Apple rootstock tissue culture seedling raising method

Abstract

The invention discloses an apple rootstock tissue culture seedling raising method, and relates to the technical field of tissue culture. The method comprises the following steps: (1) washing annual robust branches with tender shoots in a dormancy period with running water, soaking with soapy water, cleaning, obliquely shearing into stem segments, soaking with alcohol, washing, sterilizing, washing, absorbing moisture, and shearing off wounds to obtain pretreated explants; (2) inoculating the pretreated explant into a differential culture medium, and culturing to obtain an adventitious bud; (3) transferring the adventitious buds into a proliferation culture medium, and performing dark culture and light culture to obtain adventitious bud clusters; (4) inoculating the adventitious bud clusters into a rooting culture medium, and culturing to obtain tissue culture seedlings; and (5) hardening the tissue culture seedlings, washing off the culture medium, disinfecting, putting into a culture medium, culturing to obtain apple seedlings, and transplanting to outdoor soil. According to the method, the seedlings capable of being transplanted outdoors can be rapidly obtained, and the problems of infectious microbe pollution, low survival rate, poor rooting effect and the like in existing tissue culture seedling culture are effectively solved.

Description

Technical Field

[0001] The invention relates to the technical field of tissue culture, and in particular to a method for raising seedlings through tissue culture of apple rootstock. Background Art

[0002] Apple is a deciduous tree with oval leaves and white flowers with red blush. The fruit is round and tastes sweet or slightly sour. It is a common fruit with rich nutrients and has dietary and auxiliary therapeutic functions. It is one of the four major fruits in the world. Apples are usually red, but they can also be yellow or green. They are the fruit of plants. Apples are native to Europe, Central Asia, West Asia and Turkey, and were introduced to China in the 19th century. Apples are common fruits and the most common fruits. They are rich in vitamins and nutrients and are the top of the four major fruits in the world (apples, grapes, oranges and bananas).

[0003] Apple tissue culture uses sterile culture technology to culture organs such as stem tips, axillary buds, leaves and their tissue sections from excellent plants in vitro, so as to obtain a large number of genetically consistent individuals in a short period of time. This method has the advantages of short cycle, easy artificial control of culture conditions, fast reproduction speed, small space occupation and high economic benefits. At the same time, it also accelerates the promotion and application of new apple varieties.

[0004] Plant tissue culture has the technical advantage of providing a large number of seedlings in a short period of time and is easy to achieve mass production, so it is one of the main production methods for a variety of fruit seedlings. However, in the existing research on apple rootstock tissue culture, multiple culture media are often contaminated by bacteria, which affects the differentiation, proliferation and rooting of rootstock tissues, resulting in a low survival rate after transplantation, which leads to low production efficiency and increased production costs. There is also a problem of poor rooting effect during rootstock tissue culture. Summary of the invention

