Cultivation method for improving cold resistance of apple plants

Abstract

The invention relates to the technical field of fruit tree cultivation, and particularly discloses a cultivation method for improving cold resistance of apple plants, which comprises the following steps: after apples grow to a dormancy period, spraying 80-120mg / L of inositol galactoside solution on the whole tree for 3-4 times every 10-11 days. According to the method provided by the invention, the cold resistance of the apple plants is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of fruit tree cultivation, and in particular to a cultivation method for improving the cold resistance of apple plants. Background Art

[0002] Apple (Malus domestica Borkh.) is a fruit crop with rich nutrition and high economic value, and has become a model of horticultural crops. Apple fruit is rich in minerals and vitamins and is one of the most commonly consumed fruits. The nutrients in apples are highly soluble and easily absorbed by the human body, so they are known as "living water" and are conducive to dissolving sulfur elements, making the skin smooth and tender. Apples contain copper, iodine, manganese, zinc, potassium and other elements, which have the effects of lowering cholesterol, calming the mind and promoting sleep, and relieving fatigue.

[0003] As a temperate deciduous fruit tree, apples are often subjected to various abiotic and biotic stresses, among which low temperature damage is one of the important abiotic stresses that restricts the development of the apple industry in Northeast my country. After plants experience low temperature stress, they will undergo changes in morphology, physiology, and biochemical processes to cope with the damage caused by low temperature stress, and these changes will affect the growth and development of plants. Therefore, it is of great significance to develop a cultivation method to improve the cold tolerance of apples. Summary of the invention

[0004] In order to develop a method for improving the cold resistance of apple plants, the present invention provides a cultivation method for improving the cold resistance of apple plants. The method provided by the present invention significantly improves the cold resistance of apple plants.

[0005] The present invention provides a cultivation method for improving the cold resistance of apple plants, comprising the following steps:

[0006] When the apples reach dormancy, spray the whole tree with 80mg / L-120mg / L inositol galactoside solution 3-4 times every 10-11 days, with a spraying amount of 15ml-20ml per tree.

[0007] The present invention increases the thickness and proportion of the xylem and phloem of apple plant branches by spraying an exogenous inositol galactoside solution, alleviates low-temperature damage to apples during dormancy, significantly improves the tolerance of apple plants to low-temperature environments, and improves the cold resistance of apple plants.

[0008] Furthermore, the concentration of the galactoside solution is 100 mg / L. Spraying with the 100 mg / L galactoside solution significantly improves the cold resistance of apples more than other concentrations.

[0009] Furthermore, the inositol galactoside solution is obtained by dissolving inositol galactoside with distilled water and diluting it to a concentration of 80 mg / L to 120 mg / L.

[0010] Furthermore, the apple plants are obtained by grafting through high-position grafting.

[0011] Further, the GM-256 stock is grafted at a distance of 40 cm to 60 cm from the ground from the base stock. After the GM-256 stock grows to a semi-lignified lower part, about 2 months after the spring grafting, the lower 10 cm to 15 cm of the GM-256 stock is cut off, and 3 to 4 new shoots grown are selected according to different directions, and the others are thinned out. When pruning in winter the following year, each new shoot is cut off to 20 cm to 25 cm and the apple seedlings are grafted.

[0012] The grafting platform formed by high-position grafting of GM-256 rootstock by grafting technology can be reused repeatedly. After the upper part of the rootstock has been stably bearing fruit for 2 years, the scion part can be cut off and reused for a new round of superior line selection, thus realizing cold-resistant apple variety breeding in a short period of time. Therefore, the high-position grafting method of the present invention shortens the cycle of cold-resistant apple variety breeding.

[0013] Furthermore, the anvil is selected from a Malus or a Stator with a thickness greater than 3 cm.

[0014] Furthermore, the process of grafting the GM-256 rootstock onto the base stock is as follows: a grafting port is opened on the base stock at a distance of 40 cm to 60 cm from the ground, and then a GM-256 single bud branch segment is grafted onto the grafting port.

[0015] Furthermore, the apple seedling progeny are selected lines after natural wintering elimination and screening.

[0016] Further, an apple seedling is grafted onto each new shoot.

[0017] Furthermore, the apple seedling offspring variety is: 'Hanfu' or 'Changfu 2'.

