Application of Propyl Gallate in Reducing the Fruit Drop Rate of Carya cathayensis

Abstract

The invention discloses the application of propyl gallate in reducing the fruit drop rate of pecan, belongs to the technical field of plant growth regulation, and provides the application of propyl gallate in reducing the fruit drop rate of pecan. Provided is a method for reducing the fruit drop rate of pecan by using the propyl gallate, comprising the following steps: applying a propyl gallate solution to the connection between pecan fruit and branches at the early stage of pecan fruit development, applying 2 to 3 times, each time at an interval of 7 to 10 days. The invention proposes the application of propyl gallate in reducing the fruit drop rate of pecan, analyzes the role of propyl gallate in fruit drop control through statistical analysis, pecan morphological anatomy and tissue staining, uses it to reduce the fruit drop rate of pecan, and slows down the occurrence of fruit drop in Zhejiang pecan.

Description

Technical Field

[0001] The invention belongs to the technical field of plant growth regulation, and particularly relates to application of propyl gallate in reducing the pecan fruit drop rate. Background Art

[0002] Zhejiang walnut is a species of the genus Carya ( Carya ) Juglandaceae Juglandaceae ) is a species widely planted in Lin'an and Chun'an, Zhejiang Province. It is an important economic forest and characteristic industry in the local area. Affected by the natural environment and growth habits, the phenomenon of pecan flower and fruit drop is serious. Under normal growth conditions, the flower and fruit drop rate is over 60%, which has the adverse effect of reducing production and income. Therefore, the law of pecan fruit drop and control technology have always been a concern of growers and scientific researchers.

[0003] The fruit drop mechanism of plants is closely related to the formation of abscission layer. After receiving the abscission signal, the fruit forms a small and dense abscission zone. Then, 1 to 2 layers of cells in the abscission zone separate under the action of enzymatic hydrolysis, and then form an abscission zone (AZ) with the branches, and the fruit drops under the action of external force. Plants can form abscission layers to actively remove underdeveloped fruits, avoid nutrient competition and water loss, and prevent interference from pests and diseases. The abscission layer can be produced in three situations: when the fruit is ripe or aging, when the fruit is underdeveloped and nutrient competition occurs, and when it is subjected to environmental stress. Its formation process is subject to both physiological regulation of hormones and enzymes and molecular regulation of fruit drop genes.

[0004] Reactive oxygen is a secondary metabolite produced by plant cells and is also a signal molecule for plants to respond to stress. It plays an important role in plant cell aging and apoptosis. Reactive oxygen usually occurs in the form of superoxide anions (O2 -), hydroxyl radicals (·OH), hydrogen peroxide (H2O2), etc. exist in plants. However, excessive accumulation of reactive oxygen can damage plant cells and tissues, affecting plant growth and development and the pattern of flower and fruit drop. The formation of abscission layer is a process of plant cell death, and reactive oxygen is closely related to the appearance of abscission layer. Studies have shown that when abscission zone occurs in the petiole of pear leaves, reactive oxygen accumulates, leading to leaf shedding; H2O2 promotes cell dedifferentiation during the shedding of eucalyptus bark, participates in the formation of cell walls and the initiation of plant cell senescence during the formation of the cork layer, induces ethylene production and accelerates the separation of abscission layer cells. In addition, reactive oxygen has different degrees of influence on the storage, processing and transportation of common fruits and vegetables such as apples, blueberries and cucumbers. In agriculture and industry, some reactive oxygen scavengers are often used to maintain the balance of reactive oxygen and preserve fruits and vegetables, such as polyphenols, polysaccharides, flavonoids, vitamins, antioxidant enzymes and other reducing compounds. Propyl gallate (PG) is a natural compound with antioxidant properties. It has the functions of scavenging free radicals and inhibiting lipid peroxidation. It is often used in food preservation research. It has a clear antioxidant effect and can be used as a reactive oxygen scavenger to study the mechanism of pecan fruit drop. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes the application of propyl gallate in reducing the fruit drop rate of pecans. Through statistical analysis, pecan morphological anatomy and tissue staining, the role of propyl gallate in fruit drop control is analyzed, and the appropriate reagent concentration is determined to reduce the pecan fruit drop rate, thereby slowing down the occurrence of pecan fruit drop in Zhejiang.

