Apple growth promoting preparation and application thereof in promoting growth of apple seedlings and improving apple quality and wax content

Abstract

The invention provides an apple growth promoting preparation and application thereof in promoting growth of apple seedlings and improving apple quality and wax content, and belongs to the technical field of fruit tree planting. The apple growth promoting preparation disclosed by the invention is prepared from a tea residue extract or epigallocatechin gallate, the concentration of the epigallocatechin gallate is 2 to 200 [mu] mol / L. By adopting the tea residue extract or EGCG, the plant height of the apple seedlings can be remarkably increased, the net photosynthesis of the apple seedlings is improved, and the growth of the apple seedlings and the content of wax in leaves are promoted. EGCG is sprayed in the growth period of the fruit trees, so that the leaf area of the fruit trees can be increased, the content of H2O2 in leaves is reduced, and the content of ester substances in leaf wax is increased; the fruit transverse diameter, the fruit longitudinal diameter and the single fruit weight can be increased, the fruit growth is promoted, the surface glossiness is improved, the appearance quality of the fruit is further improved, and the content of primary alcohol and ester substances in the fruit wax is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the technical field of fruit tree planting, and in particular relates to an apple growth-promoting preparation and an application of the preparation in promoting the growth of apple seedlings and improving apple quality and wax content. Background Art

[0002] Catechins are the main pharmacologically active ingredients in tea, accounting for 8% to 26% of the dry weight of tea. Catechins belong to flavanols in flavonoid compounds. Among the catechins isolated from tea, the most common are epicatechin (EC), epigallocatechin (ECG), epicatechin gallate (EGC) and epigallocatechin gallate (EGCG), with EGCG being the main component, and its content can reach 50% of tea flavanols. In the field of plant research, EGCG can play an important role in plant stress metabolism as a growth regulator. Low concentrations of EGCG can promote the germination of tomato seedlings and enhance the tolerance of tomatoes to salt stress by increasing the activity of antioxidant enzymes. Exogenous EGCG can enhance the tolerance of tomatoes to abiotic stress and resistance to tobacco mosaic virus. In Arabidopsis, EGCG improves the resistance of plants to gray mold through the jasmonic acid pathway. At present, the impact of EGCG on plants is mainly reflected in improving the stress resistance of plants, while the impact on apples before harvest is still unclear. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide an apple growth-promoting preparation and its application in promoting the growth of apple seedlings, improving apple quality and wax content. Through component analysis, it is clarified that epigallocatechin gallate (EGCG) is the main pharmacologically active ingredient in tea residue extract, and then the application concentration of EGCG on seedlings and fruit trees is explored, and the effect of EGCG on promoting the growth of apple seedlings and improving apple quality is clarified, which provides a reference for the recycling of tea waste on fruit trees, and is of great significance to the low-cost quality improvement and efficiency enhancement of apples.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] An apple growth-promoting preparation, comprising tea residue extract or epigallocatechin gallate;

[0006] The content of epigallocatechin gallate in the tea residue extract is 62.94-131.43 mg / g;

[0007] The concentration of the epigallocatechin gallate is 2-200 μmol / L.

[0008] Preferably, the method for preparing the tea residue extract comprises the following steps:

[0009] The tea leaves are mixed with boiling water and brewed for 2 to 5 times, and then filtered, dried, ground and sieved to obtain a tea residue extract.

[0010] Preferably, the mass volume ratio of the tea leaves to boiling water is 0.5-6 g:1 L;

[0011] The drying temperature is 60-70°C and the drying time is 18-30h;

[0012] The mesh number of the sieving is 300 to 500 meshes.

[0013] The present invention also provides application of the apple growth-promoting preparation in promoting the growth of apple seedlings.

[0014] The present invention also provides application of the apple growth-promoting preparation in increasing the wax content of leaves of apple seedlings.

[0015] Preferably, the apple growth-promoting preparation is prepared into a solution and applied to apple seedlings once a week at an application rate of 400 to 600 mL per time per plant.

[0016] The present invention also provides application of the apple growth-promoting preparation in promoting the growth of apple fruits and improving the quality of apple fruits.

[0017] The present invention also provides application of the apple growth-promoting preparation in increasing the wax content on the surface of apple fruit.