[0005] In order to solve the above technical problems, the purpose of the present invention is to provide an apple rootstock tissue culture seedling raising method, which can quickly obtain seedlings that can be transplanted outdoors through pretreatment, differentiation induction, adventitious bud proliferation, rooting culture and seedling transplanting, effectively solving the problems of bacterial contamination, low survival rate and poor rooting effect in existing tissue culture seedling raising.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: a method for raising apple rootstock seedlings through tissue culture is provided, comprising the following steps: (1) Rinse the one-year-old healthy branches with tender buds in dormant period with running water for 1-2 hours, then soak them in soapy water for 1-3 minutes, wash them with running water, cut them obliquely into stem segments with 1-2 buds and 1.5-2.5 cm in length, soak them in alcohol for 0.5-1 minute, rinse them with sterile water for 1-3 times, put them in mercuric chloride solution, add 3-6 drops of polysorbate-80 solution to sterilize for 8-10 minutes, rinse them with sterile water for 5-8 times, each time for 1-2 minutes, finally dry them with sterile filter paper, cut off the wound, and obtain the pretreated explants; (2) inoculating the pretreated explants obtained in step (1) into a differentiation medium and culturing them at 26±2°C and 25-50% humidity for 15-30 days to obtain adventitious buds; The differentiation medium is based on MS medium and also includes 0.1-0.3 mg / L naphthaleneacetic acid, 2-4 mg / L 6-benzyl adenine, 20-30 g / L sucrose, 5-10 g / L agar, 30-60 mg / L hydrolyzed casein, 2-8 mg / L nisin and 0.5-1 mg / L methylcobalamin; (3) The adventitious buds obtained in step (2) are transferred to a proliferation medium, cultured in the dark at 26±2°C and 25-50% humidity for 3-4 days, and then cultured in the light for 20-35 days to obtain adventitious bud clusters; The proliferation medium further comprises 0.5-1.2 mg / L naphthaleneacetic acid, 0.3-1 mg / L indolebutyric acid, 0.5-1 mg / L 6-benzyl adenine, 20-30 g / L sucrose, 5-10 g / L agar, 0.5-1.2 mg / L bamboo leaf extract and 0.2-0.8 mg / L yucca extract on the basis of MS medium; (4) inoculating the adventitious bud cluster obtained in step (3) into a rooting medium, and culturing at 26±2°C and 25-50% humidity for 10-30 days to obtain tissue culture seedlings; The rooting medium includes 0.1-0.8 mg / L naphthaleneacetic acid, 0.3-1 mg / L indolebutyric acid, 20-30 g / L sucrose, 5-10 g / L agar, 0.2-1 mg / L willow extract and 0.5-0.8 mg / L streptomycin on the basis of MS medium; (5) After hardening the tissue culture seedlings obtained in step (4), the culture medium is washed off, and the seedlings are sterilized with a carbendazim solution and placed in a cultivation medium. The seedlings are cultured under natural light conditions at 25-30°C for 8-15 days to obtain apple seedlings, which are then transplanted to outdoor soil.

[0007] Furthermore, in step (1), the alcohol concentration is 75 vt%.

[0008] Furthermore, in step (1), the concentration of the mercuric chloride solution is 0.05-0.2 vt%.

[0009] Furthermore, in step (1), the concentration of the polysorbate 80 solution is 0.1-0.2 vt%.

[0010] Furthermore, in step (2), the differentiation medium further comprises 0.2 mg / L naphthylacetic acid, 3 mg / L 6-benzyl adenine, 25 g / L sucrose, 7 g / L agar, 40 mg / L hydrolyzed casein, 5 mg / L nisin and 0.7 mg / L methylcobalamin on the basis of MS medium.

[0011] Furthermore, in step (2), the light intensity is maintained at 1500-2000 Lx for 7-10 h / d.

[0012] Furthermore, in step (3), the proliferation medium further comprises 0.8 mg / L naphthylacetic acid, 0.6 mg / L indolebutyric acid, 0.7 mg / L 6-benzyl adenine, 25 g / L sucrose, 8 g / L agar, 0.9 mg / L bamboo leaf extract and 0.5 mg / L yucca extract based on the MS medium.

[0013] Furthermore, in step (3), the bamboo leaf extract is prepared by the following method: fresh bamboo leaves are dried and then crushed, passed through a 30-40 mesh sieve, and then added with anhydrous ethanol to soak for 0.2-0.8 h, with a solid-liquid ratio of 1:15-30 g / mL, and then microwaved at 500-800 W and 50-70 ° C for 5-10 min, and finally reflux extracted, filtered and vacuum dried in sequence to obtain the bamboo leaf extract.

[0014] Further, reflux extraction is performed at 70-90°C for 0.5-3 h.

[0015] Furthermore, in step (3), the yucca extract is prepared by the following method: decocting the yucca with water for 2-3 times, with the amount of water added being 9-10 times the weight of the yucca, decocting for 1-3 h each time, combining the decoctions, and then concentrating to 70-80% of the original volume, adding ethanol to an alcohol content of 60-80 vt%, and then filtering, concentrating under reduced pressure and drying in sequence to obtain the yucca extract.

[0016] Furthermore, in step (3), during light culture, the light intensity is maintained at 1500-2000 Lx for 10-12 h / d.

[0017] Furthermore, in step (4), the rooting medium further comprises 0.5 mg / L naphthylacetic acid, 0.5 mg / L indolebutyric acid, 30 g / L sucrose, 8 g / L agar, 0.6 mg / L willow extract and 0.7 mg / L streptomycin on the basis of MS medium.

