Tissue culture and rapid propagation culture method for cold-resistant apple rootstock

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly discloses an apple cold-resistant rootstock tissue culture and rapid propagation culture method which comprises the following steps: preparing a culture medium containing an MS culture medium, 6-benayl aminopurine, indolebutyric acid, chitosan, polyvinylpyrrolidone and a WPM culture medium; selecting top tender shoots of the apple cold-resistant rootstock; disinfecting the obtained tender shoots, and then inoculating the tender shoots into a tissue culture bottle containing the prepared culture medium; placing the tissue culture bottle in a culture room for culturing, and observing whether the tissue culture bottle survives or not; respectively transferring the induced cluster buds into a tissue culture bottle containing a subculture multiplication culture medium and a tissue culture bottle containing a rooting culture medium for culturing, and counting the multiplication coefficient and the rooting rate of the cluster buds. According to the tissue culture and rapid propagation culture method for the cold-resistant apple rootstock, rapid and mass propagation of excellent germplasm of the cold-resistant apple rootstock is achieved, and then powerful technical support is provided for genetic improvement and industrial development of the cold-resistant apple rootstock.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method for cold-resistant apple rootstocks. Background Art

[0002] Cold-resistant apple rootstocks are a crucial component of apple cultivation. They form the rootstock of the apple tree, also known as the rootstock. Rootstocks are essential for apple tree cultivation, supporting the tree's growth, absorbing nutrients, regulating water, and transferring nutrients. Choosing the right apple rootstock variety is the first step for fruit growers to successfully cultivate their apple trees.

[0003] Apple rootstocks play a crucial role in apple cultivation. The quality and selection of rootstocks determine many aspects of apple tree growth, fruit yield, and quality. They also play a crucial role in the tree's disease resistance, cold tolerance, and adaptability. Improper selection can not only reduce the quality and yield of the fruit, but can also lead to poor fruit quality on many trees.

[0004] When choosing a suitable apple rootstock variety, you need to consider many factors, including the growth conditions, climatic conditions, geographical conditions of the apple tree, and your own cultivation techniques.

[0005] As a preferred apple rootstock in my country, cold-resistant apple rootstocks are in high demand, and their breeding and promotion are crucial to the development of the entire industry. Therefore, research on cold-resistant apple rootstocks urgently requires a more efficient and rapid breeding method, and asexual propagation is one of the important ways to solve this problem.

[0006] Numerous studies have been conducted on asexual propagation of cold-resistant apple rootstocks, but methods such as grafting and cuttings are still unable to rapidly produce a large number of high-quality plants. Plant tissue culture can be used as a method to rapidly induce sterile seedlings.

[0007] Therefore, establishing an efficient and stable rapid propagation system for cold-resistant apple rootstocks using superior cold-resistant apple rootstocks as material is of vital importance for the genetic improvement of cold-resistant apple rootstocks and the development and application of new variety breeding. Therefore, tissue culture research on this superior cold-resistant apple rootstock is being conducted to achieve rapid and large-scale propagation of superior cold-resistant apple rootstock germplasm, thereby providing strong technical support for the genetic improvement and industrial development of cold-resistant apple rootstocks. Summary of the Invention

[0008] The purpose of the present invention is to provide a tissue culture rapid propagation method for cold-resistant apple rootstocks, so as to achieve rapid and large-scale reproduction of excellent germplasm of cold-resistant apple rootstocks, thereby providing strong technical support for the genetic improvement and industrial development of cold-resistant apple rootstocks.

[0009] To achieve the above object, the present invention provides a method for rapid propagation of cold-resistant apple rootstocks through tissue culture, comprising the following steps:

[0010] Step 1, preparation and sterilization of culture medium: prepare a culture medium containing MS medium, 6-benzylaminopurine, indolebutyric acid, chitosan, polyvinylpyrrolidone, and WPM medium, adjust the pH value to between 5.8 and 6.0, and sterilize in an autoclave at 121° C. for 20 minutes. After sterilization, set aside for use;

[0011] Step 2, select tender shoots for processing: select tender shoots from the top of cold-resistant apple rootstocks and clean them with distilled water;

[0012] Step 3, sprout disinfection and inoculation: disinfect the sprouts obtained in step 2, and then inoculate them into a tissue culture bottle containing the culture medium prepared in step 1;

[0013] Step 4: Calculate the survival rate: Place the tissue culture bottle in the culture room and observe whether it survives;

[0014] Step 5: The induced clustered buds were transferred into tissue culture bottles containing subculture proliferation medium and rooting medium respectively for culture, and the proliferation coefficient and rooting rate of the clustered buds were calculated.

