Apple asexual propagation seedling raising method

Abstract

The invention relates to the technical field of plant propagation, and discloses an apple asexual propagation seedling raising method which comprises the following steps: taking 10-15cm annual semi-lignified branches, reserving 2-3 leaves on the tops, soaking the base parts of the branches in 0.1%-0.2% carbendazim for 10-15 minutes, and cleaning; preparing a specific culture medium with the pH value of 5.8-6.0, inserting the culture medium into the base part of the branch, culturing for 2-3 weeks under the conditions of 22-25 DEG C, 1500-2000lx illumination and 12-14 hours per day, and inducing the callus. The method has the advantages that stable and excellent genetic information is provided for vegetative propagation from the source, branches which are high in propagation activity and capable of maintaining self growth are obtained, an internal nutrition environment beneficial to growth and propagation is created for the branches, and normal growth and development of the branches are prevented from being affected by pathogenic bacteria. The problem that excellent characters of female parents cannot be stably kept is solved.

Description

Technical Field

[0001] The invention relates to the technical field of plant propagation, in particular to an apple asexual propagation seedling raising method. Background Art

[0002] In the field of plant propagation technology, apple seedling cultivation is an important basic link in the development of the apple industry. At present, there are many problems in traditional apple seedling cultivation methods that are difficult to ignore. The phenomenon of trait separation of seedling offspring propagated by seeds is serious, which greatly destroys the stability of the excellent traits of the mother parent, resulting in uneven fruit quality, yield and stress resistance, and cannot meet the market demand for consistency in apple quality. The commonly used asexual propagation methods, such as grafting, can maintain the characteristics of the mother parent to a certain extent, but the grafting operation has high technical requirements. The survival rate after grafting is affected by many factors such as grafting time, affinity between rootstock and scion, environmental conditions, etc., and the growth cycle is long. It takes a long time from grafting to seedling, which greatly increases the cost of seedling cultivation. Cutting propagation is also difficult in apple seedling cultivation. Rooting is difficult, and auxiliary means such as rooting agents are often required. The rooting rate of cuttings of different varieties of apples varies significantly, and the overall rooting rate is low, which is difficult to apply on a large scale in actual production. These problems have seriously restricted the efficient and stable development of the apple seedling industry, and innovative seedling cultivation technology is urgently needed to break through the difficulties. Summary of the invention

[0003] In view of the shortcomings of the prior art, the present invention provides an apple asexual propagation seedling raising method, which solves the problems existing in the prior apple seedling raising method, such as the inability to efficiently and stably maintain the excellent traits of the mother plant, the low survival rate, the long growth cycle and the high cost.

[0004] To achieve the above objectives, the present invention is implemented through the following technical scheme: a method for asexual propagation and seedling raising of apples, comprising the following steps: S1. Mother plant selection and pretreatment: Select the best mother plant, take 10-15cm one-year-old semi-lignified branches, leave 2-3 leaves on the top, soak the base of the branches with 0.1%-0.2% carbendazim for 10-15 minutes and then wash; S2. Callus induction: Prepare a specific culture medium with pH 5.8-6.0, insert it into the base of the branch, and culture it for 2-3 weeks at 22-25°C, 1500-2000lx light, 12-14 hours / day to induce callus; S3, adventitious bud differentiation: When the callus is 0.5-1.0 cm, transfer to the corresponding culture medium with pH 5.8-6.0, and culture for 3-4 weeks at 23-25°C, 2000-2500lx light, 14-16 hours / day to differentiate adventitious buds; S4, rooting culture: select 3-5cm adventitious buds, transfer to rooting medium pH 5.8-6.0, use yeast fermentation to provide carbon, and culture for 2-3 weeks at 20-22℃, 1000-1500lx light, 10-12 hours / day for rooting; S5. Hardening and transplanting: Soak the small plants in medicine for 10-15 minutes and then plant them in nutrient soil seedling pots. Harden them in a greenhouse at 18-22℃ and 70%-80% humidity for 1-2 weeks. Transplant them into the field after strengthening light and ventilation.

[0005] Preferably, the S1 specifically includes: S101: Select adult apple plants that are healthy, free of pests and diseases, have excellent traits, good fruit quality, high yield, and strong stress resistance as mother plants; S102: Select one-year-old, semi-lignified branches from the mother plant, with a length of 10-15 cm, retain 2-3 leaves on the top of the branches, and remove the remaining leaves; S103: Soak the base of the selected branches in a solution containing 0.1%-0.2% carbendazim for 10-15 minutes for disinfection, and then rinse with clean water.

[0006] Preferably, S2 specifically includes: S201: preparing a callus induction medium, the components of which include MS basic medium, 1.0-2.0 mg / L 6-benzylaminoadenine, 0.5-1.0 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, and adjusting the pH value to 5.8-6.0; S202: inserting the disinfected branch base into callus induction medium, culturing for 2-3 weeks under the conditions of temperature of 22-25°C, light intensity of 1500-2000lx, and light time of 12-14 hours / day, to induce callus formation at the branch base.

[0007] Preferably, S3 specifically includes: S301. When the callus grows to a certain size with a diameter of about 0.5-1.0 cm, transfer it to an adventitious bud differentiation medium, wherein the adventitious bud differentiation medium comprises MS basic medium, 1.5-2.5 mg / L kinetin, 0.3-0.5 mg / L indolebutyric acid, 30 g / L sucrose and 7 g / L agar, and the pH value is adjusted to 5.8-6.0; S302. Cultivate for 3-4 weeks at a temperature of 23-25°C, a light intensity of 2000-2500 lx, and a light duration of 14-16 hours / day to promote callus differentiation into adventitious buds.

