity, the effects of different natural polysaccharide treatments on POD activity in tomato leaves after grafting varied significantly. Overall, POD activity in all treatment groups showed a fluctuating trend of first increasing and then decreasing. However, the xanthan gum treatment group maintained a high enzyme activity level at all time points, with the fastest increase and the most prominent peak, significantly superior to the xanthan gum, pectin, and tea brown gum treatment groups. The xanthan gum treatment group was second, while the pectin and tea brown gum treatment groups had relatively lower levels, with a more significant decrease in the later stages. The control group had generally low POD activity with a smaller increase. These results indicate that xanthan gum treatment can more effectively induce an increase in POD activity in grafted plants, enhance antioxidant capacity, thereby alleviating oxidative damage caused by graft union stress and promoting the healing process.

[0063] 2.3 Changes in soluble sugar content refer to Figure 6Based on the soluble sugar content determination results, the effects of different natural polysaccharide treatments on the soluble sugar level of tomato leaves after grafting varied significantly. In terms of overall trends, the soluble sugar content in the control group remained at a high level at all stages, while the polysaccharide treatment groups showed a decrease in the early stages of grafting, followed by varying degrees of recovery. The xanthan gum treatment group exhibited the highest soluble sugar content at all time points, significantly superior to the xanthan gum, pectin, and brown gum treatment groups; the brown gum treatment group was second, while xanthan gum and pectin showed similar effects but were slightly lower than brown gum. The pectin treatment group showed a larger decrease in the later stages, indicating weaker recovery ability. These results suggest that xanthan gum is most effective in maintaining and restoring the soluble sugar content of grafted plants, providing sufficient osmotic regulators and carbon sources for the graft union healing process, thereby promoting healing.

[0064] 2.4 Changes in superoxide dismutase (SOD) activity refer to Figure 7 The effects of different natural polysaccharide treatments on SOD activity in tomato leaves after grafting varied significantly. Overall, SOD activity in all groups showed a fluctuating trend of first increasing and then decreasing. In the early grafting stage, SOD activity in xanthan gum and the control group increased rapidly and reached a peak, exhibiting a strong instantaneous antioxidant response, but the activity decreased rapidly and lacked persistence. In the early stage, the SOD activity increase in the xanthan gum treatment group was relatively gradual, without a significant peak, while the enzyme activities in the pectin and brown gum treatment groups remained at a generally low level. In the mid-to-late grafting stage, SOD activity in xanthan gum and the control group dropped sharply, while the xanthan gum treatment group maintained a relatively stable enzyme activity, ensuring the continuous operation of the plant's antioxidant system. These results indicate that xanthan gum cannot rapidly induce a short-term surge in SOD activity, but it can maintain the stable operation of the antioxidant system, reduce the continuous accumulation of reactive oxygen species, and work synergistically with other antioxidant enzymes to alleviate oxidative damage caused by grafting stress.

[0065] 2.5 Changes in catalase (CAT) activity refer to Figure 8 Different natural polysaccharide treatments significantly affected CAT activity in grafted tomato leaves. Overall, CAT activity in all treatment groups showed a fluctuating upward trend. The xanthan gum treatment group showed the fastest increase in the mid-grafting stage, with the most prominent enzyme activity peak in the later stage, significantly outperforming both the pectin and control groups throughout the process. The tea brown gum treatment group showed a similar pattern to the xanthan gum treatment group, reaching a similarly high level in the later grafting stage. The xanthan gum treatment group maintained a stable, moderate enzyme activity. The pectin treatment group showed relatively low overall activity, while the control group maintained the lowest CAT activity throughout the process. These results indicate that xanthan gum treatment can continuously induce an increase in CAT activity in grafted plants, synergistically scavenge hydrogen peroxide, alleviate oxidative stress at the graft union, and effectively promote wound healing.