[0018] Furthermore, in step (4), the willow extract is prepared by the following method: the willow branches are dried and crushed, and then added into water at a solid-liquid ratio of 1:4-6 g / mL, followed by the addition of cellulase and pectinase, and enzymolysis at 40-60°C for 1-3 h, and after the enzyme is inactivated, ethanol is added to an alcohol content of 60-70 vt%, and then extracted, filtered, and the filtrate is dried to obtain the willow extract.

[0019] Furthermore, the mass ratio of cellulase to pectinase is 2-5:1.

[0020] Further, extraction was performed at 40-60 °C for 2-4 h.

[0021] Furthermore, in step (4), the light intensity is maintained at 3000-4000 Lx for 10-12 h / d.

[0022] Furthermore, in step (5), the concentration of the carbendazim solution is 0.05-0.2 vt%.

[0023] Furthermore, in step (5), the cultivation medium includes peat, perlite and humus soil; and the volume ratio of peat, perlite and humus soil is 1:1:2.

[0024] The present invention has the following beneficial effects: 1. The present invention can quickly obtain seedlings that can be transplanted outdoors through pretreatment, differentiation induction, adventitious bud proliferation, rooting culture and seedling transplantation, effectively solving the problems of bacterial contamination, low survival rate and poor rooting effect in existing tissue culture seedling cultivation. In the tissue culture seedling cultivation method of the present invention, the explant is first selected and pre-treated by cleaning and sterilization, and then through differentiation induction, adventitious bud proliferation, rooting culture and seedling transplantation, seedlings that can be transplanted outdoors can be quickly obtained, and bacterial contamination is avoided in all links, the survival rate of seedlings is improved, and a good growth promotion and rooting effect is achieved, achieving the goal of rapid and high-quality reproduction, which is convenient for large-scale promotion of planting.

[0025] 2. In the differentiation medium of the present invention, naphthylacetic acid, indolebutyric acid, sucrose, agar and other components are added on the basis of MS medium. Naphthylacetic acid and the like are used as growth regulators to induce the formation of callus, improve the induction rate of adventitious buds and the number of adventitious buds, and sucrose and agar and the like provide nutrients for the medium; hydrolyzed casein contains various amino acid components, which provide sufficient nutrients for the differentiation process of explants, and components such as arginine and lysine in the hydrolyzed casein can also be used as nitrogen sources, which are conducive to the absorption and utilization of plant cells, promote cell differentiation, accelerate the differentiation process, and prevent callus from browning, and have a certain antioxidant effect; and nisin is also composed of various amino acids, while providing nutrients, it can also inhibit the growth and reproduction of bacteria, and plays an important antibacterial effect in the differentiation medium; methylcobalamin can inhibit the growth of miscellaneous bacteria, and acts together with nisin, further mentioning the antibacterial ability of the medium, avoiding the contamination of miscellaneous bacteria affecting the differentiation, and methylcobalamin can also improve the protoplasm activity of the explant wound, promote cell division, and accelerate the formation and differentiation of callus together with hydrolyzed casein.

[0026] 3. In the proliferation medium, naphthylacetic acid, indolebutyric acid, and 6-benzyl adenine are used to promote the proliferation of adventitious buds, obtain more adventitious buds, and achieve better proliferation effects. Sucrose and agar are used to provide nutrients for the culture medium. Yucca extract is also added to the proliferation medium. The extract can reduce the concentration of harmful gases such as ammonia and hydrogen sulfide in the culture medium, improve the proliferation culture environment, promote the normal growth of cells, and promote the absorption of nutrients. Improve the proliferation efficiency, and the steroid components in the yucca extract can promote the stability of the pH value and osmotic pressure of the culture medium, providing a stable culture environment for proliferation. Polyphenol ingredients also have antioxidant effects, scavenge free radicals, reduce oxidative stress damage to cells, improve cell survival rate, and also have a certain antibacterial effect; importantly, polysaccharides and other components in yucca extract can also promote differentiation and proliferation, shorten the growth time of adventitious bud clusters; flavonoids, alkaloids, and anthraquinone compounds in bamboo leaf extract have obvious antibacterial activity, further enhancing the antibacterial ability of yucca extract. The two together avoid bacterial contamination during the proliferation process, further improve proliferation efficiency and survival rate, and bamboo leaf extract also has antioxidant ability, which further enhances the antioxidant capacity of the culture medium.