[0008] Preferably, S4 specifically includes: S401. Select healthy adventitious buds with a height of 3-5 cm and transfer them to a rooting medium. The rooting medium includes 1 / 2MS basic medium, 0.5-1.0 mg / L indole-3-acetic acid, 0.2-0.3 mg / L rooting powder, 20 g / L glucose, 2 g / L yeast powder and 6 g / L vermiculite. The pH value is adjusted to 5.8-6.0. Yeast ferments glucose into ethanol and carbon dioxide under anaerobic conditions. The chemical equation is: C6H 12 O6+yeast→2C2H5OH+2CO2↑Carbon dioxide provides carbon for plant photosynthesis; S402. Cultivate for 2-3 weeks at a temperature of 20-22°C, a light intensity of 1000-1500 lx, and a light duration of 10-12 hours / day to allow the adventitious buds to take root and form complete plantlets.

[0009] Preferably, the S5 specifically includes: S501, taking the rooted plantlets out of the culture bottle, and soaking them in a solution containing 0.1%-0.2% carbendazim and 0.2%-0.3% potassium dihydrogen phosphate for 10-15 minutes; S502. Transplant the plantlets into a seedling pot filled with nutrient soil, wherein the ratio of leaf mold: perlite: vermiculite is 3:1:1, and harden the seedlings in a greenhouse at a temperature of 18-22°C and a humidity of 70%-80%. After hardening the seedlings for 1-2 weeks, gradually increase the light intensity and ventilation. When the plantlets grow strong, they can be transplanted into the field.

[0010] Preferably, S1 further comprises: after soaking the branches in carbendazim, spraying Bacillus subtilis at a concentration of 1×10^8 CFU / ML to inhibit root rot. At the same time, fumigating the soil in the garden where the mother plant is located to reduce the base number of nematodes and fungi.

[0011] Preferably, S5 further comprises: before transplanting, immersing the roots of the tissue culture seedlings in a mixture of 100 mg / L humic acid and 0.1% seaweed extract for 10 minutes to promote the development of capillary roots and enhance drought resistance. During the seedling hardening stage, gradually increase UV-B light for 2 hours per day to stimulate the plants to synthesize secondary metabolites for adaptation to the field.

[0012] The present invention provides a method for asexual propagation and seedling raising of apples, which has the following beneficial effects: 1. The present invention provides stable and excellent genetic information for asexual reproduction from the source, obtains branches with high reproductive activity and capable of maintaining their own growth, creates an internal nutritional environment conducive to growth and reproduction, and creates a relatively sterile environment for branch reproduction to avoid pathogens from invading and affecting the normal growth and development of branches. The problem of being unable to stably maintain the excellent traits of the mother plant is solved.

[0013] 2. The present invention achieves the effect of creating a suitable nutritional and physical and chemical environment for the growth, division and differentiation of cells at the base of the branch by adjusting the culture medium components and controlling the pH value, thereby promoting cell dedifferentiation to form callus tissue. This solves the problem that in the traditional seedling raising process, branches are difficult to induce to form callus tissue due to insufficient or unbalanced nutrient supply, lack of growth regulating substances and unsuitable culture medium environment.

[0014] 3. The present invention provides suitable culture medium components and environment to create favorable conditions for callus differentiation into adventitious buds, promote callus differentiation into buds, efficiently and stably promote callus differentiation into adventitious buds, and increase the quantity and quality of adventitious buds. The problem of low survival rate due to poor control of culture environment conditions is solved.

[0015] 4. The present invention provides a rooting medium with specific ingredients to provide a suitable nutritional, physical and chemical environment for adventitious buds to take root, promotes rapid and healthy rooting of adventitious buds, enables adventitious buds to take root efficiently and develop into complete plantlets, improves the survival rate of plantlets and shortens the growth cycle. This solves the problem of long growth cycle in the prior art.

[0016] 5. The present invention sterilizes the plantlets and supplements them with phosphorus and potassium nutrients to enhance the stress resistance of the plantlets, help the plantlets gradually adapt to the external environment, and improve the survival rate and growth cycle after transplanting them to the field. Since the survival rate is improved and the growth cycle is shortened, the manpower, material and time costs in the seedling raising process are reduced. At the same time, the composition of the culture medium is relatively simple and the cost is low, which is conducive to large-scale apple seedling production. The problem of high cost of use is solved. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The present invention provides a flow chart of an apple asexual propagation and seedling raising method. DETAILED DESCRIPTION

[0018] The technical solution of the present invention will be described clearly and completely below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0019] Please see attached Figure 1 The embodiment of the present invention provides a method for asexual propagation and seedling raising of apples, comprising the following steps: S1. Mother plant selection and pretreatment: Select the best mother plant, take 10-15cm one-year-old semi-lignified branches, leave 2-3 leaves on the top, soak the base of the branches with 0.1%-0.2% carbendazim for 10-15 minutes and then wash; S2. Callus induction: Prepare a specific culture medium with pH 5.8-6.0, insert it into the base of the branch, and culture it for 2-3 weeks at 22-25°C, 1500-2000lx light, 12-14 hours / day to induce callus; S3, adventitious bud differentiation: When the callus is 0.5-1.0 cm, transfer to the corresponding culture medium with pH 5.8-6.0, and culture for 3-4 weeks at 23-25°C, 2000-2500lx light, 14-16 hours / day to differentiate adventitious buds; S4, rooting culture: select 3-5cm adventitious buds, transfer to rooting medium pH 5.8-6.0, use yeast fermentation to provide carbon, and culture for 2-3 weeks at 20-22℃, 1000-1500lx light, 10-12 hours / day for rooting; S5. Hardening and transplanting: Soak the small plants in medicine for 10-15 minutes and then plant them in nutrient soil seedling pots. Harden them in a greenhouse at 18-22℃ and 70%-80% humidity for 1-2 weeks. Transplant them into the field after strengthening light and ventilation.