[0018] Preferably, the apple growth-promoting preparation is prepared into a solution, and the apple growth-promoting preparation is sprayed on the apple trees during the growth period, once every two weeks, with an application amount of 1 to 3 L / time / tree.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention adopts tea residue extract or EGCG to significantly increase the height of apple seedlings, improve the net photosynthesis of apple seedlings, promote the growth of apple seedlings and the wax content of leaves. Spraying EGCG during the growth period of fruit trees can not only increase the leaf area of ​​fruit trees, reduce the content of H2O2 in leaves, and increase the content of ester substances in leaf wax; it can also increase the horizontal diameter of fruits, the vertical diameter of fruits, and the weight of single fruits, promote the growth of fruits, improve the surface glossiness, and thus improve the appearance quality of fruits, and significantly increase the content of primary alcohols and ester substances in fruit wax. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is the EGCG content in three green tea residue extracts;

[0022] Figure 2The effect of tea residue extract on the net photosynthesis increment of apple seedlings after 7 days of treatment, the plant height increment and stem diameter increment after 26 days of treatment (where A is the growth potential of apple seedlings in different treatment groups, B is the net photosynthesis increment of apple seedlings after 7 days of treatment, C is the plant height increment after 26 days of treatment, and D is the stem diameter increment after 26 days of treatment);

[0023] Figure 3 This is the growth potential of apple seedlings after being treated with different concentrations of EGCG;

[0024] Figure 4 The effect of different concentrations of EGCG treatment on the net photosynthesis rate, intercellular CO2 concentration, stomatal conductance, and transpiration rate of 'M9T337' rootstock seedlings after 7 days (where A is the net photosynthesis rate, B is the intercellular CO2 concentration, C is the stomatal conductance, and D is the transpiration rate);

[0025] Figure 5 The effects of different concentrations of EGCG on net photosynthesis rate, intercellular CO2 concentration, stomatal conductance and transpiration rate of 'M9T337' rootstock seedlings after 15 days of treatment;

[0026] Figure 6 is the effect of different concentrations of EGCG on the wax content and the expression of wax synthesis-related genes in 'M9T337' seedlings (where A is the scanning electron microscope observation, B is the wax content, and C is the expression of wax synthesis-related genes);

[0027] Figure 7 The effect of EGCG on leaf fresh weight, leaf area, specific leaf area, H2O2 content, SOD enzyme activity, POD enzyme activity, leaf surface wax crystals and relative expression of wax synthesis-related gene MdMYB30 of 'Venus Gold' fruit trees (where A is leaf fresh weight, B is leaf area, C is specific leaf area, D is H2O2 content, E is SOD enzyme activity, F is POD enzyme activity, G and H are relative expression of leaf surface wax crystals and wax synthesis-related gene MdMYB30);

[0028] Figure 8 is the effect of EGCG on the wax components and total wax of leaves and fruits of 'Venus Gold' (where A is leaves and B is fruits);

[0029] Fig. 9 is the effect of EGCG treatment on the vertical and horizontal diameters, single fruit weight and fruit shape index of 'Venus Gold' fruit (where A is the horizontal diameter of the fruit, B is the vertical diameter of the fruit, C is the single fruit weight, and D is the fruit shape index);

[0030] Fig.10The effect of EGCG treatment on the glossiness, soluble solids content and titratable acid content of 'Venus Gold' fruit (where A is the glossiness of the fruit, B is the soluble solids content, and C is the titratable acid content). DETAILED DESCRIPTION

[0031] The present invention provides an apple growth-promoting preparation, comprising a tea residue extract or epigallocatechin gallate; the content of epigallocatechin gallate in the tea residue extract is 62.94-131.43 mg / g; the concentration of the epigallocatechin gallate is preferably 2-200 μmol / L, and further preferably 5-100 μmol / L.

[0032] In the present invention, the preparation method of the tea residue extract comprises the following steps: mixing tea leaves and boiling water and brewing for 2 to 5 times, filtering, drying, grinding, and sieving to obtain the tea residue extract. The tea leaves are preferably bamboo leaf green tea; the mass volume ratio of the tea leaves and boiling water is preferably 0.5 to 6 g: 1 L, and more preferably 1 to 5 g: 1 L; the number of brewing is preferably 3 to 4 times; the drying temperature is preferably 60 to 70°C, and more preferably 62 to 68°C; the drying time is preferably 18 to 30 hours, and more preferably 20 to 28 hours; the mesh number of the sieving is preferably 300 to 500 mesh, and more preferably 350 to 450 mesh.