[0027] 4. In the rooting medium, indolebutyric acid and naphthylacetic acid are used as growth regulators to promote cell dedifferentiation and have a certain rooting effect. Sucrose and agar provide nutrients for the culture medium. Willow extract contains polyphenols such as catechol and pyrogallol, which have the effect of promoting rooting. Salicylic acid is a natural growth hormone that can stimulate the growth of plant roots and promote the absorption of nutrients by plants. Salicylic acid can also prevent root rot and ensure the health of the root system. At the same time, willow extract also contains rooting hormones (rhizocaline, etc.), which can stimulate the synthesis and transportation of plant growth hormones, promote cell division and growth, greatly improve its rooting ability, promote root hair growth, enhance the ability of roots to absorb water and nutrients, and are beneficial to root growth. The added streptomycin is used to inhibit bacteria, actinomycetes and other miscellaneous bacteria, prevent bacterial contamination, avoid affecting the rooting effect of willow extracts, etc., and ensure the orderly progress of rooting culture. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of the adventitious bud germination rate results; Figure 2 This is a schematic diagram of the adventitious bud contamination rate results; Figure 3 This is a schematic diagram of the rooting rate results; Figure 4 Schematic diagram of survival rate results. DETAILED DESCRIPTION

[0029] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0030] Embodiment 1:

[0031] A method for raising apple rootstock seedlings through tissue culture comprises the following steps: (1) Rinse the one-year-old healthy branches with tender buds in dormant period with running water for 1 hour, then soak them in soapy water for 1 minute, wash them with running water, cut them obliquely into stem segments with 1-2 buds and 2 cm in length, soak them in 75 vt% alcohol for 0.5 min, rinse them once with sterile water, put them in 0.05 vt% mercuric chloride solution, and add 3 drops of 0.1 vt% polysorbate-80 solution to sterilize for 8 minutes, rinse them with sterile water 5 times, each time for 1 minute, finally dry them with sterile filter paper, cut off the wound, and obtain the pretreated explants; (2) The pretreated explants obtained in step (1) were inoculated into differentiation medium and cultured at 26±2°C and 30% humidity for 15 days, with a light intensity of 1500 Lx for 7 h / d to obtain adventitious buds; The differentiation medium was based on MS medium and also included 0.1 mg / L naphthylacetic acid, 2 mg / L 6-benzyl adenine, 20 g / L sucrose, 5 g / L agar, 30 mg / L hydrolyzed casein, 2 mg / L nisin, and 0.5 mg / L methylcobalamin; (3) The adventitious buds obtained in step (2) were transferred to a proliferation medium and cultured in the dark at 26±2°C and 30% humidity for 3 days, and then cultured in the light for 20 days, with a light intensity of 1500 Lx for 10 h / d to obtain adventitious bud clusters; The proliferation medium is based on MS medium and also includes 0.5 mg / L naphthaleneacetic acid, 0.3 mg / L indolebutyric acid, 0.5 mg / L 6-benzyl adenine, 20 g / L sucrose, 5 g / L agar, 0.5 mg / L bamboo leaf extract and 0.2 mg / L yucca extract; The bamboo leaf extract was prepared by the following method: fresh bamboo leaves were dried and crushed, passed through a 40-mesh sieve, and then soaked in anhydrous ethanol for 0.5 h, with a solid-liquid ratio of 1:20 g / mL, and then microwaved at 600 W and 60 °C for 8 min, refluxed at 80 °C for 1 h, filtered and vacuum dried to obtain the bamboo leaf extract; the yucca extract was prepared by the following method: yucca was decocted with water for 3 times, with the amount of water added being 10 times the weight of the yucca, and decocted for 2 h each time, the decoctions were combined, and then concentrated to 75% of the original volume, ethanol was added to the alcohol content of 70 vt%, and then filtered, concentrated under reduced pressure and dried in sequence to obtain the yucca extract; (4) inoculating the adventitious bud cluster obtained in step (3) into a rooting medium, culturing at 26±2°C and 30% humidity for 10 days, and maintaining a light intensity of 3000 Lx for 10 h / d to obtain tissue culture seedlings; The rooting medium was based on MS medium and also included 0.1 mg / L naphthaleneacetic acid, 0.3 mg / L indolebutyric acid, 20 g / L sucrose, 5 g / L agar, 0.2 mg / L willow extract, and 0.5 mg / L streptomycin; The willow extract was prepared by the following method: the willow branches were dried and crushed, and then added into water with a solid-liquid ratio of 1:5 g / mL, followed by the addition of cellulase and pectinase (mass ratio 4:1), enzymolysis at 50 °C for 2 h, and after the enzymes were inactivated, ethanol was added to an alcohol content of 70 vt%, and then extracted at 50 °C for 3 h, filtered, and the filtrate was dried to obtain the willow extract; (5) After hardening the tissue culture seedlings obtained in step (4), the culture medium is washed off, and the seedlings are disinfected with a 0.05 vt% carbendazim solution and placed in a cultivation medium (peat, perlite and humus soil in a volume ratio of 1:1:2). The seedlings are cultured under natural light at 25°C for 8 days to obtain apple seedlings, which are then transplanted to outdoor soil.