[0006] To achieve the above object, the present invention provides the use of propyl gallate in reducing the pecan fruit drop rate.

[0007] Preferably, the propyl gallate is applied at the early stage of pecan fruit development.

[0008] The present invention also provides a method for reducing the pecan fruit drop rate by using the propyl gallate, comprising the following steps: applying the propyl gallate solution to the connection between the pecan fruit and the branch at the early stage of pecan fruit development, applying 2 to 3 times, each time at an interval of 7 to 10 days.

[0009] Preferably, the concentration of the propyl gallate solution is 0.1-0.5 mM.

[0010] Preferably, the propyl gallate solution is applied by spraying, and the amount of the propyl gallate solution applied each time is 1 to 3 mL.

[0011] Preferably, the propyl gallate solution also includes Triton X-100.

[0012] Further preferably, the volume concentration of Triton X-100 in the propyl gallate solution is 0.1%.

[0013] Preferably, the propyl gallate solution is applied between 8:00 and 11:00 a.m., and the temperature at which the propyl gallate solution is applied is 25-30° C.

[0014] Preferably, the propyl gallate solution is applied on a sunny day.

[0015] The invention also provides a preparation for reducing the pecan fruit drop rate, and the preparation comprises the propyl gallate.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] The present invention uses pecans in Lin'an, Zhejiang Province as experimental materials, studies the key time of pecan fruit drop and corresponding cytological characteristics, determines the law of fruit drop and active oxygen change, statistically analyzes the pecan fruit drop rate, and determines the appropriate concentration of PG, aiming to more systematically and comprehensively understand the law of pecan fruit drop, reduce the occurrence of pecan fruit drop by applying exogenous PG solution, increase the economic benefit of pecan planting, and provide support for the research on pecan fruit preservation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The abscission layer is produced during the shedding process of pecan fruit, among which A is the abscission layer phenotype of pecan fruit grown for one month, and B is the side microscopic observation of the abscission layer of pecan fruit grown for one month, with a scale of 200 μm;

[0020] Figure 2 Schematic diagram of different positions of pecan fruit shedding, where A is a diagram of fruit stalk and branch shedding, fr represents pecan fruit, fs represents fruit stalk, br represents branch, and B is a diagram of the shape of the fruit shedding in two shedding forms;

[0021] Figure 3 The abscission layer of pecan fruit is observed under microscope, among which A is the abscission layer of normal growing fruit, the red arrow in the figure indicates the abscission layer position, and the scale is 200μm; B is the abscission layer of fallen fruit, the red arrow in the figure indicates the abscission layer position, and the scale is 200μm;

[0022] Figure 4 These are NBT staining images of stunted pecan fruits, where A is a pecan fruit with a relatively better growth state. The red arrow in the figure indicates the area where the fruit is connected to the branch, and the scale is 200 μm. B is a pecan fruit with a relatively poor growth state. The red arrow in the figure indicates the area where the fruit is connected to the branch, and the scale is 200 μm.

[0023] Figure 5 These are DAB and NBT staining images after PG solution treatment, where A is the DAB staining treatment image, the blue arrow indicates the area near the connection between the fruit and the branch, and the scale is 500μm; B is the NBT staining treatment image, the blue arrow indicates the area near the connection between the fruit and the branch, and the scale is 500μm. DETAILED DESCRIPTION

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0026] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0027] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0028] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0029] Example 1