[0020] S1 specifically includes: S101: Select adult apple plants that are healthy, free of pests and diseases, have excellent traits, good fruit quality, high yield, and strong stress resistance as mother plants; S102: Select one-year-old, semi-lignified branches from the mother plant, with a length of 10-15 cm, retain 2-3 leaves on the top of the branches, and remove the remaining leaves; S103: Soak the base of the selected branches in a solution containing 0.1%-0.2% carbendazim for 10-15 minutes for disinfection, and then rinse with clean water.

[0021] Specifically, the operation of selecting adult apple plants that are healthy, pest-free, have good fruit quality, high yield and strong stress resistance as mother plants is based on genetic theory. The characteristics of organisms are determined by genes, and excellent mother plants carry high-quality gene combinations. Using such plants as mother plants can ensure that the offspring will inherit these excellent genes to the greatest extent during the subsequent reproduction process.

[0022] Select one-year-old, semi-lignified branches with a length of 10-15cm from the mother plant, and keep 2-3 leaves on the top, and remove the rest. From the perspective of plant physiology, one-year-old semi-lignified branches have active cell division and differentiation capabilities, vigorous physiological activities inside, contain more growth hormones and nutrients, and are easier to induce callus formation and rooting, laying a good foundation for subsequent reproduction. A small number of leaves on the top are retained because leaves are the main place for plants to photosynthesize, and can use light energy to convert carbon dioxide and water into organic matter and oxygen, providing the branches with the energy and substances needed for growth; removing excess leaves can reduce water loss and nutrient consumption, and avoid affecting branch growth due to nutrient competition and excessive evaporation of water.

[0023] Soak the base of the selected branches in a solution containing 0.1%-0.2% carbendazim for 10-15 minutes, then rinse with clean water. Carbendazim is a highly effective, broad-spectrum fungicide that works by interfering with the mitosis process of fungal cells and inhibiting the growth and reproduction of pathogens. During the branch cutting process, the wound at the base is easily infected by pathogens. Soaking and disinfecting with carbendazim solution can effectively kill the pathogens on the branch surface and around the wound, reducing the risk of bacterial infection.

[0024] By providing stable and excellent genetic information for asexual reproduction from the source, and obtaining branches with high reproductive activity and capable of maintaining their own growth, and creating an internal nutritional environment conducive to growth and reproduction for them, and creating a relatively sterile environment for branch reproduction, to avoid pathogens from invading and affecting the normal growth and development of branches. The problem of being unable to stably maintain the excellent traits of the mother plant is solved, ensuring that the subsequent reproduction process has a reliable genetic basis to maintain the advantages of fruit quality, yield and stress resistance.

[0025] S2 specifically includes: S201: preparing a callus induction medium, the components of which include MS basic medium, 1.0-2.0 mg / L 6-benzylaminoadenine, 0.5-1.0 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, and adjusting the pH value to 5.8-6.0; S202: inserting the disinfected branch base into callus induction medium, culturing for 2-3 weeks under the conditions of temperature of 22-25°C, light intensity of 1500-2000lx, and light time of 12-14 hours / day, to induce callus formation at the branch base.

[0026] Specifically, the MS basic medium provides a large number of elements such as nitrogen, phosphorus, and potassium, as well as trace elements such as iron and zinc for the growth of plant tissues, meeting the basic needs of branch cell growth and metabolism. 6-Benzylaminoadenine (6-BA) and naphthylacetic acid are plant growth regulators. 6-BA can promote cell division and induce bud differentiation and formation; naphthylacetic acid plays a role in promoting cell elongation and inducing rooting. The two synergistically regulate the cell division, growth and differentiation process. Sucrose, as a carbon source, provides energy for cell respiration and maintains the osmotic pressure of the culture medium; agar is a coagulant that makes the culture medium solid, provides support for the branches, and facilitates their growth in a stable environment. The pH value is adjusted to 5.8-6.0 because within this pH range, the solubility and effectiveness of various nutrients are optimal, which is conducive to the absorption and utilization of nutrients by plant cells.

[0027] Under the temperature environment of 22-25℃, the activities of various enzymes in the cells are in a more suitable range, which can efficiently catalyze the biochemical reactions in the cells and ensure the normal growth and metabolic activities of the cells. The light intensity is 1500-2000lx and the light time is 12-14 hours / day. Such light conditions can not only meet the photosynthesis needs of the cells at the base of the branches, synthesize organic matter and energy, but also will not damage the cells due to excessive light or long time. Cultivated in this environment for 2-3 weeks, the cells at the base of the branches are given sufficient time to dedifferentiate and form callus tissue.

[0028] By adjusting the composition of the culture medium and controlling the pH value, the effect of creating a suitable nutritional and physical and chemical environment for the growth, division and differentiation of the cells at the base of the branches is achieved, which promotes the dedifferentiation of cells to form callus tissue. This solves the problem of difficulty in inducing the formation of callus tissue in branches due to insufficient or unbalanced nutrient supply, lack of growth regulators and unsuitable culture medium environment in the traditional seedling raising process.

[0029] S3 specifically includes: S301. When the callus grows to a certain size with a diameter of about 0.5-1.0 cm, transfer it to an adventitious bud differentiation medium, wherein the adventitious bud differentiation medium comprises MS basic medium, 1.5-2.5 mg / L kinetin, 0.3-0.5 mg / L indolebutyric acid, 30 g / L sucrose and 7 g / L agar, and the pH value is adjusted to 5.8-6.0; S302. Cultivate for 3-4 weeks at a temperature of 23-25°C, a light intensity of 2000-2500 lx, and a light duration of 14-16 hours / day to promote callus differentiation into adventitious buds.