[0066] 3. Effects of different treatments on tomato growth refer to Figure 9 and Figure 10 Based on the measurements of plant height and stem diameter, the effects of different natural polysaccharide treatments on the growth of grafted tomato plants varied. On day 3 post-treatment, the differences in plant height and stem diameter among the treatment groups were not yet significant, but the xanthan gum treatment group showed a trend of being superior to the control, and exhibited significant differences compared to some other treatment groups. By day 6 post-treatment, the plant height and stem diameter of the xanthan gum treatment group were significantly higher than other polysaccharide treatment groups; the xanthan gum and pectin treatment groups were next, with differences compared to the control not reaching a significant level or at an intermediate level; the brown gum treatment group was relatively weaker. Overall, xanthan gum showed the most prominent effect in promoting tomato plant height and stem diameter growth, indicating that it can effectively promote the recovery and vegetative growth of grafted plants. By day 9, the plant height of all groups steadily increased, the differences in plant height caused by different polysaccharide treatments further narrowed, and the overall growth became more uniform; the xanthan gum treatment still showed a growth advantage in stem diameter, and there was no significant differentiation in growth among the groups. By day 12, plant height continued to increase synchronously with only slight differences in growth between groups. Stem diameter, however, showed a greater difference in the later stages. The xanthan gum treatment maintained good stem diameter growth, while the brown gum treatment experienced a slower rate of stem diameter growth, showing a clear difference in growth performance. The stem diameter growth of xanthan gum, pectin, and the blank control group fell within the middle range. Observing the entire growth cycle, xanthan gum not only demonstrated a significant growth-promoting effect in the early stages but also continued to promote plant growth in the middle and later stages.

[0067] 4. Effects of different treatments on chlorophyll content and survival rate of grafted seedlings refer to Figure 11 and Figure 12 Based on the dynamic measurement results of chlorophyll content, the effects of different natural polysaccharide treatments on the chlorophyll level of tomato leaves after grafting varied significantly. The chlorophyll content of all treatment groups showed a trend of first increasing and then decreasing after grafting. However, the xanthan gum treatment group maintained a relatively high chlorophyll content throughout all stages, with the smallest decrease, especially in the later stages of grafting, significantly better than the xanthan gum, pectin, and brown gum treatment groups. The xanthan gum and pectin treatment groups were next, while the brown gum and control treatment groups had relatively low chlorophyll content, and the decrease was faster in the later stages. These results indicate that xanthan gum treatment can more effectively maintain the chlorophyll level of grafted plant leaves, delay chlorophyll degradation, and help maintain a good photosynthetic pigment state, thus providing support for plant recovery and growth.

[0068] In the early stage of grafting, the survival rate of grafted seedlings in all treatment groups was at a high level with no significant differences between groups; as the grafting time progressed, different healing agents had a differentiated impact on the survival of grafted seedlings in the later stage, and the survival rate of some treatment groups decreased slightly.

[0069] 5. Discussion 5.1 Microscopic mechanism by which natural polysaccharides promote graft union healing After grafting tomatoes, the successful healing of the graft union hinges on callus formation and effective vascular regeneration. Microscopic observation of paraffin sections revealed that the treatment groups coated with natural polysaccharides such as xanthan gum and pectin showed earlier callus formation, denser cell arrangement, and significantly faster vascular differentiation and connectivity compared to the control group. In the xanthan gum treatment group, the graft union was essentially closed by day 9, with unobstructed vascular growth. This result is similar to the role of natural polysaccharides in animal wound healing. There are two possible reasons: first, natural polysaccharides have strong film-forming and moisturizing properties, forming a protective film on the graft union surface after application, reducing water loss and creating a relatively stable and moist environment for cell division and differentiation; second, polysaccharide molecules may also act as signaling molecules or carbon sources, promoting dedifferentiation and accelerating proliferation of thin-walled cells around the graft union. In comparison, xanthan gum and pectin may be slightly weaker in film-forming and adhesive properties than xanthan gum, thus their healing effect may be slightly inferior from a microscopic perspective. In summary, pectin is most effective in promoting callus formation and vascular bundle regeneration.

[0070] 5.2 Regulatory effect of polysaccharide treatment on the antioxidant system The stress caused by grafting wounds stimulates the production of large amounts of reactive oxygen species (ROS) within the plant, leading to membrane lipid peroxidation and a significant accumulation of malondialdehyde (MDA). In this experiment, treatments with four natural polysaccharides altered the activities of three antioxidant enzymes—SOD, POD, and CAT—and significantly reduced MDA content while increasing soluble sugar content. This indicates that polysaccharides can synergistically regulate the plant's antioxidant and osmotic metabolism, effectively alleviating oxidative damage caused by grafting. This result is consistent with previous research findings. Peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD) together constitute the core defense system for the plant to scavenge ROS. It is noteworthy that the pectin-treated group maintained high POD activity throughout the observation period, with the fastest and most significant increase in activity during the early healing stage. CAT activity continued to rise with the wound healing process, significantly outperforming other treatments in the mid-healing stage and reaching its peak in the late healing stage, continuously decomposing hydrogen peroxide. The SOD regulation pattern differed from other treatments; xanthan gum only rapidly increased SOD activity in the short term of healing, demonstrating a strong instantaneous ability to scavenge superoxide anions, but its activity declined significantly in the later stages. While pectin did not form a short-term activity peak, it maintained the stability of the antioxidant system in the mid-to-late healing stages, avoiding secondary stress caused by drastic fluctuations in enzyme activity. Considering the combined performance of the three enzyme types, pectin showed the best induction effect on the entire set of antioxidant enzymes, followed by xanthan gum. Pectin and tannin showed similar antioxidant activation abilities, but neither was as effective as the former two.

