Efficient apple breeding method for improving flowering gene expression quantity and endogenous hormone content based on multi-trunk grafting

Abstract

The invention relates to a fruit tree breeding method, in particular to an efficient apple breeding method for improving flowering gene expression quantity and endogenous hormone content based on multi-trunk grafting, and belongs to the technical field of agriculture. According to the method, the dwarf rootstock capable of being subjected to multi-main-head grafting is cultivated, and hybrid seedling grafting and cultivation are carried out, so that the number of seedlings cultivated in unit land area is remarkably increased, the juvenile phase is effectively shortened, and the breeding efficiency is remarkably improved. By means of the method, a large amount of land can be saved, the breeding period can be effectively shortened, the juvenile phase of hybrid seedlings obtained through a conventional method is 7 years, the juvenile phase of hybrid seedlings obtained through the method is 4 years, and the total breeding period is shortened by 3 years. The rootstock used in the method is the same as the dwarfing rootstock used in production, so that different strains are the same as the rootstock and scion combination in actual production in the variety breeding stage, and the results and growth conditions which are the same as those in production can be shown; and the subsequent stock-scion combination evaluation work of the new variety is reduced.

Description

Technical Field

[0001] The invention relates to a fruit tree breeding method, in particular to an efficient apple breeding method based on multi-trunk grafting to increase the expression amount of flowering genes and the content of endogenous hormones, and belongs to the technical field of agriculture. Background Art

[0002] China is the world's largest apple producer and consumer, with the largest cultivated area and output in the world, and has obvious scale advantages. However, there is a lack of diversified varieties in apple production. The variety structure dominated by Fuji has led to a single market supply and insufficient competitiveness, which has restricted the sustainable development of the industry. The main reason is the lack of efficient breeding technology, which has restricted the selection and breeding of new varieties.

[0003] Hybrid breeding is the main way to select new apple varieties, but hybrid breeding requires the cultivation of a large number of hybrid seedlings. Hybrid seedlings need to go through a childhood period of about 7 years from sowing to fruiting. The breeding cycle is long. Direct planting or single-plant grafting of dwarf seedlings occupies a lot of land, resulting in high breeding costs, which restricts the selection of new varieties. Summary of the invention

[0004] In view of the problems existing in the prior art, the present invention provides an efficient apple breeding method based on multi-trunk grafting to increase the expression of flowering genes and the content of endogenous hormones. The method cultivates a dwarfing rootstock that can be grafted with multiple main heads, and carries out hybrid seedling grafting and cultivation. This not only significantly increases the number of seedlings cultivated per unit area of ​​land, but also effectively shortens the juvenile period, thereby significantly improving the breeding efficiency.

[0005] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides an efficient apple breeding method for increasing the expression of flowering genes and the content of endogenous hormones based on multi-trunk grafting, comprising the following steps: (1) Hybridization: In the spring of the first year, prepare the hybrid combination and collect the anthers of the male parent. When the female parent flowers reach the big bell flowering period, pollinate them and mark the pollinated trees or pollinated branches. (2) Obtaining hybrid seeds: In the year of pollination, when the seed coat turns brown and the fruit is physiologically mature, hybrid fruits are collected and the hybrid seeds are peeled off; (3) Rootstock planting and management: In mid-March of the first year, plant apple dwarf rootstock seedlings on the ridges of the support in the established nursery. Cut off the part of the apple dwarf rootstock seedlings above the full buds at 45-50 cm and start rootstock cultivation. (4) Cultivation of grafted shoots of rootstock: After the rootstock buds germinate, keep the new shoots from the two buds on the top and remove all other shoots; when the two new shoots grow to 25-30 cm, coil them and tie them to the wire; spray the pesticide and the tied shoots at the same time; keep the two new shoots and some secondary shoots, and remove the other secondary shoots; (5) Hybrid seedling cultivation: Place 1 seed per hole of stratified, germinated hybrid seeds in the seedling medium, evenly cover with fine soil, and then cover with plastic film; observe the germination of seeds every day, and after the seedlings emerge from the soil, puncture the plastic film, and then sprinkle water every day to ensure that the medium is moist. After the seedlings grow 4 true leaves, transplant them to the field for cultivation and perform field management to obtain hybrid seedlings; (6) Cultivation of multi-trunk trees of hybrid offspring: In late August of the second year, collect a full bud from the middle and upper part of the cultivated hybrid seedlings and graft it to the upper part of the second upright rootstock of the cultivated stock; graft a bud of a hybrid seedling on each shoot. Before budding in the spring of the third year, cut off the upper part of the upright shoot 0.3-0.5 cm above the grafted bud, and remove the buds on the upright shoot below the grafted bud as it grows. Then carry out fertilizer, water, disease and pest control management.