[0030] Specifically, when the diameter of the callus grows to 0.5-1.0cm, its cell number and metabolic level reach a certain level, and it has the basis for differentiation. MS basic medium provides the macroelements, trace elements and organic components required for cell growth and differentiation, and is the basic material source for cell growth. Kinetin can promote cell division and differentiation, especially induce the formation of buds, which can break the dormant state of cells and stimulate the differentiation potential of cells. Indolebutyric acid can promote the elongation and division of cells, and synergize with kinetin to regulate the growth and differentiation direction of cells, which is conducive to the differentiation of adventitious buds. Sucrose, as a carbon source and energy source, provides energy for the metabolism and differentiation of cells, while maintaining the osmotic pressure of the culture medium. Agar solidifies the culture medium and provides a stable growth support environment for callus. Adjusting the pH value to 5.8-6.0 can ensure the stability and effectiveness of each nutrient in the culture medium, which is conducive to the absorption and utilization of nutrients by cells.

[0031] The temperature is controlled at 23-25℃, which is the suitable temperature for various enzymes in plant cells to be active, which can ensure the efficient biochemical reactions in cells and provide the necessary material and energy metabolism basis for the differentiation of adventitious buds. The light intensity is 2000-2500lx and the light time is 14-16 hours / day. The appropriate light intensity and time can promote photosynthesis and provide cells with sufficient photosynthetic products. At the same time, light can also serve as a signal to regulate the distribution and balance of hormones in plants, affect cell differentiation and development, and promote callus differentiation into adventitious buds. Cultivated under such conditions for 3-4 weeks, the cells were given enough time to complete the differentiation process.

[0032] By providing appropriate culture medium components and environment, we can create good conditions for callus differentiation into adventitious buds, promote callus differentiation into buds, efficiently and stably promote callus differentiation into adventitious buds, and increase the quantity and quality of adventitious buds. This solves the problem of long adventitious bud differentiation time and low survival rate due to poor control of culture environment conditions.

[0033] S4 specifically includes: S401. Select healthy adventitious buds with a height of 3-5 cm and transfer them to a rooting medium. The rooting medium includes 1 / 2MS basic medium, 0.5-1.0 mg / L indole-3-acetic acid, 0.2-0.3 mg / L rooting powder, 20 g / L glucose, 2 g / L yeast powder and 6 g / L vermiculite. The pH value is adjusted to 5.8-6.0. Yeast ferments glucose into ethanol and carbon dioxide under anaerobic conditions. The chemical equation is: C6H 12 O6+yeast→2C2H5OH+2CO2↑Carbon dioxide provides carbon for plant photosynthesis; S402. Cultivate for 2-3 weeks at a temperature of 20-22°C, a light intensity of 1000-1500 lx, and a light duration of 10-12 hours / day to allow the adventitious buds to take root and form complete plantlets.

[0034] Specifically, the adventitious buds with a growth of 3-5 cm are selected because the adventitious buds at this stage are in good physiological state and have strong growth and differentiation potential. Compared with the MS basic medium, the 1 / 2MS basic medium reduces the concentration of a large number of elements and is more suitable for the nutritional needs of the adventitious bud rooting stage, avoiding the inhibition of rooting by high-concentration nutrients. Indole-3-acetic acid is a natural plant growth hormone that can promote cell elongation, division and differentiation. During the rooting process of adventitious buds, it can stimulate the formation of root primordia and the growth of the root system. Rooting powder (ABT) contains a variety of active ingredients that promote rooting, which synergize with indole-3-acetic acid to further enhance the rooting effect. Glucose provides an energy source for the growth and rooting of adventitious buds, and is also a substrate for yeast fermentation. Yeast ferments glucose into ethanol and carbon dioxide under anaerobic conditions. Carbon dioxide is used by adventitious buds as a carbon source for photosynthesis, and synthesizes more organic matter through photosynthesis, providing a material basis for rooting. Yeast powder is rich in nutrients such as amino acids, vitamins and minerals, which can provide additional nutritional support for adventitious bud rooting. Vermiculite has good air permeability and water retention, which can create a suitable physical environment for the growth of adventitious bud roots. Adjusting the pH value to 5.8-6.0 can ensure the effectiveness and stability of various nutrients in the culture medium, which is conducive to the absorption of nutrients by adventitious buds.

[0035] The temperature is controlled at 20-22℃ because within this temperature range, the various enzyme activities involved in the rooting process in the adventitious bud cells are high, which can effectively catalyze biochemical reactions related to rooting, such as cell division, hormone synthesis and transportation, and provide the necessary substances and energy for rooting. The light intensity is 1000-1500lx and the light time is 10-12 hours / day. Such lighting conditions can not only meet the photosynthesis needs of the adventitious buds, so that they can produce enough energy and organic matter for rooting through photosynthesis, but also will not cause excessive evaporation of water and physiological disorders of the adventitious buds due to excessive light or too long time. Cultivate in such an environment for 2-3 weeks, giving the adventitious buds enough time to complete the rooting process, so that they can develop from adventitious buds into small plants with complete root systems.

[0036] By providing a rooting medium with specific ingredients, a suitable nutritional, physical and chemical environment is provided for the rooting of adventitious buds, the adventitious buds are promoted to take root quickly and healthily, the adventitious buds take root efficiently and develop into complete small plants, the survival rate of the small plants is increased and the growth cycle is shortened. The problem of a long growth cycle caused by an unsuitable culture environment in the prior art is solved.