[0018] The present invention also provides an application of inositol galactoside in improving the cold resistance of apple plants, wherein inositol galactoside is prepared into an inositol galactoside solution with a concentration of 80 mg / L to 120 mg / L, and after the apples grow to a dormant period, the inositol galactoside solution is sprayed on the whole tree 3 to 4 times every 10 to 11 days, and the spraying amount is 15 ml to 20 ml per tree.

[0019] Furthermore, inositol galactoside is used to increase the thickness and proportion of the xylem and phloem of apple plant branches, alleviate low temperature damage during the dormancy period of apples, and improve the tolerance of apple plants to low temperature environments.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] The cultivation method of apple plants provided by the present invention significantly improves the cold resistance of the plants, which is specifically manifested in effectively alleviating the low temperature damage suffered by apples during the dormant period, promoting the stability of the membrane structure and the increase in the content of osmotic regulating substances, and up-regulating the enzymatic reaction mechanism and the non-enzymatic reaction mechanism, thereby improving the cold resistance of apples. In the cultivation method, exogenous spraying of 100 mg / L inositol galactoside solution has the best effect on alleviating the low temperature damage of apples during the dormant period.

[0022] The cultivation method of apple plants provided by the present invention can also be used for the selection of cold-resistant superior apple lines. The grafting platform formed during the grafting process of the apple plants of the present invention can be reused repeatedly. After the upper part of the local plant has been stably bearing fruit for 2 years, the scion part can be cut off and reused for a new round of superior line selection. Compared with the traditional method, the breeding time and cost are reduced by half. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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 or the description of the prior art 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.

[0024] Figure 1 The diagram shows the cross-sectional anatomical structure of one-year-old apple branches treated with different concentrations of inositol galactoside solution. In the figure, the upper picture is the 'Hanfu' variety, and the lower picture is the 'Changfu 2' variety. From left to right, they represent the spraying concentrations of inositol galactoside solution, which are 0 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L.

[0025] Figure 2 The figure shows the effect of different concentrations of exogenous inositol galactoside solution on the relative conductivity and malondialdehyde content of apple branches under low temperature stress; in the figure, A shows the effect of different concentrations of exogenous inositol galactoside solution on the conductivity of 'Hanfu' apple branches; B shows the effect of different concentrations of exogenous inositol galactoside solution on the conductivity of 'Changfu 2' apple branches; C shows the effect of different concentrations of exogenous inositol galactoside solution on the malondialdehyde content of 'Hanfu' apple branches; D shows the effect of different concentrations of exogenous inositol galactoside solution on the malondialdehyde content of 'Changfu 2' apple branches.

[0026] Figure 3The figures show the effects of exogenous galactoside solution on the soluble sugar, soluble protein and proline contents of apple branches under low temperature stress; in the figure, A shows the effect of exogenous galactoside solution on the soluble sugar content of 'Hanfu' apple branches under low temperature stress; B shows the effect of exogenous galactoside solution on the soluble sugar content of 'Changfu 2' apple branches under low temperature stress; C shows the effect of exogenous galactoside solution on the soluble protein content of 'Hanfu' apple branches under low temperature stress; D shows the effect of exogenous galactoside solution on the soluble protein content of 'Changfu 2' apple branches under low temperature stress; E shows the effect of exogenous galactoside solution on the proline content of 'Hanfu' apple branches under low temperature stress; F shows the effect of exogenous galactoside solution on the proline content of 'Changfu 2' apple branches under low temperature stress.

[0027] Figure 4 Effect of exogenous galactinol solution on the growth of apple branches under low temperature stress 2. - and H 2 O 2 In the figure, A shows the effect of exogenous inositol galactoside solution on the branches of 'Hanfu' apple under low temperature stress. 2 .- B is the effect of exogenous inositol galactoside solution on the growth of 'Changfu 2' apple branches under low temperature stress. 2. - C is the effect of exogenous inositol galactoside solution on H content of 'Hanfu' apple branches under low temperature stress 2 O 2 D is the effect of exogenous inositol galactoside solution on the H content of Changfu 2' apple branches under low temperature stress 2 O 2 The influence of content.