[0006] Furthermore, in step (1), the specific steps of hybridization are as follows: in the spring of the first year, a hybrid combination is prepared, anthers of the male parent are collected, dried indoors in the shade for 48 hours, and pollen is sieved; when the female parent flower reaches the big bell flowering period, petals and stamens are removed, pistils are retained, and pollen of the male parent is applied to the stigma of the female parent flower pistil, and pollination inflorescences are retained at intervals of 15-20 cm, 1-2 flowers are retained in each inflorescence for pollination, and the remaining flowers are thinned out; then, paper bags are placed on the pollinated flowers to isolate them from exogenous pollen contamination; the paper bags are removed after 72 hours of bagging; and the pollinated trees or pollinated branches are marked.

[0007] Furthermore, in step (3), the specific preparation process of the nursery is as follows: in the spring of the first year, the land is leveled; planting rows are set at a row spacing of 2.4-2.5 meters and a north-south row direction, and planting trenches are dug with a depth of 0.5 meters and a width of 0.5 meters; well-rotted organic fertilizer is applied to the planting rows at a fertilizer rate of 10,000 kg / 666.7 square meters, and the fertilizer is filled into the planting rows and mixed with the soil, and then the planting rows are backfilled and watered to make them compact; then ridges are formed on the planting rows, with a ridge height of 0.2-0.3 meters and a width of 1.0-1.2 meters; brackets are set up along the center line of the ridges and drip irrigation equipment is installed.

[0008] Furthermore, in step (3), the plant spacing of the apple dwarfing rootstock seedlings is 1.6 meters.

[0009] Furthermore, in step (3), during the rootstock cultivation process, the soil moisture content is maintained at 55-60%; the specific process of the rootstock cultivation is as follows: starting from the time when the new shoots of the apple dwarf rootstock seedlings grow to 25-30 cm, topdressing is applied once every 20 days, for a total of 3 times, with 0.05 kg of urea applied per plant each time; in August, 0.3% potassium dihydrogen phosphate solution is sprayed at intervals of 15 days, for a total of 2 times; from mid-April to mid-July, insecticides are sprayed once a month, with sulfoxaflor and spirotetramat being used alternately; starting from late June, pyraclostrobin is sprayed twice continuously at intervals of 15 days, and Bordeaux mixture is sprayed once in mid-July.

[0010] Furthermore, in step (4), the specific operation of spraying the pesticide is as follows: when the new shoots grow to 30-40 cm, spray the new shoots with 1.0 g / L 6-benzylaminopurine solution once; when the new shoots grow to 60-70 cm, spray the new shoots with 0.025 g / L cyclopropanecarboxamide solution once; the retained secondary shoots are the secondary shoots growing upright at 10-11 cm, 25-30 cm, and 45-50 cm from the main trunk.

[0011] Furthermore, in step (5), the hybrid seeds are pre-stratified, and the specific process is as follows: in the early spring of the second year, 40 days before sowing, the hybrid seeds to be bred are rinsed with clean water, soaked for 12 hours, and then mixed with clean river sand with a water content of 60% in a volume ratio of 1:3, and placed in a 4°C environment for stratification; during the stratification process, the seeds are checked every 10 days, ventilated, and water is added; 3 days before sowing, the seeds are sieved out of the sand, washed, and placed on moist gauze for germination, during which the gauze is kept moist, and the seeds are sown after germination.

[0012] Furthermore, in step (5), the seedling medium is composed of organic fertilizer with an organic matter content of 45% and fine soil in a volume ratio of 1:2; the thickness of the covering fine soil is 0.3~0.5 cm.

[0013] Furthermore, in step (6), the grafting method is a "T"-shaped bud grafting method; when the full buds in the middle and upper parts of the hybrid seedlings are grafted slightly above the second upright rootstock, the grafting position is located 15 to 20 cm from the base of the upright shoot.