[0037] S5 specifically includes: S501, taking the rooted plantlets out of the culture bottle, and soaking them in a solution containing 0.1%-0.2% carbendazim and 0.2%-0.3% potassium dihydrogen phosphate for 10-15 minutes; S502. Transplant the plantlets into a seedling pot filled with nutrient soil, wherein the ratio of leaf mold: perlite: vermiculite is 3:1:1, and harden the seedlings in a greenhouse at a temperature of 18-22°C and a humidity of 70%-80%. After hardening the seedlings for 1-2 weeks, gradually increase the light intensity and ventilation. When the plantlets grow strong, they can be transplanted into the field.

[0038] Specifically, when the rooted plantlets are taken out of the culture bottle, their roots and plant surfaces may carry microorganisms in the culture bottle, which may cause diseases in the new environment. Carbendazim is a broad-spectrum fungicide that can bind to tubulin in fungal cells and inhibit the mitosis of fungal cells, thereby effectively killing pathogens attached to the surface and roots of the plantlets and reducing the risk of pathogens invading the plantlets. Potassium dihydrogen phosphate is rich in phosphorus and potassium. Phosphorus is essential for the growth and development of plant roots, which can promote the division and elongation of root cells and enhance the absorption capacity of the roots; potassium participates in the activation process of various enzymes in plants, helps regulate cell osmotic pressure, and enhances the stress resistance of plants. Soak the plantlets in a solution containing 0.1%-0.2% carbendazim and 0.2%-0.3% potassium dihydrogen phosphate for 10-15 minutes, so that carbendazim and potassium dihydrogen phosphate can fully act on the plantlets.

[0039] Leaf mold is rich in humus, which can provide rich organic nutrients for small plants, improve soil structure, and enhance the soil's ability to retain fertilizer and water; perlite has a loose texture and can increase the air permeability of the soil; vermiculite also has good air permeability and water retention. The three are mixed in a 3:1:1 ratio to form a nutrient soil, which creates a growth matrix environment with sufficient nutrients and good air permeability and water retention for small plants. Hardening the seedlings in a greenhouse at 18-22℃ and a humidity of 70%-80%. This temperature and humidity range is close to the growth environment of small plants in culture bottles, which can reduce the stress response of small plants caused by sudden changes in the environment and gradually adapt them to the external environment. After 1-2 weeks of hardening the seedlings, gradually increase the light intensity and ventilation. Light is a necessary condition for plants to photosynthesize. Moderately increasing the light intensity can promote photosynthesis of small plants, synthesize more organic matter, and is beneficial to plant growth; ventilation can regulate the gas composition in the greenhouse, increase the oxygen content, and discharge waste gases such as carbon dioxide, providing a good gas exchange environment for small plants and promoting healthy growth of plants. When the small plants grow strong, they have stronger adaptability and can better adapt to the field environment when transplanted to the field.

[0040] By sterilizing the plantlets and supplementing them with phosphorus and potassium nutrients, the plant's stress resistance is enhanced, helping the plantlets gradually adapt to the external environment, and improving their survival rate and growth cycle after transplanting to the field. Due to the improved survival rate and shortened growth cycle, the manpower, material and time costs in the seedling raising process are reduced. At the same time, the composition of the culture medium is relatively simple and the cost is low, which is conducive to large-scale apple seedling production. The problem of high cost of use is solved.

[0041] S1 also includes: after soaking the branches in carbendazim, spraying Bacillus subtilis with a concentration of 1×10^8 CFU / ML to inhibit root rot. At the same time, fumigating the soil in the park where the mother plant is located to reduce the base number of nematodes and fungi.

[0042] Specifically, Bacillus subtilis is a beneficial microorganism that has a strong competitive advantage at a concentration of 1×10^8 CFU / ML. After the branches are soaked in carbendazim and sprayed with Bacillus subtilis, it will quickly colonize on the surface of the branches. On the one hand, Bacillus subtilis inhibits the growth and reproduction of root rot bacteria by secreting metabolites such as antibiotics and bacteriocins. These substances can destroy the cell walls and cell membranes of root rot bacteria or interfere with their metabolic processes, making them unable to grow normally. On the other hand, Bacillus subtilis competes with root rot bacteria for nutrients and living space, and occupies an advantageous position by virtue of its strong reproductive ability and efficient use of nutrients, thereby reducing the survival chances of root rot bacteria on branches.

[0043] The soil of the garden where the mother plant is located is fumigated, and the fumigants commonly used will evaporate and diffuse in the soil. The active ingredients of the fumigants can penetrate into the gaps between soil particles and come into contact with nematodes and fungi. For nematodes, fumigants can destroy their cell structure and physiological functions, interfere with their nervous system, respiratory system, etc., and cause the death of nematodes. For fungi, fumigants can inhibit their spore germination, hyphae growth and reproduction, and inactivate key biological molecules in fungal cells, such as DNA and proteins. In this way, the base number of nematodes and fungi in the soil is reduced.

[0044] By establishing a beneficial microbial defense line on the surface of the branches, the occurrence of root rot is effectively inhibited, and the number of nematodes and fungi in the soil of the mother plant garden is reduced, reducing the risk of mother plants being infected by pests and diseases. The problem of poor growth of seedlings in the process of asexual reproduction of apples is solved.

[0045] S5 also includes: Before transplanting, the roots of the tissue culture seedlings are immersed in a mixture of 100 mg / L humic acid and 0.1% seaweed extract for 10 minutes to promote the development of capillary roots and enhance drought resistance. During the seedling hardening stage, UV-B light is gradually increased to 2 hours per day to stimulate the plants to synthesize secondary metabolites for adaptation to the field.