[0030] 1 Materials and methods

[0031] 1.1 Experimental Materials and Reagents

[0032] The variety of this experiment is Zhejiang pecan, and the sampling site is at the base of Zhejiang Agricultural and Forestry University in Lin'an District, Hangzhou City, Zhejiang Province. The experiment was conducted from April to July in 2023 and 2024. Pecan trees with a height of 3-5m and good growth were selected to observe and count the fruit drop patterns of pecans. After the pecans completed pollination and began to form young fruits, they were labeled and classified, and PG solution was sprayed. The concentrations of PG solution were 0.1mM, 0.3mM, and 0.5mM, respectively. At the same time, water was sprayed as the experimental control group. PG (SIGMA, CAS121-79-9), DAB reagent (3,3-diaminobenzidine) and NBT reagent (nitro blue tetrazolium chloride) are both commonly used colorimetric reagents for active oxygen determination. DAB reacts with hydrogen peroxide to form a brown product, and NBT reacts with superoxide anions to form an insoluble blue product.

[0033] 1.2 Experimental methods

[0034] 1.2.1 Reagent treatment: The treatment time for pecans starts in mid-May, with an interval of 7 to 12 days, 2 to 3 times of treatment, and the number of pecan fruits is counted before each treatment. The treatment time is selected between 8 and 11 in the morning, the temperature is 25 to 30 ° C, and the climate is selected to avoid rainy and high temperature weather. 0.1 mL of Triton X-100 buffer is added to every 100 mL of PG solution to prevent the active ingredient from evaporating too quickly. The reagent spraying position is at the connection between the pecan fruit and the branch. Stop the treatment when the liquid begins to drip. The dosage is 2 mL.

[0035] 1.2.2 Fruit drop statistics: Before spraying PG solution, the experimental groups treated with different concentrations of reagents were classified to ensure that the number of fruits on each branch was around 10-30. One branch was counted as one replicate. The three concentration gradients of PG had more than three replicates. H2O was the control group CK, and all samples were randomly distributed.

[0036] The pecan fruit count before treatment is S0, and the first reagent treatment is carried out at the same time. After an interval of 10 days, the pecan fruit count after the first reagent treatment is S1, and so on, the pecan fruit count after the second reagent treatment is S2, and the pecan fruit count after the third reagent treatment is S3.

[0037] 1.2.3 DAB and NBT staining: Select the pecan fruits that are growing normally and have completely fallen off, collect the fruits that have been sprayed with 0.5mM PG solution and H2O for 1d and 3d, and perform DAB and NBT staining. Cut the tissue about 5mm near the abscission layer of the fruit and soak it in DAB (1mg·mL -1 ) and NBT (mass concentration of 0.1%, pH 7.8) solution, vacuum infiltration soaked for 4 hours and then placed at room temperature overnight. After staining, the fruit tissue was decolorized with 75% ethanol by volume, and the decolorization solution was changed 4 times during the period until the decolorization was completed. After decolorization, the fruit tissue samples were used as anatomical materials.

[0038] 1.2.4 Oscillating sectioning and microscopic observation: The instrument used for sectioning is Leica VT1200S oscillating sectioning machine. The decolorized tissue samples are embedded with 5% agarose and fixed on the stage with super glue. The parameters are adjusted to collect 50μm sections. Deionized water is added to the test tube to preserve the sections for subsequent microscopic observation. The instrument used for observation is Leica DM6B upright fluorescence microscope. The pecan slices are taken out and placed on a slide. An appropriate amount of deionized water is added and the slide is slowly covered. The tissue structure and coloring are observed under a microscope, and photos are taken for record.

[0039] 1.3 Data Analysis

[0040] The fruit drop rate of pecans after treatment with different concentrations of reagents was calculated, and the significant differences among different groups were analyzed by one-way analysis of variance. Excel 2016 software was used to organize the data, and SPSS27.0 software was used to perform ANOVA and Duncan's multiple comparisons on the data. GraphPad Prism 10.1.2 software was used to draw charts, and Microsoft Power Point software was used to process pictures.

[0041] The total fruit drop rate L = (the number of pecan fruits after the final treatment S-the number of pecan fruits before treatment S0) / the number of pecan fruits before treatment S0×100%.