[0033] The present invention also provides application of the apple growth-promoting preparation in promoting the growth of apple seedlings.

[0034] The present invention also provides application of the apple growth-promoting preparation in increasing the wax content of leaves of apple seedlings.

[0035] In the present invention, the apple growth-promoting preparation is prepared into a solution and applied to apple seedlings once a week, and the application amount is preferably 400-600 mL / time / plant, and more preferably 450-550 mL / time / plant.

[0036] The present invention also provides application of the apple growth-promoting preparation in promoting the growth of apple fruits and improving the quality of apple fruits.

[0037] The present invention also provides application of the apple growth-promoting preparation in increasing the wax content on the surface of apple fruit.

[0038] In the present invention, the apple growth-promoting preparation is prepared into a solution, and the apple growth-promoting preparation is applied during the growth period of the apple trees, once every two weeks, and the application amount is preferably 1 to 3 L / time / tree, and more preferably 1.5 to 2.5 L / time / tree.

[0039] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0040] Plant materials used in the present invention:

[0041] Seedlings of 'Pingyi Sweet Tea': seeds were stored in the laboratory and stratified at 4°C for 30 days before germination and growth in a tray containing a mixture of nutrient soil and vermiculite (1:1). When the third true leaf was grown, the seedlings were transplanted into 7 cm × 7 cm × 8 cm nutrient pots, one pot per seedling, and 4 seedlings per group, placed in 30 cm × 20 cm × 8 cm white plastic pots. Growth conditions: light intensity 200 μmol / m 2 / s, the ambient temperature is 25±1℃, the relative humidity is 50%-60%, the day and night cycle is 14h / 10h, and the treatment is carried out after the seedlings have been acclimatized for one week.

[0042] 'M9T337' rootstock seedlings were purchased from Shandong Yiteng Tianyuanmei Agricultural and Forestry Technology Co., Ltd. The rootstock seedlings were about 30 cm tall. The seedlings with consistent growth, similar plant height and robustness were selected and transplanted into nutrient pots, one seedling per pot, and 4 seedlings were grouped into white plastic pots. They were pre-cultivated for 7 days before being processed.

[0043] The 6-year-old 'Venus Gold' apple trees in the orchard of the Experimental Station of the South Campus of Shandong Agricultural University were used as test materials. The orchard is located in the temperate continental semi-humid monsoon climate zone with a mild climate. The city's average annual total solar radiation is 121.58 kal / cm 2 The annual average sunshine hours are 2627.1h, the average precipitation is 697mm, and the annual average temperature is 12.8℃. The soil in the orchard is sandy loam with a pH of 6.8-7.0. The ground of the orchard is covered with black mulch, and protective nets are installed above the fruit trees. The orchard carries out the same fertilizer and water management, fruit tree pruning, and pest and disease control. Zhuyeqing, Anji white tea, and West Lake Longjing tea were purchased online at 0.136 yuan / g, 0.396 yuan / g, and 0.432 yuan / g, respectively.

[0044] Example 1

[0045] Determination of EGCG content in tea residue extract

[0046] Preparation of tea residue extract: 5 g of each of Zhuyeqing, Anji white tea and West Lake Longjing tea were weighed and brewed with 1 L of 100°C hot water. When cooled to room temperature, the mixture was brewed again with 100°C hot water, and repeated three times. The mixture was then dried (65°C, 24 h), ground and sieved through a 400-mesh sieve, and stored in a refrigerator at 4°C.

[0047] EGCG content determination: The EGCG content in the tea residues of Zhuyeqing, Anji White Tea and West Lake Longjing was determined by Shandong Guocangjian Biotechnology Co., Ltd. (each type of tea residue was repeated three times).

[0048] Experimental results: Figure 1 As shown. Figure 1 It can be seen that the EGCG content in the extract of Zhuyeqing tea residue is the highest, reaching 131.43 mg / g, followed by the extract of Anji white tea residue at 85.05 mg / g, and the extract of West Lake Longjing tea residue has the least EGCG content, which is 62.94 mg / g. Therefore, the extract of Zhuyeqing tea residue was selected for research.