[0046] Specifically, humic acid contains abundant active functional groups, such as carboxyl, phenolic hydroxyl, etc. When the roots of tissue culture seedlings are immersed in 100 mg / L humic acid solution, these active functional groups can exchange and adsorb with ions on the surface of root cells, provide a variety of nutrients for the roots, stimulate the division and elongation of root cells, and thus promote the development of capillary roots. At the same time, humic acid can also improve soil structure, enhance the soil's ability to retain water and fertilizer, and indirectly create a good environment for root growth. Seaweed extract contains a large amount of plant growth regulators, such as auxin, cytokinin and gibberellin, as well as a variety of minerals and organic compounds. 0.1% seaweed extract mixture can provide comprehensive nutrition for tissue culture seedlings, in which plant growth regulators can regulate the growth and development of the root system, promote the differentiation and proliferation of root cells, and further stimulate the formation of capillary roots. In addition, some components in seaweed extract can also enhance the osmotic pressure regulation ability of plant cells, so that plants can better retain water under drought conditions, thereby enhancing drought resistance.

[0047] During the seedling hardening stage, gradually increase the UV-B light exposure by 2 hours per day. After being exposed to UV-B radiation, the plants will initiate a series of physiological response mechanisms. As an external stimulus signal, UV-B light can activate the signal transduction pathway in the plant body and prompt the plant to synthesize a variety of secondary metabolites, such as flavonoids, phenolic compounds and alkaloids. These secondary metabolites have multiple functions. Flavonoids and phenolic compounds can absorb and shield UV-B radiation, reducing its damage to plant cells; at the same time, they can also enhance the stability of plant cell walls and improve the plant's stress resistance. Secondary metabolites such as alkaloids have antibacterial and antiviral effects, which help plants resist external pathogens. By synthesizing these secondary metabolites, plants gradually adapt to UV-B light and are prepared to adapt to various stresses in the field environment.

[0048] By promoting the development of capillary roots of tissue culture seedlings and enhancing their drought resistance, the tissue culture seedlings can adapt to the new environment more quickly after transplantation, improve the transplantation survival rate, stimulate the plants to synthesize secondary metabolites, and enhance the plants' ability to adapt to stress factors such as ultraviolet rays in the field environment. This solves the problem of poor root development of tissue culture seedlings after transplantation and difficulty in adapting to the drought environment in the field. Example

[0049] A method for asexual propagation and seedling raising of apples comprises the following steps: S1. Mother plant selection and pretreatment: 10-year-old 'Ruixue' apple plants were selected as mother plants, 15 cm one-year-old semi-lignified branches were taken, 2 leaves were left on the top, the base of the branches was soaked in 0.2% carbendazim for 10 minutes and then washed; S2, callus induction: prepare a specific culture medium with pH 6.0, insert it into the base of the branch, and culture it at 25°C, 2000lx light, 14 hours / day for 2 weeks to induce callus; S3, adventitious bud differentiation: When the callus is 1.0 cm, it is transferred to the corresponding culture medium with pH 6.0 and cultured for 3 weeks at 25°C, 2500 lx light, 16 hours / day to differentiate adventitious buds; S4, rooting culture: select 5cm adventitious buds, transfer to rooting medium pH 6.0, use yeast fermentation to provide carbon, and culture for 2 weeks at 22℃, 1500lx light, 12 hours / day for rooting; S5. Hardening and transplanting: Soak the plantlets in medicine for 10 minutes and plant them in nutrient soil seedling pots. Harden them in a greenhouse at 22℃ and 80% humidity for 1 week. Transplant them into the field after strengthening light and ventilation.

[0050] S1 specifically includes: S101: Select adult apple plants that are healthy, free of pests and diseases, have excellent traits, good fruit quality, high yield, and strong stress resistance as mother plants; S102: Select one-year-old, semi-lignified branches from the mother plant, with a length of 15 cm, retain the top two leaves of the branches, and remove the remaining leaves; S103: Soak the base of the selected branches in a 0.2% carbendazim solution for 10 minutes for disinfection, and then rinse with clean water.

[0051] S2 specifically includes: S201: preparing a callus induction medium, the components of which include MS basic medium, 2.0 mg / L 6-benzylaminoadenine, 1.0 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, and adjusting the pH value to 6.0; S202: inserting the disinfected branch base into callus induction medium, culturing for 2 weeks under the conditions of temperature of 25°C, light intensity of 2000lx, and light time of 14 hours / day, to induce callus formation at the branch base.

[0052] S3 specifically includes: S301. When the callus tissue grows to a certain size with a diameter of about 1.0 cm, it is transferred to an adventitious bud differentiation medium, wherein the adventitious bud differentiation medium comprises MS basic medium, 2.5 mg / L kinetin, 0.5 mg / L indolebutyric acid, 30 g / L sucrose and 7 g / L agar, and the pH value is adjusted to 6.0; S302. Cultivate the cells at 25°C, 2500 lx of light intensity and 16 hours of light per day for 3 weeks to promote the differentiation of adventitious buds from callus tissue.

[0053] S4 specifically includes: S401. Select healthy adventitious buds with a height of 5 cm and transfer them to a rooting medium. The rooting medium includes 1 / 2MS basic medium, 1.0 mg / L indole-3-acetic acid, 0.3 mg / L rooting powder, 30 g / L glucose, 2 g / L yeast powder and 7 g / L vermiculite. The pH value is adjusted to 6.0. Yeast ferments glucose into ethanol and carbon dioxide under anaerobic conditions. The chemical equation is: C6H 12 O6+yeast→2C2H5OH+2CO2↑Carbon dioxide provides carbon for plant photosynthesis; S402. Cultivate at 22°C, 1500 lx light intensity and 12 hours / day light duration for 2 weeks to allow the adventitious buds to take root and form complete plantlets.