[0032] Embodiment 2:

[0033] A method for raising apple rootstock seedlings through tissue culture comprises the following steps: (1) Rinse the one-year-old healthy branches with tender buds in dormant period with running water for 1 hour, then soak them in soapy water for 2 minutes, wash them with running water, cut them obliquely into stem segments with 1-2 buds and 2 cm in length, soak them in 75 vt% alcohol for 0.6 minutes, rinse them with sterile water twice, put them in 0.1 vt% mercuric chloride solution, and add 5 drops of 0.2 vt% polysorbate-80 solution to sterilize for 9 minutes, rinse them with sterile water 6 times, each time for 1 minute, finally dry them with sterile filter paper, cut off the wound, and obtain the pretreated explants; (2) The pretreated explants obtained in step (1) were inoculated into differentiation medium and cultured at 26±2°C and 40% humidity for 20 days, with a light intensity of 1800 Lx for 8 h / d to obtain adventitious buds; The differentiation medium was based on MS medium and also included 0.2 mg / L naphthaleneacetic acid, 3 mg / L 6-benzyl adenine, 25 g / L sucrose, 7 g / L agar, 40 mg / L hydrolyzed casein, 5 mg / L nisin, and 0.7 mg / L methylcobalamin; (3) The adventitious buds obtained in step (2) were transferred to a proliferation medium and cultured in the dark at 26±2°C and 40% humidity for 3 days, and then cultured in the light for 30 days, with a light intensity of 1800 Lx for 11 h / d, to obtain adventitious bud clusters; The proliferation medium is based on MS medium and also includes 0.8 mg / L naphthaleneacetic acid, 0.6 mg / L indolebutyric acid, 0.7 mg / L 6-benzyl adenine, 25 g / L sucrose, 8 g / L agar, 0.9 mg / L bamboo leaf extract and 0.5 mg / L yucca extract; The bamboo leaf extract was prepared by the following method: fresh bamboo leaves were dried and crushed, passed through a 40-mesh sieve, and then soaked in anhydrous ethanol for 0.5 h, with a solid-liquid ratio of 1:20 g / mL, and then microwaved at 600 W and 60 °C for 8 min, refluxed at 80 °C for 1 h, filtered and vacuum dried to obtain the bamboo leaf extract; the yucca extract was prepared by the following method: yucca was decocted with water for 3 times, with the amount of water added being 10 times the weight of the yucca, and decocted for 2 h each time, the decoctions were combined, and then concentrated to 75% of the original volume, ethanol was added to the alcohol content of 70 vt%, and then filtered, concentrated under reduced pressure and dried in sequence to obtain the yucca extract; (4) inoculating the adventitious bud cluster obtained in step (3) into a rooting medium, culturing at 26±2°C and 40% humidity for 20 days, and maintaining a light intensity of 3500 Lx for 11 h / d to obtain tissue culture seedlings; The rooting medium was based on MS medium and also included 0.5 mg / L naphthaleneacetic acid, 0.5 mg / L indolebutyric acid, 30 g / L sucrose, 8 g / L agar, 0.6 mg / L willow extract, and 0.7 mg / L streptomycin; The willow extract was prepared by the following method: the willow branches were dried and crushed, and then added into water with a solid-liquid ratio of 1:5 g / mL, followed by the addition of cellulase and pectinase (mass ratio 4:1), enzymolysis at 50 °C for 2 h, and after the enzymes were inactivated, ethanol was added to an alcohol content of 70 vt%, and then extracted at 50 °C for 3 h, filtered, and the filtrate was dried to obtain the willow extract; (5) After hardening the tissue culture seedlings obtained in step (4), the culture medium is washed off, and the seedlings are disinfected with a 0.1 vt% carbendazim solution and placed in a cultivation medium (peat, perlite and humus soil in a volume ratio of 1:1:2). The seedlings are cultured under natural light at 25°C for 10 days to obtain apple seedlings, which are then transplanted to outdoor soil.