[0028] Figure 5 Figure 2 shows the effect of exogenous inositol galactoside solution on the activities of SOD, POD, CAT and APX in apple branches under low temperature stress; Figure 3 shows the effect of exogenous inositol galactoside solution on the activity of SOD, POD, CAT and APX in apple branches under low temperature stress; Figure 4 shows the effect of exogenous inositol galactoside solution on the activity of SOD in 'Hanfu' apple branches under low temperature stress; Figure 5 shows the effect of exogenous inositol galactoside solution on the activity of SOD in 'Changfu 2' apple branches under low temperature stress; Figure 6 shows the effect of exogenous inositol galactoside solution on the activity of POD in 'Hanfu' apple branches under low temperature stress; Figure 7 shows the effect of exogenous inositol galactoside solution on the activity of Effect of lactosidase solution on POD activity of 'Changfu 2' apple; E is the effect of exogenous inositol galactoside solution on CAT activity of 'Hanfu' apple under low temperature stress; F is the effect of exogenous inositol galactoside solution on CAT activity of 'Changfu 2' apple under low temperature stress; G is the effect of exogenous inositol galactoside solution on APX activity of 'Hanfu' apple under low temperature stress; H is the effect of exogenous inositol galactoside solution on APX activity of 'Changfu 2' apple under low temperature stress.

[0029] Figure 6 To study the effects of exogenous galactoside solution on sugar components in apple branches under low temperature stress.

[0030] In the figure, A shows the effect of exogenous galactoside solution on the sucrose content of 'Hanfu' apple under low temperature stress; B shows the effect of exogenous galactoside solution on the sucrose content of 'Changfu No. 2' apple under low temperature stress; C shows the effect of exogenous galactoside solution on the glucose content of 'Hanfu' apple under low temperature stress; D shows the effect of exogenous galactoside solution on the glucose content of 'Changfu No. 2' apple under low temperature stress; E shows the effect of exogenous galactoside solution on the fructose content of 'Hanfu' apple under low temperature stress; F shows the effect of exogenous galactoside solution on the fructose content of 'Changfu No. 2' apple under low temperature stress; G shows the effect of exogenous galactoside solution on the sorbitol content of 'Hanfu' apple under low temperature stress; H shows the effect of exogenous galactoside solution on the sorbitol content of 'Changfu No. 2' apple under low temperature stress.

[0031] Figure 7 The figure shows the effect of exogenous galactoside solution on the sugar components of apple branches under low temperature stress; in the figure, A shows the effect of different concentrations of exogenous galactoside solution on the inositol content of 'Hanfu' apple branches; B shows the effect of different concentrations of exogenous galactoside solution on the inositol content of 'Changfu 2' apple branches; C shows the effect of different concentrations of exogenous galactoside solution on the inositol content of 'Hanfu' apple branches; D shows the effect of different concentrations of exogenous galactoside solution on the inositol content of 'Changfu 2' apple branches; E shows the effect of different concentrations of exogenous galactoside solution on the raffinose content of 'Hanfu' apple branches; F shows the effect of different concentrations of exogenous galactoside solution on the raffinose content of 'Changfu 2' apple branches.

[0032] Figure 8 The figure shows the effect of exogenous galactoside solution on the activity of RFOs metabolic enzymes in apple branches under low temperature stress; in the figure, A shows the effect of different concentrations of exogenous galactoside solution on the enzyme activity of galactoside solution in 'Hanfu' apple branches; B shows the effect of different concentrations of exogenous galactoside solution on the enzyme activity of galactoside solution in 'Changfu 2' apple branches; C shows the effect of different concentrations of exogenous galactoside solution on the activity of raffinose synthase in 'Hanfu' apple branches; D shows the effect of different concentrations of exogenous galactoside solution on the activity of raffinose synthase in 'Changfu 2' apple branches. DETAILED DESCRIPTION

[0033] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0034] Example 1: A cultivation method for improving the cold resistance of apple plants.

[0035] The experiment was conducted at the base on the campus of Shenyang Agricultural University. The experimental material was 'Hanfu' apple plants. The brand of inositol galactoside was Merrill, item number: M58097-100MG, CAS number: 3687-64-7, specification: 99% (HPLC).