[0054] S5 specifically includes: S501, taking the rooted plantlets out of the culture bottle, and soaking them in a solution containing 0.2% carbendazim and 0.3% potassium dihydrogen phosphate for 10 minutes; S502. Transplant the plantlets into a seedling pot filled with nutrient soil, wherein the ratio of leaf mold: perlite: vermiculite is 3:1:1, and harden the seedlings in a greenhouse at a temperature of 22°C and a humidity of 80%. After hardening the seedlings for one week, gradually increase the light intensity and ventilation. When the plantlets grow strong, they can be transplanted into the field.

[0055] S1 also includes: after soaking the branches in carbendazim, spraying Bacillus subtilis with a concentration of 1×10^8 CFU / ML to inhibit root rot. At the same time, fumigating the soil in the park where the mother plant is located to reduce the base number of nematodes and fungi.

[0056] S5 also includes: Before transplanting, the roots of the tissue culture seedlings are immersed in a mixture of 100 mg / L humic acid and 0.1% seaweed extract for 10 minutes to promote the development of capillary roots and enhance drought resistance. During the seedling hardening stage, UV-B light is gradually increased to 2 hours per day to stimulate the plants to synthesize secondary metabolites for adaptation to the field. Example

[0057] The difference between this embodiment and the above-mentioned embodiment 1 is that: A method for asexual propagation and seedling raising of apples comprises the following steps: S1. Mother plant selection and pretreatment: 8-year-old 'Qincui' apple plants were selected as mother plants, 10 cm one-year-old semi-lignified branches were taken, 3 leaves were left on the top, the base of the branches was soaked in 0.1% carbendazim for 15 minutes and then washed; S2, callus induction: prepare a specific culture medium with pH 5.8, insert it into the base of the branch, and culture it at 22°C, 1500lx light, 12 hours / day for 3 weeks to induce callus; S3, adventitious bud differentiation: When the callus is 0.5 cm, it is transferred to the corresponding culture medium with pH 5.8, and cultured for 4 weeks at 23°C, 2000 lx light, 14 hours / day to differentiate adventitious buds; S4, rooting culture: select 3cm adventitious buds, transfer to rooting medium pH 5.8, use yeast fermentation to provide carbon, and culture for 3 weeks at 20℃, 1000lx light, 10 hours / day for rooting; S5. Hardening and transplanting: Soak the plantlets in medicine for 15 minutes and then plant them in nutrient soil seedling pots. Harden them in a greenhouse at 18℃ and 70% humidity for 2 weeks. Transplant them into the field after enhanced light and ventilation. Example

[0058] The difference between this embodiment and the above-mentioned embodiment 1 is that: 1. A method for asexual propagation and seedling raising of apples, comprising the following steps: S1. Mother plant selection and pretreatment: Select 5-year-old 'Yanfu No. 6' apple plants as mother plants, take 12 cm one-year-old semi-lignified branches, keep the top 2 leaves, soak the base of the branches in 0.15% carbendazim for 12 minutes and then wash; S2, callus induction: prepare a specific culture medium with pH 5.9, insert it into the base of the branch, and culture it at 23°C, 1800lx light, 13 hours / day for 2.5 weeks to induce callus; S3, adventitious bud differentiation: When the callus was 0.8 cm, it was transferred to the corresponding culture medium with pH 5.9 and cultured at 24°C, 2200 lx light, 15 hours / day for 3.5 weeks to differentiate adventitious buds; S4, rooting culture: select 4 cm adventitious buds, transfer to rooting medium pH 5.9, use yeast fermentation to provide carbon, and culture for 2.5 weeks at 21 ° C, 1200 lx light, 11 hours / day for rooting; S5. Hardening and transplanting: Soak the plantlets in medicine for 12 minutes and then plant them in nutrient soil seedling pots. Harden them in a greenhouse at 20℃ and 75% humidity for 1.5 weeks. Transplant them into the field after strengthening light and ventilation.

[0059] Table 1: contrast Example Traditional grafting Cutting propagation The degree of good traits of the mother Maximum value: 98% Middle value: 95% Minimum value: 92% Maximum value: 90% Middle value: 85% Minimum value: 80% Maximum value: 80% Middle value: 60% Minimum value: 40% Survival rate Maximum value: 95% Middle value: 87.7% Minimum value: 80% Maximum value: 85% Middle value: 70% Minimum value: 60% Maximum value: 55% Middle value: 40% Minimum value: 30% Growth cycle Maximum: 12 weeks Median: 10 weeks Minimum: 8 weeks Maximum: 24 weeks Median: 21 weeks Minimum: 18 weeks Maximum: 20 weeks Median: 18 weeks Minimum: 16 weeks Seedling cost Maximum: 2500 yuan Middle: 2300 yuan Minimum: 2100 yuan Maximum: 3500 yuan Middle: 3300 yuan Minimum: 3100 yuan Maximum: 3000 yuan Middle: 2500 yuan Minimum: 2300 yuan The comparison in the above table is the existing method. Through the above comparison table, it can be known that the apple asexual propagation seedling raising method of the present invention has different requirements from traditional grafting propagation and cutting propagation in terms of operation process, key technical links and environmental conditions.

[0060] It achieves the effect of maintaining the excellent traits of the mother plant in a more stable and efficient manner, improving the survival rate of apple seedlings, shortening the seedling growth cycle and reducing the cost of seedlings.

[0061] It solves the problems of fluctuating survival rates, unstable traits and long growth cycles in traditional grafting propagation caused by factors such as the affinity between rootstock and scion, difficulty of grafting technology and environmental influences. It also solves the problems of rooting difficulties, low survival rates and actually high seedling costs due to the low survival rate in cutting propagation.