[0015] Preferably, in step 1, the MS medium includes the following components: NH4NO3, CaCl2·2H2O, MgSO4·7H2O, KH2PO4, MnSO4·4H2O, ZnSO4·7H2O, H3BO3, NaMoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O, KI, niacin, vitamin B1, glycine, vitamin B6, inositol, FeSO4·7H2O, Na2-EDTA, sucrose, agar and distilled water.

[0016] Preferably, the concentrations of the components of the MS medium are: NH4NO3 0.3-0.5g / L, CaCl2·2H2O 0.067-0.078g / L, MgSO4·7H2O 0.46-0.78g / L, KH2PO4 0.43-0.56g / L, MnSO4·4H2O 20.5-22.5mg / L, ZnSO4·7H2O 9.4-9.6mg / L, H3BO3 5.9mg / L, NaMoO4·2H2O 0.34-0.45mg / L, CuSO4·5H2O 0.029-0.032mg / L, CoCl2·6H2O 0.025mg / L, KI 0.03-0.04mg / L, niacin 0.48-0.56mg / L, vitamin B11.4-1.5mg / L, glycine 2.5-3.5mg / L, vitamin B6 0.6mg / L, inositol 100mg / L, FeSO4·7H2O27.85mg / L, Na2-EDTA 35.25mg / L, sucrose 20-30g / L, agar 6-7g / L.

[0017] Preferably, in step 1, the concentration of 6-benzylaminopurine is 0.4-1 mg / L, and the concentration of indolebutyric acid is 0.1-1 mg / L.

[0018] Preferably, in step 2, the cold-resistant apple rootstock buds are selected buds from plants that are healthy and free of diseases and insect pests.

[0019] Preferably, in step 3, the sprouts are disinfected in a clean bench, first disinfected with 75% alcohol for 30 seconds, rinsed with sterile water three times, then disinfected with 0.1% mercuric chloride for 6 minutes, rinsed with sterile water three times, and then the disinfected sprouts are placed on sterilized filter paper to absorb moisture on the surface of the sprouts. The disinfected sprouts are inoculated in tissue culture bottles, with one sprout inoculated in each bottle.

[0020] Preferably, in step 4, the survival rate is calculated after culturing for 7-14 days.

[0021] Preferably, in step 5, the subculture proliferation medium uses WPM medium as the basic medium, supplemented with 6-benzylaminopurine, indolebutyric acid, gibberellin, humic acid, sucrose, and agar, and the pH value is adjusted to 5.6-5.8.

[0022] Preferably, in step 5, the rooting medium uses 1 / 2 MS medium as the basic medium, is supplemented with indolebutyric acid, naphthaleneacetic acid, activated carbon, seaweed extract, sucrose, and agar, and the pH value is adjusted to 5.6-5.8.

[0023] Preferably, in step 5, one value-added bud is inoculated into each tissue culture bottle, and the culture conditions are a temperature of 25±2° C., a humidity of 50-70%, and a culture period of 20-35 days.

[0024] The advantages and beneficial effects of the present invention using the above-mentioned apple cold-resistant rootstock tissue culture rapid propagation method are:

[0025] 1. The culture medium for obtaining regenerated plants from cold-resistant apple rootstocks of the present invention can be used for plant breeding, quickly obtaining high-quality seedlings, shortening seedling raising time, and reducing costs. The terminal bud differentiation induction survival rate is as high as over 80%, the seedling proliferation coefficient is over 4, and the rooting rate can reach over 95%. The present invention proposes adding 6-benzylaminopurine and indolebutyric acid to the WPM culture medium, which can promote the successful tissue culture of apple cold-resistant rootstock seedlings and achieve rapid propagation of apple cold-resistant rootstocks.

[0026] 2. The primary culture medium of the present invention contains an appropriate amount of polyvinylpyrrolidone, which can effectively inhibit the browning of explants and improve the callus induction rate. Chitosan has antibacterial properties and can further reduce the explant contamination rate. It can also promote cell division and growth, which is beneficial to the initiation and growth of explants. The secondary proliferation culture medium is rationally proportioned with plant growth regulators such as 6-benzylaminoadenine, indolebutyric acid, and gibberellins, and the added humic acid can promote the differentiation and proliferation of adventitious buds, significantly improving the reproduction coefficient. Humic acid can provide a variety of nutrients and physiologically active substances, promote the growth and proliferation of adventitious buds, and improve the reproduction coefficient. The rooting culture medium contains an appropriate amount of indolebutyric acid, naphthaleneacetic acid, and activated carbon. The synergistic effect of indolebutyric acid and naphthaleneacetic acid promotes the formation of root primordia and the growth of the root system. The activated carbon adsorbs harmful substances in the culture medium and improves the rhizosphere environment. The addition of seaweed extract further promotes root development, improving the rooting rate and root quality. Seaweed extract is rich in plant growth regulators, amino acids, etc., which can promote the development of the root system and improve the quality and quantity of roots.