[0036] In January 2022, one-year-old branches of 'Hanfu' apple were collected and stored in sand as spare for apple seedlings. In mid-April 2022, a five-year-old crabapple was used as the base stock with a thickness of about 6 cm. The base stock was cut off 50 cm from the ground, and two GM-256 rootstock scions were grafted onto the five-year-old crabapple base stock by bark grafting. In the year of grafting (about 2 months after grafting), when the GM-256 rootstock grows more than 30 cm and the lower part is semi-lignified, the lower 15 cm of the GM-256 rootstock is retained and cut off. When it sprouts, only the new shoots spread out in three directions are retained, and the others are thinned out. In the next year, three branches (new shoots in three different directions selected in the previous year) are cut to 20 cm and each is grafted with an apple seedling;

[0037] When the apples grow to dormancy, spray the whole tree with 100 mg / L inositol galactoside solution three times every 10 days. The spraying degree is based on the leaves being wet but no droplets condense and fall. The spraying amount is about 20 ml per tree.

[0038] The 100 mg / L inositol galactoside solution was obtained by dissolving 50 mg of inositol galactoside in 500 mL of distilled water.

[0039] Example 2: A cultivation method for improving the cold resistance of apple plants.

[0040] The experimental steps are the same as those in Example 1. The difference is that the experimental material is the 'Changfu No. 2' apple plant.

[0041] Example 3: A cultivation method for improving the cold resistance of apple plants.

[0042] The experimental steps are the same as those in Example 1. The difference is that the concentration is 80 mg / L. The 80 mg / L galactoside solution is obtained by dissolving 40 mg galactoside in 500 mL distilled water.

[0043] Example 4: A cultivation method for improving the cold resistance of apple plants.

[0044] The experimental steps are the same as those in Example 1. The difference is that the concentration of the galactoside solution is 120 mg / L. The 120 mg / L galactoside solution is obtained by dissolving 60 mg of galactoside in 500 mL of distilled water.

[0045] Example 5: A cultivation method for improving the cold resistance of apple plants.

[0046] The experimental steps are the same as those in Example 1. The difference is that the grafting point of the crabapple rootstock is opened at 40 cm from the ground.

[0047] Example 5: A cultivation method for improving the cold resistance of apple plants.

[0048] The experimental steps are the same as those in Example 1. The difference is that the grafting point of the crabapple rootstock is opened at 60 cm from the ground.

[0049] Comparative Example 1: A cultivation method for improving the cold resistance of apple plants.

[0050] The experimental steps are the same as those in Example 1. The difference is that the concentration of the galactoside solution is 50 mg / L. The 50 mg / L galactoside solution is obtained by dissolving 25 mg of galactoside in 500 mL of distilled water.

[0051] Comparative Example 2: A cultivation method for improving the cold resistance of apple plants.

[0052] The experimental steps are the same as those in Example 1, except that the concentration of the galactoside solution is 200 mg / L. The 200 mg / L galactoside solution is obtained by dissolving 100 mg of galactoside in 500 mL of distilled water.

[0053] Comparative Example 3: A cultivation method for improving the cold resistance of apple plants.

[0054] The experimental steps are the same as those in Example 2, except that the concentration of the galactoside solution is 50 mg / L.

[0055] Comparative Example 4: A cultivation method for improving the cold resistance of apple plants.

[0056] The experimental steps are the same as those in Example 2, except that the concentration of the galactoside solution is 200 mg / L.

[0057] Comparative Example 5: A cultivation method for improving the cold resistance of apple plants.

[0058] The experimental steps are the same as those in Example 1, except that the galactoside solution is replaced by an equal amount of distilled water, which is recorded as CK.

[0059] 1. Study the effect of inositol galactoside on the cold resistance of apple plants.

[0060] Taking Examples 1 to 2 and Comparative Examples 1 to 5 as the research objects, field sampling was carried out 40 days after the spraying of the inositol galactoside solution, and 24 one-year-old branches with good and uniform growth were collected respectively. The wounds were smeared with a strong wound healing agent (brand: Baoguoyou, net content: 2000 grams) provided by Zhangjiakou Guofeng Agricultural Technology Co., Ltd. and wrapped with a film to prevent infection and water loss and drying. The branches of each variety of 'Hanfu' and 'Changfu No. 2' apple plants were divided into 8 groups on average, with 3 branches in each group, of which 1 group was not subjected to low temperature treatment as the control, and the remaining 7 groups were placed in a low temperature refrigerator for -15°C low temperature treatment, maintained for 1 day after reaching the set temperature, and then placed in a 4°C low temperature refrigerator for 1 day to thaw. Rinse with tap water and deionized water respectively, wipe dry with gauze, cut the thawed one-year-old branches of each variety into branches of about 0.5cm to 1cm with pruning shears, avoid buds, quickly freeze in liquid nitrogen, and finally store in a -80℃ ultra-low temperature refrigerator. In addition, take another part of the branches for measuring relative conductivity and observing anatomical structure. The relevant detection methods are as follows:

[0061] 1. Hard tissue sections

[0062] Before the first spraying of galactoside solution and 40 days after the third spraying of galactoside solution, select pest-free, upright and uniform one-year-old branches, avoid the buds in the middle and take 1 cm branch segments, and fix them with FAA fixative. Place the sliced ​​branches in ethylene glycol ether acetate, soak at 37 ° C for 6 hours and then overnight; expose to 25 ° C room temperature for 30 minutes. Then soak in anhydrous ethanol for 20 minutes, 95% ethanol for 10 minutes, 90% ethanol for 10 minutes, 80% ethanol for 10 minutes, and finally wash with deionized water. Place the sliced ​​branches in safranin dye solution for 5 seconds, wash the excess dye with deionized water, and place the slices in 50%, 70%, and 80% gradient alcohol for 8 seconds each. Then place the sliced ​​branches in fast green dye solution for 6 seconds and dehydrate with anhydrous ethanol. Place the slices in xylene for 5 minutes and finally seal with neutral gum. The cross sections were observed using a Nikon Eclipse E100 upright optical microscope and a Nikon DS-U3 imaging system, and the anatomical characteristics of the branches were analyzed using Case Viewer. The statistical method for vessel density was to draw a 200*200 pixel square frame in Adobe Photoshop software and count the number of vessels in the frame. If the area of ​​the vessel at the border of the box was greater than 1 / 2, it was counted; otherwise, it was not counted.

[0063] 2. Determination of physiological indicators related to stress resistance

[0064] The relative conductivity was determined by referring to the method in the journal (ABA is involved in regulation of cold stress response in bermudagrass. Frontiers in Plant Science, 2017, 8: 1613). The MDA content was determined by the thiobarbituric acid method, referring to the method in the journal published by Du et al. (The identification of novel and differentially expressed apple-tree genes under low-temp erature stress using high-throughput illuminase quencing. Molecular Biology Reports, 42 (3): 569-580). The soluble sugar content was determined by the anthrone reagent colorimetric method, referring to the method in the journal published by Xu et al. (Physiological and structural changes in apple tree branches of different varieties during dormancy. Horticulturae 2023, 9, 947). The soluble protein content was determined by the Coomassie Brilliant Blue G-250 method, referring to the journal published by DeSousa et al. (Increase in dry tolerance in maize plants induced by H 2 O 2 The proline content was determined by the salicylic acid colorimetric method, referring to the method in the journal published by He et al. (ZB A transcriptomic network underlies microstructural and physiological responses to cadmium in Populus × canescens. Plant Physiol. 2013, 162, 424-439). 2 .-The content was determined according to the method in the journal published by Farooq (Oxidative in jury and antioxidant enzymes regulation in arsenic-exposed seeds off our Brassicanapus L. cultivars. Environmental Science and Pollution Research International, 22 (14): 10699-10712). 2 O 2 The method for determining the content is specifically based on the method published by He et al. (Overexpression of bacterial γ-glutamylcysteinesyn the tase mediates changes in cadmium influx, allocation and detoxification in poplar. New Phytologist). The enzyme solution extraction method is consistent with the soluble protein extraction method. The SOD activity was determined according to the method published by Afzal et al. (Cadmium tolerance in rice cultivars associated with anti oxidant enzyme activities and Fe / Zn concentrations. International Journal of Environmental Science and Technology); the POD activity was determined according to the method published by Zhang et al. (Cadmium induction of lipid peroxidation and effects on root tip cells and anti oxidant enzyme activities in Vicia faba L. Ecotoxicology); the CAT activity was determined according to the method published by Emamverdian et al. (Silicon di oxide nano particles improve plant growth by enhancing anti oxidant enzyme capacity in bamboo (Pleioblastus pygmaeus); the APX activity was determined according to the method published by Emamverdian et al. The method was used to determine the soil moisture content in barley.