[0049] Example 2

[0050] Treatment of apple seedlings with tea residue extract

[0051] The sieved Zhuyeqing tea residue extract prepared in Example 1 was configured into a 1g / L tea residue suspension, and the two-month-old 'Pingyi sweet tea' seedlings were treated respectively. The treatment conditions were as follows: the tea residue suspension was sprayed (T1) and irrigated (T2) on the 'Pingyi sweet tea' seedlings, and distilled water spraying (CK1) and irrigation (CK2) were used as controls. T1, T2, CK1, and CK2 were treated once every 7 days, with a dosage of 500mL per pot, and watered once after each treatment for 3 days (4 seedlings per pot were treated as one treatment, repeated 3 times). The plant height, stem thickness, and net photosynthetic rate of the 'Pingyi sweet tea' seedlings in different treatment groups were measured.

[0052] Plant height and stem diameter: The height of the above-ground part of the seedlings was measured with an electronic vernier caliper as the plant height, and the diameter of the stem 1 cm from the soil was the stem diameter, with an accuracy of 0.01 mm. Twelve seedlings were tested in each treatment and control group.

[0053] Photosynthesis measurement: In a glass greenhouse between 9 and 11 a.m. on a clear day, a CIRAS-3 portable photosynthesis meter (CIRAS-3, PP Systems, UK) was used to measure fully developed leaves at the same position of the seedlings. The CO2 concentration was set to 390 mol CO2 / μmol, and the intercellular CO2 concentration (Ci), stomatal conductance (Gs), net photosynthetic rate (Pn), and transpiration rate (Tr) of the leaves were measured. Other conditions: light intensity was 200 μmol / m 2 / s, leaf temperature was 25±1℃, relative air humidity was 60%-70%, and 12 seedling leaves were tested for each treatment and control.

[0054] Experimental results: Figure 2 The growth rate and net photosynthesis rate of plants directly reflect the strength of plant growth. Figure 2It can be seen that after 7 days of spraying and irrigation with tea residue extract, T1 was compared with CK1, and T2 was compared with CK2 ( Figure 2 A), the increment of net photosynthesis rate increased by 128.44% and 102.22% respectively, among which the spraying treatment had the best effect on improving net photosynthesis ( Figure 2 B). After 26 days of tea residue extract spraying and irrigation, there was no significant difference in plant height increment between T1 and CK1, and the plant height increment of T2 was 1.61 times that of CK2. There was no significant difference in stem diameter among different treatments ( Figure 2 The results showed that both spraying and watering with tea residue extract could increase the net photosynthesis of seedlings, and the spraying treatment had a more significant effect, while watering with tea residue extract could significantly increase the height of seedlings.

[0055] Example 3

[0056] EGCG treatment of apple seedlings

[0057] 1. EGCG was purchased from Shanghai Solaibao Biotechnology Co., Ltd. with a purity of 98%. The seedlings of Pingyi Sweet Tea were treated with different concentrations of EGCG, and five large concentration gradients of 0 μmol / L, 5 μmol / L, 50 μmol / L, 100 μmol / L, and 200 μmol / L were set. The treatment was carried out once every 7 days, and watered once after each treatment for 3 days. The leaf phenotype was analyzed, and the concentration with the best seedling growth was selected according to the growth of the seedlings.

[0058] Experimental results: Figure 3 As shown. Figure 3 It can be seen that the seedlings treated with 5 μmol / L had more leaves and a larger leaf area than those treated with 0 μmol / L and 200 μmol / L, indicating that the application of low-concentration EGCG can significantly promote the growth of seedlings.

[0059] 2. The concentration of 5μmol / L was further refined on the 'M9T337' rootstock seedlings with a plant height of 30 cm, and was divided into 0μmol / L, 2μmol / L, 5μmol / L, 7μmol / L, 10μmol / L, 20μmol / L, and 50μmol / L. The treatment was also irrigated once every 7 days, with 500mL per pot, and watered once after 3 days of each treatment (4 seedlings per pot were treated as one treatment, and repeated 3 times).

[0060] (1) The photosynthesis indexes of 'M9T337' rootstock seedlings were measured after treatment with different concentrations of EGCG for 7 days. The specific measurement method is shown in Example 2.