[0034] Embodiment 3:

[0035] A method for raising apple rootstock seedlings through tissue culture comprises the following steps: (1) Rinse the one-year-old healthy branches with tender buds in dormant period with running water for 2 h, then soak them in soapy water for 3 min, wash them with running water, cut them obliquely into stem segments with 1-2 buds and 2 cm in length, soak them in 75 vt% alcohol for 1 min, rinse them with sterile water 3 times, put them in 0.2 vt% mercuric chloride solution, and add 6 drops of 0.1 vt% polysorbate-80 solution to sterilize for 10 min, rinse them with sterile water 8 times, each time for 2 min, finally dry them with sterile filter paper, cut off the wound, and obtain the pretreated explants; (2) The pretreated explants obtained in step (1) were inoculated into differentiation medium and cultured at 26±2°C and 50% humidity for 30 days, with a light intensity of 2000 Lx for 10 h / d to obtain adventitious buds; The differentiation medium was based on MS medium and also included 0.3 mg / L naphthylacetic acid, 4 mg / L 6-benzyl adenine, 30 g / L sucrose, 10 g / L agar, 60 mg / L hydrolyzed casein, 8 mg / L nisin, and 1 mg / L methylcobalamin; (3) The adventitious buds obtained in step (2) were transferred to a proliferation medium and cultured in the dark at 26±2°C and 50% humidity for 4 days, and then cultured in the light for 35 days, with a light intensity of 2000 Lx for 12 h / d, to obtain adventitious bud clusters; The proliferation medium is based on MS medium and also includes 1.2 mg / L naphthaleneacetic acid, 1 mg / L indolebutyric acid, 1 mg / L 6-benzyl adenine, 30 g / L sucrose, 10 g / L agar, 1.2 mg / L bamboo leaf extract and 0.8 mg / L yucca extract; The bamboo leaf extract was prepared by the following method: fresh bamboo leaves were dried and crushed, passed through a 40-mesh sieve, and then soaked in anhydrous ethanol for 0.5 h, with a solid-liquid ratio of 1:20 g / mL, and then microwaved at 600 W and 60 °C for 8 min, refluxed at 80 °C for 1 h, filtered and vacuum dried to obtain the bamboo leaf extract; the yucca extract was prepared by the following method: yucca was decocted with water for 3 times, with the amount of water added being 10 times the weight of the yucca, and decocted for 2 h each time, the decoctions were combined, and then concentrated to 75% of the original volume, ethanol was added to the alcohol content of 70 vt%, and then filtered, concentrated under reduced pressure and dried in sequence to obtain the yucca extract; (4) inoculating the adventitious bud cluster obtained in step (3) into a rooting medium, culturing at 26±2°C and 50% humidity for 30 days, and maintaining a light intensity of 4000 Lx for 12 h / d to obtain tissue culture seedlings; The rooting medium was based on MS medium and also included 0.8 mg / L naphthaleneacetic acid, 1 mg / L indolebutyric acid, 30 g / L sucrose, 10 g / L agar, 1 mg / L willow extract, and 0.8 mg / L streptomycin; The willow extract was prepared by the following method: the willow branches were dried and crushed, and then added into water with a solid-liquid ratio of 1:5 g / mL, followed by the addition of cellulase and pectinase (mass ratio 4:1), enzymolysis at 50 °C for 2 h, and after the enzymes were inactivated, ethanol was added to an alcohol content of 70 vt%, and then extracted at 50 °C for 3 h, filtered, and the filtrate was dried to obtain the willow extract; (5) After hardening the tissue culture seedlings obtained in step (4), the culture medium is washed off, and the seedlings are disinfected with a 0.2 vt% carbendazim solution and placed in a cultivation medium (peat, perlite and humus soil in a volume ratio of 1:1:2). The seedlings are cultured under natural light conditions at 30°C for 15 days to obtain apple seedlings, which are then transplanted to outdoor soil.