[0065] 3. Determination of sugar components

[0066] The contents of sucrose, fructose, glucose, sorbitol, inositol galactoside and raffinose family oligosaccharides (RFOs) in the branches were determined by high performance liquid chromatography (HPLC) and ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS). Weigh 0.2g of sample, extract with 3mL 80% ethanol in 80℃ water bath for 30 minutes, centrifuge at 8,000rpm for 10 minutes at room temperature, collect the supernatant and extract repeatedly, combine the supernatant and mix well, take 5mL and evaporate to viscous state, add 1mL ultrapure water to dissolve, centrifuge again at 10,000rpm for 10 minutes, filter and use as the test solution. Take another 1g sample, extract with 3mL 95% ethanol in 80℃ water bath for 10 minutes, mix every 2-3 minutes, cool and centrifuge at 12,000g for 5 minutes, take the supernatant and extract repeatedly with 50% ethanol and ultrapure water, combine the supernatant and filter to obtain the crude total sugar extract. 5 mL of crude extract was taken and dried by rotary evaporation and 1 mL of ultrapure water was added to dissolve it. Impurities were removed by C18 solid phase extraction column, and the purified total sugar extract was collected for testing. HPLC analysis was performed using Agilent 1260 system, with ultrapure water as the mobile phase, column temperature of 80 °C, and flow rate of 0.6 mL min - 1. UPLC-MS / MS analysis used 0.08% ammonia and acetonitrile as mobile phase, column temperature 35°C, flow rate 0.2 mL min - 1.

[0067] 4. Determination of galactinol synthase and raffinose synthase activities

[0068] The activities of galactinol synthase and raffinose synthase were determined using ELISA kits from Nanjing Herb Source Biotechnology Co., Ltd.

[0069] 5. Data statistics and analysis

[0070] The experimental data were statistically analyzed using Statgraphics 18 software. One-way ANOVA was used for variance analysis and multiple comparisons (P<0.05). Origin 8.0 software was used for drawing.

[0071] 2. Experimental Results

[0072] 1. Effects of exogenous galactinol on the anatomical structure of apple branches under low temperature

[0073] The results are shown in Table 1, Table 2 and Figure 1 As shown in the figure, the differences in branch anatomical structure and microscopic observation results between 'Hanfu' and 'Changfu 2' ( Figure 1 ) It can be seen that the cold resistance of apple branches is closely related to the tissue structure. After exogenous inositol galactoside treatment, the branch tissue structure changed significantly. The anatomical structure of apple one-year-old branches is pith, xylem, cambium, phloem, and periderm from the inside to the outside. The thickness of the xylem and phloem of the one-year-old branches of 'Hanfu' apple are significantly higher than those of 'Changfu 2'. Exogenous spraying of different concentrations of inositol galactoside solution significantly increased the thickness and proportion of the xylem and phloem of the branches of 'Hanfu' and 'Changfu 2', among which the 100 mg / L treatment had the most obvious effect ( Figure 1 The vessel density and diameter in the xylem of branches will affect the water transport efficiency, and thus affect the cold resistance of plants. As shown in Table 1, the number of vessels per unit area in the xylem of 'Hanfu' is significantly higher than that of 'Changfu 2', while the vessel unit area is significantly lower than that of 'Changfu 2'. Exogenous spraying of galactoside solution has no significant effect on the vessel density and vessel unit area (Tables 1 and 2).

[0074] Table 1 Anatomical structure indexes of cross sections of one-year-old branches of 'Hanfu' apple

[0075]

[0076] Table 2 Anatomical structure indexes of cross sections of one-year-old branches of 'Changfu 2' apple

[0077]

[0078] 2. Effects of exogenous galactinol on relative conductivity and MDA content of apple branches under low temperature

[0079] from Figure 2 It can be seen that compared with the control group at normal temperature, the relative conductivity and MDA content of apple branches under low temperature conditions increased, and the relative conductivity and MDA content of 'Changfu 2' were always higher than those of 'Hanfu'. Exogenous spraying of different concentrations of inositol galactoside solution under low temperature stress can reduce the relative conductivity and MDA content of 'Hanfu' and 'Changfu 2' branches to varying degrees, with the 100 mg / L treatment having the most significant effect, with the relative conductivity reduced by 29.35% and 30.21%, and the MDA content reduced by 19.58% and 17.78%, respectively. There was no significant difference between the 50 mg / L and 200 mg / L treatments.