[0061] Experimental results: Figure 4 As shown. Figure 4It can be seen that similar to the results of tea residue extract treatment, EGCG treatment for 7 days significantly increased the net photosynthesis of 'M9T337' seedlings, with a significant increase at a concentration of 10 μmol / L ( Figure 4 A), the promotion effect of the 0-50μmol / L treatment showed an overall trend of first increasing and then decreasing, and the transpiration rate and stomatal conductance also showed the same trend ( Figure 4 C and D). After the seedlings were treated with 0-20μmol / L concentrations, there was no significant difference in the intercellular CO2 concentration, while the 50μmol / L treatment was significantly lower than the other concentrations ( Figure 4 B).

[0062] (2) After the 'M9T337' rootstock seedlings were treated with different concentrations of EGCG for 15 days, the photosynthesis indexes of the seedlings were detected. For the specific determination method, see Example 2.

[0063] Experimental results: Figure 5 As shown. Figure 5 It can be seen that the 2μmol / L, 5μmol / L, 7μmol / L, 10μmol / L, 20μmol / L and 50μmol / L treatments increased by 24.62%, 23.08%, 16.92%, 22.08%, 11.54% and 24.04% respectively compared with the control, and the intercellular CO2 concentration, transpiration rate and stomatal conductance still showed an overall promoting effect compared with the control.

[0064] 3. Wax plays an important role in plant growth and development. Therefore, we further explored the effect of EGCG on the surface wax of apple seedlings.

[0065] (1) The surface of 'M9T337' seedlings was observed by scanning electron microscopy.

[0066] Experimental results: It was found that the number of wax crystals on the leaves of seedlings treated with EGCG was greater than that of the control group ( Figure 6 A).

[0067] (2) The total wax content of seedling leaves treated with different concentrations was tested. The specific steps are as follows:

[0068] ① Leaf wax extraction was performed according to the method of Yang et al. (2017) (Yang, Y., Zhou, B., Zhang, J., Wang, C., Liu, C., Liu, Y., Zhu, X., and Ren, X. Relationships between cuticular waxes and skin greasiness of apples during storage. Postharvest Biology and Technology, 2017, 131: 55-67.) with some modifications. The whole leaf was placed in a beaker and soaked in 30 mL of chloroform for 30 s at room temperature. Each sample was extracted three times. After extraction, the three extracts were mixed, filtered, and transferred to a rotary evaporator. Chloroform was evaporated into a waste liquid bottle by a vacuum condenser. 4 mL of chloroform and 1 mL of methanol were added to the evaporator to dissolve the white wax crystals. The dissolved concentrate was transferred to a 25 mL serum bottle. The serum bottle was blown dry with stable nitrogen gas on a nitrogen blower and then stored in a -20°C refrigerator. The treatment and control were repeated 3 times each.

[0069] ② Wax derivatization: Dissolve the wax in the bottle in a ratio of chloroform: methanol = 10:1, aspirate 1 mL and blow dry, add 200-400 μL of derivatization (BSTFA) reagent in a 70°C oven, immediately seal the mouth with film, and shake to mix. Derivatize in a 70°C metal bath for 40 minutes, shake once every 10 minutes, and blow dry with nitrogen under heating conditions at 50°C. Add 500 μL of chromatographic grade chloroform and 25 μL of n-tetracosane as internal standard. Use a 0.45 mm organic filter membrane to filter the sample into a brown loading bottle.

[0070] ③GC-MS loading analysis: GC-MS analysis was performed using a triple quadrupole gas chromatograph-mass spectrometer (Shimadzu, Japan). The GC parameters were set to an injection port temperature of 280°C and a column box temperature of 70°C. Other settings were based on the method of Wang Yongxu (2019). The MS parameters were set to an ion source temperature of 200°C, an interface temperature of 250°C, and a solvent delay time of 7 min.

[0071] ④ Wax component analysis: The retrieval database used was NIST17. The chemical structural formulas of the substances compared by computer retrieval were manually interpreted, and classified and analyzed according to the different functional groups of the chemical structural formulas. The relative substance content was calculated using the peak area normalization method and the internal standard quantification method, and the wax component content per unit area was calculated based on the surface area of ​​the leaves.