[0014] The conditions for the apple dwarf rootstock seedlings used in the present invention are that they grow robustly, have complete root systems, and have normal and full buds 45 to 50 centimeters above the ground. During the nursery preparation process, the support is composed of cement poles, steel wires, ground anchors, etc. The length of the cement pole used for the support is 3.5 meters, standing vertically on the ground, with 3.0 meters exposed and 0.5 meters buried. The spacing between the cement poles is 10 meters, and steel wires are set at 0.5 meters, 1.6 meters, and 2.6 meters above the ground and fixed on the cement poles. The outer cement poles are fixed with inclined steel wires and ground anchors, and the angle between the steel wires and the ground is 70 degrees.

[0015] The present invention cultivates a dwarf rootstock capable of multi-head grafting to carry out hybrid seedling grafting and cultivation, which not only significantly increases the number of seedlings cultivated per unit land area, but also effectively shortens the juvenile period and significantly improves breeding efficiency.

[0016] Beneficial effects of the present invention: 1. It can save a lot of land. Under the same conditions, this method can cultivate 1,328 hybrid seedlings per 666.7 square meters, while the conventional method can cultivate 533 seedlings, which is 2.49 times the conventional method, and can save a lot of land resources.

[0017] 2. It can effectively shorten the breeding period. The juvenile period of hybrid seedlings using conventional methods is 7 years, while the juvenile period of hybrid seedlings using this method is 4 years, which shortens the total breeding cycle by 3 years.

[0018] 3. It can reduce the workload of evaluating the stock and scion combination for breeding new varieties. The rootstock used in this method is the same as the dwarfing rootstock used in production, so that different strains can have the same stock and scion combination as in actual production during the variety breeding stage, and can show the same results and growth conditions as in production, reducing the subsequent evaluation work of the stock and scion combination of new varieties.

[0019] 4. The operation is simple. This method for breeding hybrid offspring is easy to operate, has no special steps, and uses conventional instruments and equipment without special requirements.

[0020] 5. The raw materials are easy to obtain. The rootstock cultivation materials used are all commercially available, without special preparations, no toxic side effects, no ingredients that are detrimental to the growth of the plant itself, and are cheap and easy to obtain. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To implement the cultivation of hybrid offspring seedlings and the collection of scions of the present invention; Figure 2 To implement the method for cultivating rootstock of the present invention; Figure 3 To implement the hybrid offspring grafting and multi-trunk tree cultivation of the present invention; Figure 4 The steps for using conventional breeding methods; Figure 5 To implement the breeding method of the present invention and the expression level of flowering-related genes using conventional methods; Figure 6 To implement the breeding method of the present invention and to use conventional methods related to the content of endogenous hormones. DETAILED DESCRIPTION

[0022] The above contents of the present invention are further described in detail below in conjunction with specific examples of embodiments, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following embodiments.

[0023] Example 1 Methods for improving the efficient breeding of apple hybrid offspring (e.g. Figure 1-Figure 3 ), specifically including the following steps: (1) Implementation of orchard The method of the present invention was implemented in the apple breeding nursery of the Tianping Lake Experimental Demonstration Base of Shandong Fruit Research Institute, Daiyue District, Tai'an City, Shandong Province from 2019 to 2024. The breeding nursery area of ​​the experimental garden is 10 mu, and the dwarfing rootstock used is M9T337.

[0024] (2) Hybridization The hybrid combination "'Fuji' X 'Luli'" was configured. On April 3, 2019, the anthers of the male parent 'Luli' were collected and dried indoors for 48 hours. The pollen was sieved and stored away from wind, light and moisture. When the female parent 'Fuji' flowers reached the big bell flowering period, the petals and stamens were removed, the pistils were retained, and the male parent pollen was point-pollinated to the stigma of the female parent's pistil. The pollinated inflorescences were retained at a spacing of 20 cm, and one flower was retained in each inflorescence for pollination, and the rest of the flowers were thinned out. Then the pollinated flowers were covered with paper bags to isolate exogenous pollen contamination. The paper bags were removed 72 hours after bagging. The pollinated trees or pollinated branches were marked.

[0025] (3) Obtaining hybrid seeds Carry out soil, fertilizer and water management for pollination trees, and control diseases and pests. Collect hybrid fruits when the seed coat color turns brown and the fruit is physiologically mature in 2019, peel the hybrid seeds, wash them with clean water, dry them in the shade, and store them in a cool and ventilated place.