[0080] 3. Effect of exogenous galactinol on the content of osmotic regulating substances in apple branches under low temperature

[0081] from Figure 3 A~ Figure 3 It can be seen from the F that under low temperature stress, the content of osmotic regulating substances in the branches of 'Hanfu' and 'Changfu 2' increased significantly. Exogenous spraying of different concentrations of inositol galactoside solution further promoted the accumulation of soluble sugars, soluble proteins and proline in the branches of 'Hanfu' and 'Changfu 2'. With the gradual increase in concentration, when the concentration reached 100 mg / L, the content of various osmotic regulating substances reached the highest value, and then decreased, with an increase of 13.6%, 15.42%, 24.49% ('Hanfu') and 24.39%, 23.19%, 35.29% ('Changfu 2').

[0082] 4. Effect of exogenous inositol galactoside on the growth of apple branches under low temperature 2. - and H 2 O 2 The influence of content

[0083] from Figure 4 A~ Figure 4 It can be seen that compared with the normal temperature control, low temperature stress induced the O 2 .- and H 2 O 2 Compared with 'Changfu 2', 'Hanfu' always maintains a lower level of O under low temperature treatment. 2 .- and H 2 O 2 Exogenous addition of different concentrations of inositol galactoside solution significantly reduced the content of O in the branches of 'Hanfu' and 'Changfu 2'. 2 .- and H 2 O 2 content, among which O 2 .- and H 2 O 2 The production rate was the lowest, and the decrease was the most obvious compared with other treatments, which were 9.93%, 10.27% ('Hanfu') and 12.88%, 15.74% ('Changfu 2').

[0084] 5. Effects of exogenous galactinol on antioxidant enzyme activities in apple branches under low temperature

[0085] from Figure 5It can be observed that after applying different concentrations of exogenous inositol galactoside solution, under low temperature stress conditions, the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) in the branches of 'Hanfu' and 'Changfu 2' were significantly lower than those in the branches of 'Hanfu' and 'Changfu 2'. 2 The activities of the four antioxidant enzymes in the 100 mg / L treatment group were higher than those in the H2O treatment group, but the activities in the 50 mg / L treatment group were not significantly improved. 2 The O treatment increased by 20.25%, 13.16%, 14.36% and 19.02% ('Hanfu'), and 17.4%, 14.8%, 17.43% and 12.33% ('Changfu 2').

[0086] 6. Effects of exogenous galactoside solution on sugar components in apple branches under low temperature stress

[0087] The results are as follows Figure 6 and Figure 7 As shown in Figure 2, under low temperature stress, after applying different concentrations of exogenous inositol galactoside, the contents of sucrose, glucose, fructose, sorbitol, inositol, inositol galactoside and raffinose in the branches of 'Hanfu' and 'Changfu 2' were significantly lower than those of the branches only sprayed with H 2 O treatments all showed different degrees of improvement. Especially in the 100 mg / L treatment group, the contents of sucrose, glucose, fructose, sorbitol, inositol, galactoinositol and raffinose reached the highest values, among which the increases of 'Hanfu' were 15.92%, 25.42%, 26.05%, 36.51%, 20.08%, 26.04% and 14.96%, respectively, while those of 'Changfu 2' were 16.36%, 27.94%, 26.04%, 38.86%, 24.26%, 28.45% and 20.75%, respectively.

[0088] 7. Effects of exogenous galactinol on the activity of RFOs metabolic enzymes in apple branches under low temperature

[0089] from Figure 8 It can be seen that after the exogenous inositol galactosidase solution of different concentrations was applied, under low temperature stress, the activities of inositol galactosidase synthase and raffinose synthase in the branches of 'Hanfu' and 'Changfu 2' were higher than those in the branches of 'Hanfu' and 'Changfu 2'. 2 O treatment was improved, except for the 50 mg / L treatment, and the activities of galactinol synthase and raffinose synthase reached the highest values ​​in the 100 mg / L treatment, which were higher than those in the exogenous spraying of H 2 The O treatment increased by 75.82% and 28.1% ('Hanfu') and 53.33% and 31.87% ('Changfu 2').