[0071] Correspondingly, the group treated with fructose had the lowest MDA accumulation and the least oxidative damage to cell membranes throughout the entire treatment cycle. This difference may be related to the molecular structure, viscosity, and affinity of the polysaccharide itself for the plant cell wall. Fructose possesses suitable macromolecular properties and good biocompatibility, making it easier to form a stable film on the graft union surface, slowly releasing active ingredients and thus continuously inducing antioxidant enzymes to function. Furthermore, soluble sugars are both osmotic regulators and sources of carbon skeletons, playing a crucial role in graft union healing. In this study, the soluble sugar content in the fructose-treated group was consistently the highest, which is beneficial for maintaining intracellular osmotic balance and providing sufficient energy and substrate for callus differentiation. Overall, natural polysaccharides, through synergistic regulation of antioxidant enzyme activity, reduction of membrane lipid peroxidation, and maintenance of soluble sugar levels, promote rapid graft union healing in tomato seedlings from multiple physiological levels, including scavenging reactive oxygen species, protecting cell membranes, stabilizing cell osmotic pressure, and supplying energy for healing.

[0072] 5.3 Effects of polysaccharide treatment on tomato plant growth and chlorophyll content The effectiveness of graft union healing not only affects the local repair but also directly impacts the subsequent growth of the entire tomato plant. Based on the growth indicators measured in this experiment, the treatment group coated with natural polysaccharides showed better growth in plant height and stem diameter compared to the control group, with xanthan gum showing the most significant promoting effect. This is likely because the graft union heals quickly, making it less susceptible to pathogen infection. The plant also avoids prolonged stress, consuming less energy and allowing more photosynthetic products to be used for growth.

[0073] Regarding chlorophyll content, all four polysaccharide treatments increased leaf chlorophyll content, but the increase from xanthan gum was significantly greater than that from xanthan gum, pectin, and brown gum. The increase in chlorophyll content is related to several factors: firstly, better healing reduced overall oxidative stress in the plant and stabilized chloroplast structure; secondly, increased accumulation of soluble sugars may, in turn, promote chlorophyll synthesis; and thirdly, polysaccharide treatment may have regulated the balance of endogenous hormones, indirectly affecting chlorophyll metabolism. Overall, the xanthan gum treatment group showed the fastest healing and best physiological recovery, and also exhibited the best subsequent growth and chlorophyll content.

[0074] 5.4 Analysis of differences in polysaccharide effects among different treatments In this experiment, among the four natural polysaccharides, xanthan gum showed the best effect in promoting graft union healing and subsequent growth in tomatoes, followed by xanthan gum and pectin, while brown gum was the least effective. This difference may be due to the following reasons: First, differences in molecular structure and fluidity. Xanthan gum has suitable viscosity and film-forming properties, forming a durable and uniform protective film on the graft union surface after application, with stronger water retention and pathogen isolation capabilities. While xanthan gum has high viscosity, its film-forming toughness is slightly weaker; pectin has good gelling properties but poor durability; brown gum has a relatively low molecular weight, resulting in the weakest adhesion and film-forming durability. Second, differences in biological activity. Different polysaccharides may induce different signaling pathways in the body, such as varying binding abilities to receptors on plant cell walls, leading to different response intensities of downstream antioxidant enzymes and hormones. Third, differences in cell affinity. Pectin, as a component naturally present in cell walls, may be more easily recognized and utilized by wound cells, but as an exogenous substance applied topically, its stability is not as good as xanthan gum. Based on the results of this experiment, xanthan gum showed the best overall performance and is recommended as the first choice for grafting healing agents between tomato varieties; xanthan gum and pectin can be considered as alternatives.