[0042] Single fruit drop rate L n =(Number of walnut fruits after the n+1th reagent treatment S n+1 -Number of walnut fruits after the nth reagent treatment S n ) / Number of pecan fruits after the nth reagent treatment S n ×100%.

[0043] 2 Results and analysis

[0044] 2.1 Pecan fruit drop phenomenon

[0045] 2.1.1 Fruit forms abscission layer during abscission

[0046] Observation of pecan fruits that have grown for one month revealed that during their natural shedding process, an obvious separation layer or separation zone (such as Figure 1 After observation, it can be seen that the cells in the abscission layer are smaller than the surrounding cells and are arranged more closely. DAB staining shows that there is a difference in staining between the abscission layer cells and the surrounding cells (such as Figure 1 (as shown in B), indicating that the shedding process of pecan is related to the formation of abscission layer.

[0047] 2.1.2 The location of the abscission layer when pecan fruit falls off is different

[0048] Through observation and records, it was found that during the natural shedding process of pecan fruits, the abscission layer mainly appeared at the connection between the fruit stalk and the branch, and between the fruit and the branch (such as Figure 2 Middle A and Figure 2 These two tissues are newly grown structures with soft texture and poor firmness, indicating that the formation of fruit drop may be related to tissue structure and cell compactness, which affect the formation of abscission layer.

[0049] 2.2 Pecan fruit drop rate

[0050] Statistics on pecan fruit drop rates in 2023 and 2024. The initial statistical time in Lin'an, Zhejiang in 2023 was May 13, with a 10-day interval between each treatment, for a total of 3 treatments. The initial statistical time in Lin'an, Zhejiang in 2024 was May 18, with a 10-day interval between each treatment, for a total of 3 treatments.

[0051] Table 1 Fruit drop rate of pecan after treatment with different reagents

[0052] ;

[0053] Note: Different letters in the same column indicate significant differences at the P≤0.05 level.

[0054] From the data in Table 1, it can be seen that the natural fruit drop of pecans was simulated under H2O treatment. The average fruit drop rate in Lin'an area around June 2023 and 2024 was as high as 80%. The statistical data of this experiment is consistent with the existing technical records of the fruit drop rate of pecans in June, which is 60%~90%. After spraying PG solutions of different concentrations, the overall fruit drop rate decreased. The average fruit drop rate of 0.5mM PG solution in 2023 and 2024 was 51.85% and 45.77%, respectively, and its fruit drop rate was significantly lower than that of the control group in both statistics, indicating that 0.5mM PG solution is more suitable as an inhibitor of pecan fruit drop. In summary, pecans were selected and further processed to explore the differences in the changes in the level of active oxygen in pecans.

[0055] 2.3 Study on the differences in reactive oxygen species in fallen pecan fruits

[0056] Since abscission layers are produced during the fruit shedding process, DAB-stained sections of normally growing pecan fruits that had not fallen and those that had fallen were sliced ​​and their coloring differences were observed under a microscope.

[0057] The results are as follows Figure 3 Middle A and Figure 3 As shown in B, there is no significant difference in active oxygen staining in normally growing fruits, and the color of the fallen fruits is darker at the abscission layer, proving that the active oxygen level at the abscission layer is higher after the fruits have fallen.

[0058] In order to study the status of active oxygen in pecan fruit when it is not completely shed, thin, dark yellow and poorly grown fruits were collected for NBT staining and sectioning. Figure 4 Middle A and Figure 4 As shown in B, it was observed that there was an obvious color difference in the area where the fruit was connected to the branch, indicating that the increase in the level of reactive oxygen may be one of the chemical signals for the formation of abscission layer, that is, the level of reactive oxygen in the fruit increased, the abscission layer cells died, and the fruit and the branch formed a separation zone until it fell off completely.

[0059] 2.4 PG treatment reduces the level of reactive oxygen species in pecans

[0060] By counting the pecan fruit drop rate, the normally grown pecan fruits were treated with 0.5 mM PG solution for 1 day and 3 days respectively. The fruits were collected at the same time and stained with DAB and NBT, and the color difference was observed by sectioning.