[0072] Statistical analysis Experimental data were recorded using Microsoft Excel 2019, graphing software was Graphpadprism 9, and gene differential expression heatmaps were produced using OriginPro 202 Learning Edition software. Data processing and analysis were performed using IBM SPSS Statisticsdan one-way analysis of variance and Graphpad prism 9 one-way analysis of variance and T-test to test the significant differences of variables. *(P<0.05), **(P<0.01), ***(P<0.001) and different lowercase letters (P<0.05) were used to indicate significant differences between groups.

[0073] Experimental results: Except for 2μmol / L and 20μmol / L, the other concentrations of EGCG can increase the wax content of leaves. 5μmol / L, 7μmol / L, 10μmol / L, and 50μmol / L increased the wax content by 37.86%, 44.65%, 57.18%, and 38.33%, respectively (e.g. Figure 6 B).

[0074] (3) Detection of the expression levels of wax synthesis-related genes MdECH, MdKCS1, and MdCER1. The total RNA in the plant was extracted using an RNA extraction kit purchased from Beijing Kangwei Biological Company. The RNA was then reverse transcribed into DNA using a cDNA reverse transcription kit purchased from Beijing Kangwei Biological Company. The real-time quantitative qRT-PCR method was then used to detect the expression levels of wax synthesis and transport-related genes in seedlings treated with different concentrations of EGCG using cDNA templates, with 18S RNA as the internal reference gene. The primer sequences of 18S and MdECH, MdKCS1, and MdCER1 genes refer to Table 1.

[0075] Table 118S, MdCER1, MdMYB30, MdKCS1, MECH gene primer sequences

[0076]

[0077] Experimental results: 2μmol / L EGCG treatment had no significant effect on the transcription levels of these three genes, while 5μmol / L treatment significantly increased the transcription levels of MdECH, MdKCS1, and MdCER1 ( Figure 6 The results showed that low concentrations of EGCG can increase the wax content of apple seedling leaves.

[0078] Example 4

[0079] EGCG treatment of 'Venus Gold' apple trees

[0080] On June 18, the 'Venus Gold' fruit trees were sprayed with 5μmol / L EGCG for the first time, and distilled water was sprayed as a control. The spraying was carried out once every two weeks, with a dosage of 1.5L per fruit tree, until the apples were picked on October 6, a total of 6 sprayings. Due to the heavy rainfall in August, no spraying treatment was carried out in August (5 trees were treated and 5 trees were treated in each control). The 'Venus Gold' fruit trees were sprayed with EGCG, and the leaves and fruits were collected at the same time after the fruits matured for the determination of relevant indicators.

[0081] 1. Determination of leaf-related indicators

[0082] (1) Leaf weight: Weigh using a 1 / 1000 electronic balance. Weigh each group of 5 leaves once, and weigh 100 leaves each for the treatment and control groups.

[0083] (2) Leaf area: The leaf area of ​​leaves was measured using an AM200 leaf area meter. Five leaves were grouped together for measurement, and 100 leaves were measured for each treatment and control group.

[0084] (3) Dry weight and specific leaf area: 5 leaves were placed in a 103℃ oven for 30-35 min, and then dried in a 65℃ oven for at least 3 days. The dry weight was measured. The ratio of the single surface area of ​​each group of leaves to its dry weight was the specific leaf area. 100 leaves were measured for each treatment and control.

[0085] (4) Detection of SOD enzyme activity, POD enzyme activity, and H2O2 content: The SOD enzyme activity, POD enzyme activity, and H2O2 content in the leaves were determined using a kit method. The kit was produced by Suzhou Keming Biotechnology Co., Ltd. The determination was performed according to the instructions, and 20 leaves were tested for each treatment and control.

[0086] Experimental results: The leaf area of ​​the fruit trees sprayed with EGCG increased by 13.67% compared with the control (Figure Figure 7 B), but did not affect the fresh weight of leaves ( Figure 7 The specific leaf area of ​​fruit trees after EGCG treatment was significantly higher than that of the control ( Figure 7 C in the leaves). The H2O2 content in the leaves was measured, and the results showed that the H2O2 content in the leaves was reduced by 23.52% after EGCG treatment ( Figure 7 D in the leaves), but had no significant effect on the activities of SOD and POD enzymes in the leaves ( Figure 7 E and F in Figure 3). It is speculated that EGCG, as an antioxidant, has the function of scavenging H2O2.

[0087] (5) Use scanning electron microscopy to observe the wax crystals on the leaves of treated and control fruit trees.