[0026] (4) Nursery site preparation In early March 2019, the land was leveled. The planting rows were set with a row spacing of 2.5 meters and a north-south direction, and the planting trenches were dug with a depth of 0.5 meters and a width of 0.5 meters. Decomposed organic fertilizer was applied to the planting rows at a rate of 10,000 kg / 666.7 square meters, and the fertilizer was filled into the planting rows and mixed with the soil. The planting rows were then backfilled and watered to make them solid. Ridges were then built on the planting rows, with a height of 0.2 meters and a width of 1.0 meters. Brackets were set up along the center line of the ridges. The brackets consisted of cement poles, steel wires, and ground anchors. The length of the cement poles was 3.5 meters, standing vertically on the ground, with 3.0 meters exposed and 0.5 meters buried. The spacing between the cement poles was 10 meters, and steel wires were set up at 0.5 meters, 1.6 meters, and 2.6 meters above the ground and fixed on the cement poles. The outer cement poles were fixed with inclined steel wires and ground anchors, with an angle of 70 degrees between the steel wires and the ground. Drip irrigation equipment was installed.

[0027] (5) Rootstock planting and management Select apple dwarf rootstock seedlings with healthy growth and complete root system, and the buds of the seedlings are normal and full 50 cm above the ground. In mid-March 2019, in the built nursery, dwarf rootstocks were planted along the support ridges with a spacing of 1.6 meters. Water thoroughly after planting. Cut off the part of the rootstock seedlings above 30 cm full buds. Normal water management, maintain soil moisture content at 60%. From the time the new shoots of the seedlings grow to 30 cm, topdress once every 20 days, for a total of 3 times, and apply 0.05 kg of urea per plant each time. In August, spray 0.3% potassium dihydrogen phosphate solution at an interval of 15 days, for a total of 2 times. From April 10 to July 10, spray insecticides once on the 10th of each month, alternating between sulfoxaflor and spirotetramat. Spray pyraclostrobin twice on June 15 and June 30, and spray Bordeaux mixture once on July 15 to prevent and control leaf diseases.

[0028] (6) Grafted shoot cultivation After the rootstock buds sprout, keep the new shoots of the two upper buds, and remove all other shoots. When the two retained new shoots grow to 30 cm, coil them and tie them to a steel wire 0.5 meters above the ground, so that the new shoots grow horizontally along the steel wire. When the new shoots grow to 40 cm, spray them with 1.0g / L 6-benzylaminopurine solution once, and when the new shoots grow to 70 cm, spray them with 0.025g / L cyclopropanecarboxamide solution once to promote the germination of secondary new shoots from leaf buds, and tie up the shoot heads at the same time. Keep two secondary shoots that grow upright at 10 cm, 30 cm, and 50 cm from the main trunk, and remove other secondary shoots.

[0029] (7) Hybrid seed stratification On January 15, 2020, 40 days before sowing, the hybrid seeds to be bred were rinsed with clean water, soaked for 12 hours, and then mixed with clean river sand with a water content of 60% at a volume ratio of 1:3, placed in a clean foam box, and placed in a 4°C cold storage for stratification. During the stratification process, the seeds were checked every 10 days, properly ventilated, and supplemented with appropriate amounts of water. Three days before sowing, the seeds were sieved out of the sand, washed, and placed on moist gauze for germination, and the gauze was kept moist during the period. Sowing was done after the seeds germinated.

[0030] (8) Hybrid seedling cultivation On February 25, 2020, organic fertilizer with an organic matter content of 45% was mixed with fine soil in a volume ratio of 1:2 as a seedling medium. The matrix was loaded into the seedling tray and the bottom water was irrigated. The hybrid seeds that had been stratified and germinated were placed on the tray with the matrix loaded, with 1 seed per hole, and 0.3 cm of fine soil was evenly covered on it, and then covered with mulch to keep it moist. Observe the germination of the seeds every day. After the seedlings emerge from the soil, puncture the mulch, and then sprinkle water every day to ensure that the matrix is ​​moist. After the seedlings emerge from the soil, spray the fungicide once. After the seedlings grow 4 true leaves, transplant them to the field for cultivation. The field management method is the same as the above rootstock seedling cultivation.

[0031] (9) Cultivation of multi-trunk trees of hybrid offspring In late August 2020, a full bud was collected from the middle and upper part of the hybrid seedlings, and grafted to the upper part of the cultivated rootstock using the "T" bud grafting method. The grafting site was 20 cm from the base of the upright shoot. Each shoot was grafted with a bud of a seedling. Before budding in mid-March 2021, the upper part of the upright shoot was cut off 0.5 cm above the grafted bud, and the buds on the upright shoot below the grafted bud were removed as they grew. Then, fertilizer, water, disease and pest control management were carried out, and the methods were the same as those for the cultivation of the rootstock seedlings above. All grafted strains will bear fruit in 2023.