[0062] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for asexual propagation and seedling raising of apples, characterized in that: The following steps are involved: S1. Mother plant selection and pretreatment: Select the best mother plant, take 10-15cm one-year-old semi-lignified branches, leave 2-3 leaves on the top, soak the base of the branches with 0.1%-0.2% carbendazim for 10-15 minutes and then wash; S2. Callus induction: Prepare a specific culture medium with pH 5.8-6.0, insert it into the base of the branch, and culture it for 2-3 weeks at 22-25°C, 1500-2000lx light, 12-14 hours / day to induce callus; S3, adventitious bud differentiation: When the callus is 0.5-1.0 cm, transfer to the corresponding culture medium with pH 5.8-6.0, and culture for 3-4 weeks at 23-25°C, 2000-2500lx light, 14-16 hours / day to differentiate adventitious buds; S4, rooting culture: select 3-5cm adventitious buds, transfer to rooting medium pH 5.8-6.0, use yeast fermentation to provide carbon, and culture for 2-3 weeks at 20-22℃, 1000-1500lx light, 10-12 hours / day for rooting; S5. Hardening and transplanting: Soak the small plants in medicine for 10-15 minutes and then plant them in nutrient soil seedling pots. Harden them in a greenhouse at 18-22℃ and 70%-80% humidity for 1-2 weeks. Transplant them into the field after strengthening light and ventilation.

2. The method for asexual propagation and seedling raising of apple according to claim 1, characterized in that: The S1 specifically includes: S101: Select adult apple plants that are healthy, free of pests and diseases, have excellent traits, good fruit quality, high yield, and strong stress resistance as mother plants; S102: Select one-year-old, semi-lignified branches from the mother plant, with a length of 10-15 cm, retain 2-3 leaves on the top of the branches, and remove the remaining leaves; S103: Soak the base of the selected branches in a solution containing 0.1%-0.2% carbendazim for 10-15 minutes for disinfection, and then rinse with clean water.

3. The method for asexual propagation and seedling raising of apple according to claim 1, characterized in that: The S2 specifically includes: S201: preparing a callus induction medium, the components of which include MS basic medium, 1.0-2.0 mg / L 6-benzylaminoadenine, 0.5-1.0 mg / L naphthaleneacetic acid, 30 g / L sucrose and 7 g / L agar, and adjusting the pH value to 5.8-6.0; S202: inserting the disinfected branch base into callus induction medium, culturing for 2-3 weeks under the conditions of temperature of 22-25°C, light intensity of 1500-2000lx, and light time of 12-14 hours / day, to induce callus formation at the branch base.

4. The method for asexual propagation and seedling raising of apple according to claim 1, characterized in that: The S3 specifically includes: S301. When the callus grows to a certain size with a diameter of about 0.5-1.0 cm, transfer it to an adventitious bud differentiation medium, wherein the adventitious bud differentiation medium comprises MS basic medium, 1.5-2.5 mg / L kinetin, 0.3-0.5 mg / L indolebutyric acid, 30 g / L sucrose and 7 g / L agar, and the pH value is adjusted to 5.8-6.0; S302. Cultivate for 3-4 weeks at a temperature of 23-25°C, a light intensity of 2000-2500 lx, and a light duration of 14-16 hours / day to promote callus differentiation into adventitious buds.

5. The method for asexual propagation and seedling raising of apple according to claim 1, characterized in that: The S4 specifically includes: S401. Select healthy adventitious buds with a height of 3-5 cm and transfer them to a rooting medium. The rooting medium includes 1 / 2MS basic medium, 0.5-1.0 mg / L indole-3-acetic acid, 0.2-0.3 mg / L rooting powder, 20 g / L glucose, 2 g / L yeast powder and 6 g / L vermiculite. The pH value is adjusted to 5.8-6.

0. Yeast ferments glucose into ethanol and carbon dioxide under anaerobic conditions. The chemical equation is: C6H 12 O6+yeast→2C2H5OH+2CO2↑Carbon dioxide provides carbon for plant photosynthesis; S402. Cultivate for 2-3 weeks at a temperature of 20-22°C, a light intensity of 1000-1500 lx, and a light duration of 10-12 hours / day to allow the adventitious buds to take root and form complete plantlets.

6. The method for asexual propagation and seedling raising of apple according to claim 1, characterized in that: The S5 specifically includes: S501, taking the rooted plantlets out of the culture bottle, and soaking them in a solution containing 0.1%-0.2% carbendazim and 0.2%-0.3% potassium dihydrogen phosphate for 10-15 minutes; S502. Transplant the plantlets into a seedling pot filled with nutrient soil, wherein the ratio of leaf mold: perlite: vermiculite is 3:1:1, and harden the seedlings in a greenhouse at a temperature of 18-22°C and a humidity of 70%-80%. After hardening the seedlings for 1-2 weeks, gradually increase the light intensity and ventilation. When the plantlets grow strong, they can be transplanted into the field.

7. The method for asexual propagation and seedling raising of apple according to claim 1, characterized in that: The step S1 also includes: after soaking the branches in carbendazim, spraying Bacillus subtilis with a concentration of 1×10^8 CFU / ML to inhibit root rot. At the same time, fumigating the soil in the garden where the mother plant is located to reduce the base number of nematodes and fungi.

8. The method for asexual propagation and seedling raising of apple according to claim 1, characterized in that: The S5 also includes: before transplanting, immersing the roots of the tissue culture seedlings in a mixture of 100 mg / L humic acid and 0.1% seaweed extract for 10 minutes to promote the development of capillary roots and enhance drought resistance. During the seedling hardening stage, gradually increase UV-B light for 2 hours per day to stimulate the plants to synthesize secondary metabolites for adaptation to the field.

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