[0088] Experimental results: The number of wax crystals on leaves treated with EGCG was significantly higher than that of the control ( Figure 7G in.

[0089] (6) The transcription level of wax synthesis-related gene MdMYB30 after EGCG treatment was 10.2 times that of the control ( Figure 7 H in the leaves). This indicates that EGCG has a promoting effect on the synthesis of leaf waxes.

[0090] (7) In order to clarify the effect of EGCG on the wax components of 'Venus Gold' fruit tree leaves, the fatty acids, alkanes, primary alcohols, esters, ketones and total wax contents of different wax components on the leaf surface were measured and analyzed. For specific measurement methods, see Example 3.

[0091] Experimental results: The specific content of each component is shown in Table 2.

[0092] Table 2 Effect of EGCG on the content of different components of wax in leaves and fruits of "Venus Gold"

[0093]

[0094]

[0095] Note: The wax components of leaves were grouped together for significant difference analysis, the wax components of fruits were grouped together for significant difference analysis, and the total wax of leaves and fruits were analyzed for significant difference separately. Different lowercase letters indicate that the differences between different treatments reached the 5% significant level.

[0096] No aldehydes were detected in the leaves. It was found that EGCG had a greater effect on esters in leaf wax. The content of esters increased significantly by 35.87% after EGCG application, while there were no significant differences in other components and total wax ( Figure 8 A).

[0097] 2. Determination of fruit-related indicators

[0098] In order to further explore the effect of EGCG on fruit quality, EGCG was sprayed during the apple growth period, and the fruits were picked after they were ripe, and their quality indicators were tested.

[0099] (1) Apple longitudinal and transverse diameters and single fruit weight: Twenty fruits of similar size were randomly selected from each treatment and control group. An electronic vernier caliper (with an accuracy of 0.01 mm) was used to measure the diameter of the apple at the equator as the transverse diameter, and the distance from the bottom of the apple to the height of the fruit shoulder as the longitudinal diameter. A 1 / 1000 electronic balance was used to determine the single fruit weight.

[0100] Experimental results: Compared with the control, EGCG treatment significantly increased the fruit diameter, fruit diameter, and single fruit weight by 4.66%, 4.08%, and 13.23%, respectively. Fig. 9A, B, C in the figure) had no significant effect on the fruit shape index that affects the aesthetics ( Fig. 9 D), which indicates that EGCG can act as a growth agent to promote the enlargement and weight gain of fruits.

[0101] (2) Fruit gloss: The gloss meter (3nh, Shenzhen, China) was used to measure the fruit surface perpendicular to the fruit surface. Three different positions of each fruit were selected for measurement. Twenty apples were tested for each treatment and control.

[0102] Experimental results: EGCG treatment significantly increased the glossiness of the apple surface at a 60° reflection angle, which was 12.1% higher than that of the control ( Fig.10 A).

[0103] (3) Fruit hardness: The equatorial plane of the apple was placed at the test point, and the fruit hardness was tested using a TA.XTplus texture analyzer (MicroSystems, UK). Each apple was tested three times. The parameters that the texture analyzer can detect include: skin strength, skin rupture depth, brittleness, skin toughness, average flesh firmness, and flesh homogeneity.

[0104] Experimental results: as shown in Table 3.

[0105] Table 3 Skin strength, skin rupture depth, brittleness, skin toughness, average flesh firmness, and flesh homogeneity of EGCG-treated “Venus Gold” apples

[0106]

[0107] Note: The differences among different treatments did not reach the 5% significant level.

[0108] As shown in Table 3, EGCG treatment did not affect the six hardness indices including skin strength, skin rupture depth, brittleness, skin toughness, average firmness of the flesh, and flesh homogeneity.

[0109] (4) Soluble solids content: Take the pulp from three different positions of the apple fruit, squeeze out the juice and drip it into a saccharimeter, and record the value displayed by the saccharimeter. Each fruit was tested three times, and 20 apples were tested for each treatment and control.

[0110] Experimental results: After EGCG treatment, the soluble solids decreased by 6.67%, but the content still reached 19.30±0.30% ( Fig.10 B).