[0027] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 A is a schematic diagram of the inoculation of tender shoots into the culture medium, B is a schematic diagram of the rooting culture of clustered shoots, and C is a schematic diagram of the rooting survival of clustered shoots. DETAILED DESCRIPTION

[0029] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0030] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0031] Unless otherwise defined, the reagents, equipment and other materials used in the present invention are all commercially available.

[0032] Example 1

[0033] A method for rapid propagation of cold-resistant apple rootstocks through tissue culture, comprising the following steps:

[0034] Step 1, preparation and sterilization of culture medium: prepare a culture medium containing MS medium, 0.6 mg / L 6-benzylaminopurine, 0.6 mg / L indolebutyric acid, 70 mg / L chitosan, 350 mg / L polyvinylpyrrolidone, and WPM medium, adjust the pH to 5.8, and sterilize in an autoclave at 121°C for 20 min. Set aside for use after sterilization.

[0035] The concentrations of the components of MS medium are as follows: NH4NO3 0.5 g / L, CaCl2·2H2O 0.067 g / L, MgSO4·7H2O 0.78 g / L, KH2PO4 0.56 g / L, MnSO4·4H2O 22.5 mg / L, ZnSO4·7H2O 9.6 mg / L, H3BO3 5.9 mg / L, NaMoO4·2H2O 0.45 mg / L, CuSO4·5H2O 0.032 mg / L, CoCl2·6H2O 0.025 mg / L, KI 0.04 mg / L, niacin 0.56 mg / L, vitamin B1 1.5 mg / L, glycine 3.5 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25mg / L, sucrose 20g / L, agar 6g / L.

[0036] Step 2, select tender shoots for processing: select the tender shoots at the top of the cold-resistant apple rootstock, select the tender shoots from plants that are healthy and free of diseases and pests, and wash the tender shoots with distilled water.

[0037] Step 3, sprout disinfection and inoculation: The sprouts obtained in step 2 are disinfected and then inoculated into a tissue culture bottle containing the culture medium prepared in step 1; the sprout disinfection is carried out in a clean bench, first disinfected with 75% alcohol for 30 seconds, rinsed with sterile water three times, then disinfected with 0.1% mercuric chloride for 6 minutes, rinsed with sterile water three times, and then the disinfected sprouts are placed on sterilized filter paper to absorb the moisture on the surface of the sprouts, and the disinfected sprouts are inoculated into the tissue culture bottles, with one sprout inoculated per bottle. Figure 1 As shown in A.

[0038] Step 4, calculate the survival rate: place the tissue culture bottle in the culture room and observe whether it survives; calculate the survival rate after 14 days of culture.

[0039] Step 5: The induced cluster buds were transferred into tissue culture bottles containing subculture proliferation medium and rooting medium for culture, and the proliferation coefficient and rooting rate of the cluster buds were counted. One value-added bud was inoculated into each tissue culture bottle, and the culture conditions were temperature 25±2℃, humidity 50-70%, and culture for 20 days. The subculture proliferation medium used WPM medium as the basic medium, and added 6-benzylaminoadenine, indolebutyric acid, gibberellin, humic acid, sucrose, agar, and the pH value was adjusted to 5.7. The rooting medium used 1 / 2MS medium as the basic medium, and added indolebutyric acid, naphthaleneacetic acid, activated carbon, seaweed extract, sucrose, agar, and the pH value was adjusted to 5.6, as shown in the following figure: Figure 1 Middle B and Figure 1 As shown in C.

[0040] Example 2

[0041] A method for rapid propagation of cold-resistant apple rootstocks through tissue culture, comprising the following steps:

[0042] Step 1, preparation and sterilization of culture medium: prepare a culture medium containing MS medium, 0.4 mg / L 6-benzylaminopurine, 1 mg / L indolebutyric acid, 70 mg / L chitosan, 350 mg / L polyvinylpyrrolidone, and WPM medium, adjust the pH to 5.9, and sterilize in an autoclave at 121°C for 20 min. Set aside for use after sterilization.