[0090] The above results indicate that low temperature stress will increase the relative conductivity and MDA content of one-year-old apple branches and promote the production of reactive oxygen species. Exogenous application of inositol galactoside can effectively alleviate the low temperature damage suffered by dormant apples, promote the stability of membrane structure and the increase in the content of osmotic regulating substances, upregulate the enzymatic reaction mechanism and non-enzymatic reaction mechanism, thereby improving the cold resistance of apples. Exogenous spraying of 100 mg / L inositol galactoside solution has the best effect on alleviating low temperature damage to apples during dormancy, while too high a concentration will weaken the mitigation effect.

[0091] Application example: The method of Example 1 is used to screen cold-resistant superior apple lines.

[0092] The method of Example 1 takes up less space, saves resources and labor, and can produce fruit in the second year after grafting. The preliminary superior line can be determined after 2-3 years. The same method can also be used to select superior lines, thereby shortening the time for regional trials.

[0093] The grafting platform formed in Example 1 can be reused repeatedly. After the upper part of the local stable fruiting is obtained for 2 years, the scion part can be cut off after the potential superior line fruit is evaluated, and reused for a new round of superior line selection. In order to reduce the fruiting height and facilitate investigation, after the hybrid offspring scion bears fruit, the scion branches with mature flower bud differentiation ability can be cut again from the upper part of the hybrid offspring part (scion) and grafted to the lower GM256 to reduce the fruiting height. This grafting method reduces the breeding time and cost by half compared with the traditional method.

[0094] In summary, the cultivation method provided by the present invention not only significantly improves the cold resistance of apple plants, but also can be used to screen cold-resistant superior apple lines, and the screening process is shortened by at least half compared with the traditional method, providing a practical basis for the efficient screening of cold-resistant apples.

[0095] Although preferred embodiments of the present invention have been described, additional changes and modifications may occur to these embodiments once those skilled in the art are aware of the basic inventive concepts.

[0096] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A cultivation method for improving the cold resistance of apple plants, characterized in that: After the apple plants grow to the dormant period, spray the whole tree with 80mg / L-120mg / L inositol galactoside solution 3-4 times every 10d-11d, with a spraying amount of 15ml-20ml per tree.

2. The cultivation method for improving the cold resistance of apple plants according to claim 1, characterized in that: The concentration of the galactoside solution is 100 mg / L.

3. The cultivation method for improving the cold resistance of apple plants according to claim 1, characterized in that: The inositol galactoside solution is obtained by dissolving inositol galactoside with distilled water and diluting the solution to a concentration of 80 mg / L to 120 mg / L.

4. The cultivation method for improving the cold resistance of apple plants according to claim 1, characterized in that: The apple plants are obtained by grafting through high-position grafting.

5. The cultivation method for improving the cold resistance of apple plants according to claim 4, characterized in that: The high-position grafting method is as follows: grafting the GM-256 rootstock at a distance of 40 cm to 60 cm from the ground to the base rootstock, and after the GM-256 rootstock grows to a semi-lignified lower part, cutting off the lower 10 cm to 15 cm of the GM-256 rootstock, selecting 3 to 4 new shoots grown according to different directions, and thinning out the others, and when pruning in winter the following year, cutting off each new shoot to 20 cm to 25 cm and grafting apple seedlings.

6. The cultivation method for improving the cold resistance of apple plants according to claim 5, characterized in that: The base anvil is selected from Malus or Malus multiflora.

7. The cultivation method for improving the cold resistance of apple plants according to claim 5, characterized in that: The process of grafting GM-256 rootstock onto the base stock is as follows: a grafting port is opened on the base stock at a distance of 40 cm to 60 cm from the ground, and then the GM-256 single bud branch segment is grafted onto the grafting port.

8. The cultivation method for improving the cold resistance of apple plants according to claim 5, characterized in that: The apple seedling progeny are selected from the superior lines after natural overwintering elimination and screening.

9. The cultivation method for improving the cold resistance of apple plants according to claim 5, characterized in that: An apple seedling is grafted onto each new shoot.

10. The cultivation method for improving the cold resistance of apple plants according to claim 9, characterized in that: The apple seedling offspring variety is: 'Hanfu' or 'Changfu No. 2'.

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