[0075] 6. Conclusion This study used tomato seedlings as experimental material. Four natural polysaccharide healing agents—xanthan gum, pectin, tannin, and brown gum—were applied after oblique grafting. Their effects on the graft union healing process, leaf physiological and biochemical indicators, plant growth, and chlorophyll content were measured. The results showed that all four natural polysaccharides promoted the healing of tomato graft union wounds to varying degrees. Microscopic observation indicated that callus formation was earlier and vascular bundle regeneration was faster and more complete after polysaccharide application. Tannin showed the best effect, with the graft union essentially closed by the 9th day after treatment; xanthan gum and pectin were next. Natural polysaccharide treatment had a certain effect on alleviating oxidative stress caused by graft union wounds. Compared with the control group, the malondialdehyde content in the leaves of each polysaccharide treatment group was significantly reduced, while peroxidase activity and soluble sugar content increased. In terms of various physiological indicators, the tannin treatment group performed best, followed by xanthan gum and pectin, with brown gum showing relatively weaker effects. This indicates that tannin is most prominent in enhancing the plant's antioxidant capacity and osmotic regulation ability. Applying natural polysaccharide wound healing agents has a positive impact on the later-stage growth and chlorophyll content of tomatoes. All polysaccharide treatment groups showed better plant height, stem diameter, and relative chlorophyll content (SPAD value) than the control group. Yellow pectin showed the most significant improvement, followed by xanthan gum and pectin, while brown gum had a relatively weaker effect. In a comprehensive comparison of the four natural polysaccharides' effects on promoting grafting wound healing in tomatoes, yellow pectin performed best, followed by xanthan gum and pectin, while brown gum had a relatively limited effect. Therefore, yellow pectin is recommended as a highly effective and green wound healing agent for grafting between tomato varieties.

[0076] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. The application of natural polysaccharides in the preparation of a healing agent that promotes the healing of grafted tomato wounds, characterized in that, The natural polysaccharide is selected from one or more of xanthan gum, pectin, and flavocarpus.

2. The application of the natural polysaccharide according to claim 1 in the preparation of a healing agent that promotes the healing of grafted tomato wounds, characterized in that, The natural polysaccharide is xanthan gum.

3. The application of the natural polysaccharide according to claim 1 in the preparation of a healing agent that promotes the healing of grafted tomato wounds, characterized in that, The healing agent contains 1% to 5% (w / v) of natural polysaccharides by mass volume.

4. The application of the natural polysaccharide according to claim 3 in the preparation of a healing agent that promotes the healing of grafted tomato wounds, characterized in that, The healing agent contains 2% to 3% (w / v) of natural polysaccharides by mass volume.

5. The application of the natural polysaccharide according to claim 1 in the preparation of a healing agent that promotes the healing of grafted tomato wounds, characterized in that, The preparation method of the yellow pectin healing agent is as follows: Mix xanthan gum and pectin at a mass ratio of 0.5~1.5:1; Add deionized water to prepare a gel-like consistency. The total mass of xanthan gum and pectin to the volume ratio of deionized water is 1.5~2.5g:40mL.

6. The application of the natural polysaccharide according to claim 5 in the preparation of a healing agent that promotes the healing of grafted tomato wounds, characterized in that, The mass ratio of xanthan gum to pectin is 0.8:1, and the total mass to the volume ratio of deionized water is 1.8g:40mL.

7. A method for promoting the healing of grafted wounds in tomatoes, characterized in that, The steps are as follows: (1) Take tomato rootstock and scion, and graft them using the oblique cutting method: make an oblique cut at 45° 2-3 cm above the base of the rootstock stem, cut a matching oblique surface at the corresponding part of the scion, and align the two together. (2) Apply the natural polysaccharide healing agent evenly to the surface of the grafting interface and the surrounding 2 mm area to ensure complete coverage; (3) Use grafting clips to fix the grafting interface so that the cut surfaces fit tightly together.

8. A natural polysaccharide healing agent for tomato grafting, characterized in that, The natural polysaccharide healing agent comprises natural polysaccharides and water, wherein the natural polysaccharides are selected from one or more of xanthan gum, pectin, and tartrate gum.

9. The application of the natural polysaccharide healing agent for tomato grafting as described in claim 8 in promoting the survival of tomato grafted seedlings.

Images