[0061] The results are as follows Figure 5 Middle A and Figure 5 As shown in B, by comparing the staining of the area near the connection between the fruit and the branch, it can be seen from the treatments of 1d and 3d that the PG treatment is lighter in color than the H2O treatment, and the level of active oxygen in the body is lower. The scavenging effect of PG on active oxygen is further verified by DAB and NBT staining.

[0062] 3 Conclusion

[0063] By collecting fallen fruits and fruits that grow normally on the tree for DAB and NBT staining, it was found that the level of reactive oxygen in the abscission layer of fallen fruits was higher than that of fruits that grow normally, indicating that the occurrence of pecan fruit drop is accompanied by an increase in the level of reactive oxygen in the abscission layer. The fruit drop rate statistics also show that fruits with higher reactive oxygen levels have a higher fruit drop rate. Pecan fruits were treated with 0.5mM PG and then stained and sliced. Compared with the control group treated with H2O, the degree of staining of tissue sections after 0.5mM PG treatment was lighter, which means that the level of reactive oxygen in the fruit is lower. Through the action of PG, the overall level of reactive oxygen in pecan is reduced, which reduces the occurrence of pecan fruit drop and increases pecan yield.

[0064] The fruit drop phenomenon of Zhejiang pecan has both the influence of external environment and the influence of internal factors. The natural fruit drop rate of May to June is 72.22% to 95.00% according to the statistics of the present invention. Other pecan varieties such as thin-shelled pecans also have a higher fruit drop rate, indicating that pecan fruit drop mainly occurs in the early stage of young fruit formation. Poor pollination and nutritional competition are the main causes of fruit drop. The connection position and fruit support form of pecans may also affect the occurrence of its fruit drop. The accumulation of active oxygen substances can cause damage to plants, leading to cell death, causing the phenomenon of falling flowers, leaves and fruits. When pecans fall fruits, the active oxygen level at the abscission layer is higher than that of fruits grown normally, and active oxygen scavengers can reduce the active oxygen level in the body. Spraying 0.5mM PG reduces the fruit drop rate of pecans. The preparation and suitable concentration for controlling fruit drop have been screened out, which has laid a foundation for the research on the fruit drop law of Zhejiang pecans and is of great significance to pecan planting management.

[0065] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The application of propyl gallate in reducing the fruit drop rate of pecan, characterized in that: In the early stage of pecan fruit development, propyl gallate solution is applied to the connection between pecan fruit and branch for 2 to 3 times, with an interval of 7 to 10 days each time; the concentration of the propyl gallate solution is 0.1 to 0.5 mM; and the application amount of the propyl gallate solution each time is 1 to 3 mL.

2. A method for reducing the pecan fruit drop rate using propyl gallate as claimed in claim 1, characterized in that: The following steps are involved: At the early stage of pecan fruit development, apply propyl gallate solution to the junction of pecan fruit and branches, 2 to 3 times, with an interval of 7 to 10 days between each application; The concentration of the propyl gallate solution is 0.1-0.5 mM; The dosage of the propyl gallate solution is 1-3 mL each time.

3. The method for reducing the pecan fruit drop rate according to claim 2, characterized in that: The propyl gallate solution is applied by spraying, and the amount of the propyl gallate solution applied each time is 1-3 mL.

4. The method for reducing the pecan fruit drop rate according to claim 2, characterized in that: The propyl gallate solution also includes Triton X-100.

5. The method for reducing the pecan fruit drop rate according to claim 4, characterized in that: The volume concentration of Triton X-100 in the propyl gallate solution is 0.1%.

6. The method for reducing the pecan fruit drop rate according to claim 2, characterized in that: The propyl gallate solution is applied between 8 am and 11 am, and the temperature of the propyl gallate solution is 25-30° C.

7. The method for reducing the pecan fruit drop rate according to claim 2, characterized in that: The propyl gallate solution was applied on a sunny day.

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