[0036] Comparative Example 1: The difference between Comparative Example 1 and Example 2 is that the differentiation medium does not contain hydrolyzed casein.

[0037] Comparative Example 2: The difference between Comparative Example 2 and Example 2 is that the differentiation medium does not contain nisin.

[0038] Comparative Example 3: The difference between Comparative Example 3 and Example 2 is that the differentiation medium does not contain methylcobalamin.

[0039] Comparative Example 4: The difference between Comparative Example 4 and Example 2 is that the proliferation culture medium does not contain bamboo leaf extract.

[0040] Comparative Example 5: The difference between Comparative Example 5 and Example 2 is that the proliferation culture medium does not contain yucca extract.

[0041] Comparative Example 6: Comparative Example 6 is different from Example 2 in that the rooting medium does not contain willow extract.

[0042] Comparative Example 7: The difference between Comparative Example 7 and Example 2 is that the rooting medium does not contain streptomycin.

[0043] During the apple rootstock tissue culture seedling raising process of Examples 1-3 and Comparative Examples 1-7, the growth conditions were statistically analyzed, and the results are shown in Tables 1 and Figure 1-Figure 4 .

[0044] Table 1 Growth of apple rootstock tissue culture seedlings

[0045] From Table 1 and Figure 1-Figure 4 It can be seen that the germination rate, rooting rate and survival rate of adventitious buds in the apple rootstock tissue culture seedling raising method of the present invention are all at a high level, and the adventitious bud contamination rate is low. Bacterial contamination is avoided in all links, the survival rate of seedlings is improved, and better growth promotion and rooting effects are achieved. Seedlings that can be transplanted outdoors can be quickly obtained, and the problems of bacterial contamination, low survival rate and poor rooting effect in existing tissue culture seedling raising are effectively solved, which is convenient for large-scale promotion of planting.

[0046] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for raising seedlings by tissue culture of apple rootstock, characterized in that: The following steps are involved: (1) Rinse the one-year-old healthy branches with tender buds in dormant period with running water for 1-2 hours, then soak them in soapy water for 1-3 minutes, wash them with running water, cut them obliquely into stem segments with 1-2 buds and 1.5-2.5 cm in length, soak them in alcohol for 0.5-1 minute, rinse them with sterile water for 1-3 times, put them in mercuric chloride solution, add 3-6 drops of polysorbate-80 solution to sterilize for 8-10 minutes, rinse them with sterile water for 5-8 times, each time for 1-2 minutes, finally dry them with sterile filter paper, cut off the wound, and obtain the pretreated explants; (2) inoculating the pretreated explants obtained in step (1) into a differentiation medium and culturing them at 26±2°C and 25-50% humidity for 15-30 days to obtain adventitious buds; The differentiation medium further comprises 0.1-0.3 mg / L naphthylacetic acid, 2-4 mg / L 6-benzyl adenine, 20-30 g / L sucrose, 5-10 g / L agar, 30-60 mg / L hydrolyzed casein, 2-8 mg / L nisin and 0.5-1 mg / L methylcobalamin on the basis of MS medium; (3) The adventitious buds obtained in step (2) are transferred to a proliferation medium, cultured in the dark at 26±2°C and 25-50% humidity for 3-4 days, and then cultured in the light for 20-35 days to obtain adventitious bud clusters; The proliferation medium further comprises 0.5-1.2 mg / L naphthaleneacetic acid, 0.3-1 mg / L indolebutyric acid, 0.5-1 mg / L 6-benzyl adenine, 20-30 g / L sucrose, 5-10 g / L agar, 0.5-1.2 mg / L bamboo leaf extract and 0.2-0.8 mg / L yucca extract on the basis of MS medium; (4) inoculating the adventitious bud cluster obtained in step (3) into a rooting medium, and culturing at 26±2°C and 25-50% humidity for 10-30 days to obtain tissue culture seedlings; The rooting medium further comprises 0.1-0.8 mg / L naphthaleneacetic acid, 0.3-1 mg / L indolebutyric acid, 20-30 g / L sucrose, 5-10 g / L agar, 0.2-1 mg / L willow extract and 0.5-0.8 mg / L streptomycin on the basis of MS medium; (5) After hardening the tissue culture seedlings obtained in step (4), the culture medium is washed off, and the seedlings are sterilized with a carbendazim solution and placed in a cultivation medium. The seedlings are cultured under natural light conditions at 25-30°C for 8-15 days to obtain apple seedlings, which are then transplanted to outdoor soil.