[0043] The concentrations of the components of MS medium are as follows: NH4NO3 0.3 g / L, CaCl2·2H2O 0.078 g / L, MgSO4·7H2O 0.46 g / L, KH2PO4 0.43 g / L, MnSO4·4H2O 20.5 mg / L, ZnSO4·7H2O 9.4 mg / L, H3BO3 5.9 mg / L, NaMoO4·2H2O 0.34 mg / L, CuSO4·5H2O 0.029 mg / L, CoCl2·6H2O 0.025 mg / L, KI 0.03 mg / L, niacin 0.48 mg / L, vitamin B1 1.4 mg / L, glycine 2.5 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25mg / L, sucrose 25g / L, agar 7g / L.

[0044] Step 2, select tender shoots for processing: select the tender shoots at the top of the cold-resistant apple rootstock, select the tender shoots from plants that are healthy and free of diseases and pests, and wash the tender shoots with distilled water.

[0045] Step 3, sprout disinfection and inoculation: the sprouts obtained in step 2 are disinfected, and then inoculated into a tissue culture bottle containing the culture medium prepared in step 1; the sprout disinfection is carried out in a clean bench, first disinfected with 75% alcohol for 30 seconds, rinsed three times with sterile water, then disinfected with 0.1% mercuric chloride for 6 minutes, rinsed three times with sterile water, and then the disinfected sprouts are placed on sterilized filter paper to absorb moisture from the sprout surface, and the disinfected sprouts are inoculated into the tissue culture bottle, with one sprout inoculated into each bottle.

[0046] Step 4, calculate the survival rate: place the tissue culture bottle in the culture room and observe whether it survives; calculate the survival rate after 7 days of culture.

[0047] Step 5: The induced clustered buds were transferred to tissue culture flasks containing subculture proliferation medium and rooting medium, respectively, for culture. The proliferation coefficient and rooting rate of the clustered buds were calculated. One proliferating bud was inoculated into each tissue culture flask and cultured at a temperature of 25±2°C and a humidity of 50-70% for 25 days. The subculture proliferation medium used WPM medium as the basic medium, supplemented with 6-benzylaminoadenine, indolebutyric acid, gibberellin, humic acid, sucrose, and agar, and the pH was adjusted to 5.7. The rooting medium used 1 / 2MS medium as the basic medium, supplemented with indolebutyric acid, naphthaleneacetic acid, activated carbon, seaweed extract, sucrose, and agar, and the pH was adjusted to 5.6.

[0048] Example 3

[0049] A method for rapid propagation of cold-resistant apple rootstocks through tissue culture, comprising the following steps:

[0050] Step 1, preparation and sterilization of culture medium: prepare a culture medium containing MS medium, 0.7 mg / L 6-benzylaminopurine, 0.1 mg / L indolebutyric acid, 70 mg / L chitosan, 350 mg / L polyvinylpyrrolidone, and WPM medium, adjust the pH to 6.0, and sterilize in an autoclave at 121°C for 20 min. Set aside for use after sterilization.

[0051] The concentrations of the components of MS medium are as follows: NH4NO3 0.4 g / L, CaCl2·2H2O 0.071 g / L, MgSO4·7H2O 0.58 g / L, KH2PO4 0.5 g / L, MnSO4·4H2O 21.5 mg / L, ZnSO4·7H2O 9.5 mg / L, H3BO3 5.9 mg / L, NaMoO4·2H2O 0.40 mg / L, CuSO4·5H2O 0.031 mg / L, CoCl2·6H2O 0.025 mg / L, KI 0.035 mg / L, niacin 0.5 mg / L, vitamin B1 1.45 mg / L, glycine 3 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85 mg / L, Na2-EDTA 35.25mg / L, sucrose 30g / L, agar 6.5g / L.

[0052] Step 2, select tender shoots for processing: select the tender shoots at the top of the cold-resistant apple rootstock, select the tender shoots from plants that are healthy and free of diseases and pests, and wash the tender shoots with distilled water.

[0053] Step 3, sprout disinfection and inoculation: the sprouts obtained in step 2 are disinfected, and then inoculated into a tissue culture bottle containing the culture medium prepared in step 1; the sprout disinfection is carried out in a clean bench, first disinfected with 75% alcohol for 30 seconds, rinsed three times with sterile water, then disinfected with 0.1% mercuric chloride for 6 minutes, rinsed three times with sterile water, and then the disinfected sprouts are placed on sterilized filter paper to absorb moisture from the sprout surface, and the disinfected sprouts are inoculated into the tissue culture bottle, with one sprout inoculated into each bottle.