[0111] (5) Titratable acid content: Use NaOH titration method to detect the titratable acid content in the fruit. Weigh 5 g of fruit sample, grind it in a mortar, transfer it to a 50 mL volumetric flask, make up to volume with distilled water and shake well, let it stand for 30 minutes and then filter. Take 20 mL of filtrate, add 2 drops of phenolphthalein indicator, and titrate with the prepared NaOH solution. Titrate until the solution turns pink and does not fade within 30 seconds, and record the amount of NaOH titrant used (titrate distilled water with NaOH solution as a control). Test 20 apples for each treatment and control. According to the calculation formula: titratable acid content = V×c×(V1-V0)×f / (VS×m)×100% V: total volume of fruit extract (mL); c: concentration of sodium hydroxide titration (mol / L); V1: volume of sodium hydroxide consumed during titration (mL); V0: volume of sodium hydroxide consumed for titration of distilled water (mL); Vs: volume of filtrate taken during titration (mL); m: sample mass (g); f: refractive index of malic acid 0.067g / mmol.

[0112] Experimental results: The titratable acid content did not change significantly ( Fig.10 C in the figure). These results indicate that EGCG can promote fruit growth, increase surface glossiness, and thus improve the appearance quality of 'Venus Gold' fruit.

[0113] (6) The wax content of the 'Venus Gold' apple surface was analyzed and compared. The fruit wax extraction, derivatization, GC-MS sampling and analysis were carried out according to Example 3. Before the wax extraction, the fruit diameter and longitudinal diameter were measured to calculate the surface area of ​​the fruit. The fruit was extracted in 600 mL of chloroform three times, each time for 1 min.

[0114] Experimental results: As shown in Table 1, fatty acids, aldehydes, alkanes, primary alcohols, esters, and ketones were detected on the fruit surface, and EGCG treatment significantly increased the content of primary alcohols and esters in the fruit wax, with primary alcohols and esters increasing by 179.36% and 41.79%, respectively ( Figure 8 B). Overall, the wax content in different organs was significantly different, and the effects of EGCG on the wax components of different organs were also different.

[0115] From the above examples, it can be seen that the tea residue extract or EGCG used in the present invention can significantly increase the height of apple seedlings, improve the net photosynthesis of apple seedlings, promote the growth of apple seedlings and the wax content of leaves. Spraying EGCG during the growth period of fruit trees can not only increase the leaf area of ​​fruit trees, reduce the content of H2O2 in leaves, and increase the content of ester substances in leaf wax; it can also increase the horizontal diameter of the fruit, the vertical diameter of the fruit, and the weight of a single fruit, promote the growth of the fruit, improve the surface gloss, and thus improve the appearance quality of the fruit, and significantly increase the content of primary alcohols and ester substances in fruit wax.

[0116] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An apple growth-promoting preparation, characterized in that: including tea residue extract or epigallocatechin gallate; The content of epigallocatechin gallate in the tea residue extract is 62.94-131.43 mg / g; The concentration of the epigallocatechin gallate is 2-200 μmol / L.

2. The apple growth-promoting preparation according to claim 1, characterized in that: The preparation method of the tea residue extract comprises the following steps: The tea leaves are mixed with boiling water and brewed for 2 to 5 times, and then filtered, dried, ground and sieved to obtain a tea residue extract.

3. The apple growth-promoting preparation according to claim 2, characterized in that: The mass volume ratio of the tea leaves to the boiling water is 0.5-6 g:1 L; The drying temperature is 60-70°C and the drying time is 18-30h; The mesh number of the sieving is 300 to 500 meshes.

4. Use of the apple growth-promoting preparation according to any one of claims 1 to 3 in promoting the growth of apple seedlings.

5. Use of the apple growth-promoting preparation according to any one of claims 1 to 3 in increasing the wax content in leaves of apple seedlings.

6. The use according to claim 4 or 5, characterized in that: The apple growth-promoting preparation is prepared into a solution and then applied to apple seedlings once a week at an application rate of 400 to 600 mL per time per plant.

7. Use of the apple growth-promoting preparation according to any one of claims 1 to 3 in promoting the growth of apple fruits and improving the quality of apple fruits.

8. Use of the apple growth-promoting preparation according to any one of claims 1 to 3 in increasing the wax content on the surface of apple fruit.

9. The use according to claim 7 or 8, characterized in that: The apple growth-promoting preparation is prepared into a solution, and the apple growth-promoting preparation is applied during the growth period of the apple tree, once every two weeks, with an application amount of 1 to 3 L / time / tree.

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