2. The apple stock tissue culture seedling raising method according to claim 1, wherein: In step (2), the differentiation medium further comprises 0.2 mg / L naphthylacetic acid, 3 mg / L 6-benzyl adenine, 25 g / L sucrose, 7 g / L agar, 40 mg / L hydrolyzed casein, 5 mg / L nisin and 0.7 mg / L methylcobalamin in addition to the MS medium.

3. The apple stock tissue culture seedling raising method according to claim 1, wherein: In step (2), maintain a light intensity of 1500-2000 Lx for 7-10 h / d.

4. The apple stock tissue culture seedling raising method according to claim 1, wherein: In step (3), the proliferation medium further comprises 0.8 mg / L naphthylacetic acid, 0.6 mg / L indolebutyric acid, 0.7 mg / L 6-benzyl adenine, 25 g / L sucrose, 8 g / L agar, 0.9 mg / L bamboo leaf extract and 0.5 mg / L yucca extract based on MS medium.

5. The apple rootstock tissue culture seedling raising method according to claim 1, characterized in that: In step (3), the bamboo leaf extract is prepared by the following method: fresh bamboo leaves are dried and then crushed, passed through a 30-40 mesh sieve, and then added with anhydrous ethanol to soak for 0.2-0.8 h, with a solid-liquid ratio of 1:15-30 g / mL, and then microwaved at 500-800 W and 50-70 ° C for 5-10 min, and finally reflux extracted, filtered and vacuum dried in sequence to obtain the bamboo leaf extract.

6. The apple rootstock tissue culture seedling raising method according to claim 1, characterized in that: In step (3), the yucca extract is prepared by the following method: decocting the yucca with water for 2-3 times, with the amount of water added being 9-10 times the weight of the yucca, decocting for 1-3 h each time, combining the decoctions, and then concentrating to 70-80% of the original volume, adding ethanol to an alcohol content of 60-80 vt%, and then filtering, concentrating under reduced pressure and drying in sequence to obtain the yucca extract.

7. The apple stock tissue culture seedling raising method according to claim 1, characterized in that: In step (3), during light culture, the light intensity is maintained at 1500-2000 Lx for 10-12 h / d.

8. The apple rootstock tissue culture seedling raising method according to claim 1, characterized in that: In step (4), the rooting medium further comprises 0.5 mg / L naphthaleneacetic acid, 0.5 mg / L indolebutyric acid, 30 g / L sucrose, 8 g / L agar, 0.6 mg / L willow extract and 0.7 mg / L streptomycin on the basis of MS medium.

9. The apple rootstock tissue culture seedling raising method according to claim 1, wherein: In step (4), the willow extract is prepared by the following method: the willow branches are dried and crushed, and then added into water with a solid-liquid ratio of 1:4-6 g / mL, followed by the addition of cellulase and pectinase, and enzymolysis at 40-60°C for 1-3 h, and after the enzyme is inactivated, ethanol is added to an alcohol content of 60-70 vt%, and then extracted, filtered, and the filtrate is dried to obtain the willow extract.

10. The apple rootstock tissue culture seedling raising method according to claim 1, characterized in that: In step (5), the cultivation medium includes peat, perlite and humus soil; and the volume ratio of peat, perlite and humus soil is 1:1:2.

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