[0032] Effect Example The conventional method used in apple breeding is: in the first year, the same hybrid combination 'Fuji' x 'Luli' is prepared. After obtaining hybrid seeds, stratification, sowing and seedling raising are carried out in the second year. The cultivated seedlings are directly planted in the field with a row spacing of 2.5 meters and a plant spacing of 0.5 meters. In the third year, only about 1% of the plants will bloom and bear fruit. In the fifth year, about 50% of the plants will bloom and bear fruit. In the seventh year, all the plants will bloom and bear fruit (such as Figure 4 As shown in the figure), the method for cultivating apple hybrid offspring provided by Example 1 of the present invention is compared with the conventional method, and the results are as follows: (1) After implementing this method, compared with the conventional method, the number of hybrid strains cultivated per 666.7 square meters of land by this method is 2.49 times that of the conventional method. Cultivating the same hybrid seedlings can greatly save land resources (Table 1). Conventional method: Hybrid seedlings are directly planted, with a row spacing of 2.5 meters and a plant spacing of 0.5 meters.

[0033] (2) After the implementation of this method, compared with the conventional method, the juvenile period of hybrid seedlings of this method was 4 years, while the juvenile period of hybrid seedlings of the conventional method was 7 years, and the total breeding cycle was shortened by 3 years (Table 1).

[0034] Table 1 (3) After the implementation of this method, compared with the conventional method, in 2022, which was the third year after the conventional method was used to plant the hybrid seedlings and the second year after the grafting by this method, the terminal buds of the same strains propagated by the two methods were collected in mid-July when the new shoots stopped growing, and the expression levels of flowering-related genes and the contents of related endogenous hormones were determined. This method significantly promoted the expression of flowering-related genes in the hybrid seedlings. MdSOC1 (MD07G1123600), MdFT1 (MD12G1262000), LqCy (MD06G1129500), MdAP1 (MD16G1058500) up-regulated expression ( Figure 5 ), and reduce auxin (IAA) and gibberellins (GA 1+3 , GA 4+7 ) and other flowering-inhibiting hormones, and promotes the synthesis of flowering-inducing hormones zeatin (ZR) and abscisic acid (ABA) ( Figure 6 ), which has a significant promoting effect on early flowering and fruiting of hybrid seedlings.

Claims

1. An efficient apple breeding method for increasing the expression of flowering genes and the content of endogenous hormones based on multi-trunk grafting, characterized in that: The following steps are involved: (1) Hybridization: In the spring of the first year, prepare the hybrid combination and collect the anthers of the male parent. When the female parent flowers reach the big bell flowering period, pollinate them and mark the pollinated trees or pollinated branches. (2) Obtaining hybrid seeds: In the year of pollination, when the seed coat turns brown and the fruit is physiologically mature, the hybrid fruits are collected and the hybrid seeds are peeled off; (3) Rootstock planting and management: In mid-March of the first year, plant apple dwarf rootstock seedlings on the ridges of the support in the established nursery. Cut off the part of the apple dwarf rootstock seedlings above the full buds at 45-50 cm and start rootstock cultivation. (4) Cultivation of grafted shoots of rootstock: After the rootstock buds germinate, keep the new shoots from the two buds on the top and remove all other shoots; when the two new shoots grow to 25-30 cm, coil them and tie them to the wire; spray the pesticide and the tied shoots at the same time; keep the two new shoots and some secondary shoots, and remove the other secondary shoots; (5) Hybrid seedling cultivation: Place 1 seed per hole of stratified, germinated hybrid seeds in the seedling medium, evenly cover with fine soil, and then cover with plastic film; observe the germination of seeds every day, and after the seedlings emerge from the soil, puncture the plastic film, and then sprinkle water every day to ensure that the medium is moist. After the seedlings grow 4 true leaves, transplant them to the field for cultivation and perform field management to obtain hybrid seedlings; (6) Cultivation of multi-trunk trees of hybrid offspring: In late August of the second year, collect a full bud from the middle and upper part of the cultivated hybrid seedlings and graft it to the upper part of the second upright rootstock of the cultivated stock; graft a bud of a hybrid seedling on each shoot. Before budding in the spring of the third year, cut off the upper part of the upright shoot 0.3-0.5 cm above the grafted bud, and remove the buds on the upright shoot below the grafted bud as it grows. Then carry out fertilizer, water, disease and pest control management.

2. The efficient apple breeding method according to claim 1, characterized in that: In step (1), the specific steps of hybridization are as follows: in the spring of the first year, a hybrid combination is prepared, anthers of the male parent are collected, dried indoors in the shade for 48 hours, and pollen is sieved; when the female parent flower reaches the big bell flowering period, petals and stamens are removed, pistils are retained, and pollen of the male parent is applied to the stigma of the female parent flower pistil, and pollination inflorescences are retained at intervals of 15-20 cm, 1-2 flowers are retained in each inflorescence for pollination, and the remaining flowers are thinned out; then, paper bags are placed on the pollinated flowers to isolate them from exogenous pollen contamination; the paper bags are removed after 72 hours of bagging; and the pollinated trees or pollinated branches are marked.

3. The efficient apple breeding method according to claim 1 or 2, characterized in that: In step (3), the specific preparation process of the nursery is as follows: in the spring of the first year, the land is leveled; planting rows are set at a row spacing of 2.4-2.5 meters and a north-south row direction, and planting trenches are dug with a depth of 0.5 meters and a width of 0.5 meters; decomposed organic fertilizer is applied to the planting rows at a fertilizer rate of 10,000 kg / 666.7 square meters, and the fertilizer is filled into the planting rows and mixed with the soil, and then the planting rows are backfilled and watered to make them solid; then ridges are formed on the planting rows, with a ridge height of 0.2-0.3 meters and a width of 1.0-1.2 meters; brackets are set up along the center line of the ridges and drip irrigation equipment is installed.

4. The efficient apple breeding method according to claim 1 or 3, characterized in that: In step (3), the plant spacing of the apple dwarfing rootstock seedlings is 1.6 meters.

5. The efficient apple breeding method according to claim 1 or 4, characterized in that: In step (3), during the rootstock cultivation process, the soil moisture content is maintained at 55-60%; the specific process of the rootstock cultivation is as follows: starting from the time when the new shoots of the apple dwarf rootstock seedlings grow to 25-30 cm, topdressing is applied once every 20 days, for a total of 3 times, with 0.05 kg of urea applied per plant each time; in August, 0.3% potassium dihydrogen phosphate solution is sprayed at intervals of 15 days, for a total of 2 times; from mid-April to mid-July, insecticides are sprayed once a month, with sulfoxaflor and spirotetramat being used alternately; starting from late June, pyraclostrobin is sprayed twice continuously at intervals of 15 days, and Bordeaux mixture is sprayed once in mid-July.

6. The efficient apple breeding method according to claim 1 or 5, characterized in that: In step (4), the specific operation of spraying the pesticide is as follows: when the new shoots grow to 30-40 cm, spray the new shoots with 1.0 g / L 6-benzylaminopurine solution once; when the new shoots grow to 60-70 cm, spray the new shoots with 0.025 g / L cyclopropanecarboxamide solution once; the retained secondary shoots are the secondary shoots growing upright at 10-11 cm, 25-30 cm, and 45-50 cm from the main trunk.

7. The efficient apple breeding method according to claim 1 or 6, characterized in that: In step (5), the hybrid seeds are pre-stratified, and the specific process is as follows: in the early spring of the second year, 40 days before sowing, the hybrid seeds to be bred are rinsed with clean water, soaked for 12 hours, and then mixed with clean river sand with a water content of 60% in a volume ratio of 1:3, and placed in a 4°C environment for stratification; during the stratification process, the seeds are checked every 10 days, ventilated, and water is added; 3 days before sowing, the seeds are sieved out of the sand, washed, and placed on moist gauze for germination, during which the gauze is kept not dry, and the seeds are sown after germination.

8. The efficient apple breeding method according to claim 1 or 7, characterized in that: In step (5), the seedling medium is composed of organic fertilizer with an organic matter content of 45% and fine soil in a volume ratio of 1:2; the thickness of the covering fine soil is 0.3~0.5 cm.

9. The efficient apple breeding method according to any one of claims 1 to 8, characterized in that: In step (6), the grafting method is a "T"-shaped bud grafting method; when the full buds in the middle and upper parts of the hybrid seedlings are grafted slightly above the second upright rootstock, the grafting position is located 15 to 20 cm from the base of the upright shoot.

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