[0054] Step 4, calculate the survival rate: place the tissue culture bottle in the culture room and observe whether it survives; calculate the survival rate after 10 days of culture.

[0055] Step 5: The induced bud clusters were transferred to tissue culture flasks containing secondary proliferation medium and rooting medium, respectively, for culture. The proliferation coefficient and rooting rate of the bud clusters were calculated. One bud was inoculated into each tissue culture flask and cultured at a temperature of 25±2°C and a humidity of 50-70% for 35 days. The secondary proliferation medium used WPM medium as the base medium, supplemented with 6-benzylaminoadenine, indolebutyric acid, gibberellin, humic acid, sucrose, and agar, and the pH was adjusted to 5.8. The rooting medium used 1 / 2MS medium as the base medium, supplemented with indolebutyric acid, naphthaleneacetic acid, activated carbon, seaweed extract, sucrose, and agar, and the pH was adjusted to 5.8.

[0056] Comparative Example 1

[0057] A method for rapid propagation of cold-resistant apple rootstocks through tissue culture, which differs from Example 1 in that:

[0058] Step 1, preparation and sterilization of culture medium: prepare a culture medium containing MS culture medium and WPM culture medium, adjust the pH to 5.8, and sterilize in an autoclave at 121°C for 20 minutes. The remaining contents are the same as in Example 1.

[0059] Comparative Example 2

[0060] A method for rapid tissue culture propagation of cold-resistant apple rootstocks, different from that in Example 1, is provided in step 5: a subculture proliferation medium using WPM medium as a basic medium supplemented with 6-benzylaminoadenine, indolebutyric acid, sucrose, and agar, and the pH value is adjusted to 5.7. A rooting medium using 1 / 2MS medium as a basic medium supplemented with naphthaleneacetic acid, activated carbon, sucrose, and agar, and the pH value is adjusted to 5.6.

[0061] Comparative Example 3

[0062] A method for rapid propagation of cold-resistant apple rootstocks through tissue culture, which differs from Example 1 in that:

[0063] Step 1, Preparation and Sterilization of Culture Medium: Prepare a culture medium containing MS medium, 0.6 mg / L 6-benzylaminopurine, and 0.6 mg / L indolebutyric acid, adjust the pH to 5.8, and sterilize in an autoclave at 121°C for 20 minutes. The remaining steps are the same as in Example 1.

[0064] The rooting rate and terminal bud differentiation survival rate of apple cold-resistant rootstocks cultured according to the culture methods of Examples 1-3 and Comparative Examples 1-3 are shown in Table 1. Experimental treatment: 20 tissue culture bottles were treated in each group, with three replicates, and the average rooting rate and average terminal bud differentiation survival rate were taken.

[0065] Table 1 Statistical results

[0066]

[0067] Comparative Example 1 did not add 6-benzylaminopurine, indolebutyric acid, chitosan and polyvinyl pyrrolidone, which affected the rooting rate and proliferation coefficient. It shows that polyvinyl pyrrolidone can effectively inhibit the browning of explants and improve the callus induction rate. Chitosan has antibacterial properties and can further reduce the explant contamination rate. It can also promote cell division and growth, which is beneficial to the initiation and growth of explants. Comparative Example 2 subculture proliferation culture medium did not add gibberellins and humic acid, and the rooting culture medium did not add indolebutyric acid and seaweed extract, which affected the terminal bud differentiation survival rate, rooting rate and proliferation coefficient. It shows that indolebutyric acid and naphthaleneacetic acid synergistically promoted the formation of root primordium and the growth of root system. At the same time, the addition of seaweed extract further promoted root development and improved root system quality. Comparative Example 3 did not add WPM culture medium, chitosan and polyvinyl pyrrolidone, and the proliferation coefficient, rooting rate and terminal bud differentiation survival rate were poor compared with Examples 1-3.

[0068] Therefore, the present invention adopts the above-mentioned apple cold-resistant rootstock tissue culture rapid propagation method to achieve rapid and large-scale reproduction of excellent apple cold-resistant rootstock germplasm, thereby providing strong technical support for the genetic improvement and industrial development of apple cold-resistant rootstock.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A method for rapid propagation of cold-resistant apple rootstock tissue culture, characterized in that: The steps include: Step 1, preparation and sterilization of culture medium: prepare a culture medium containing MS medium, 6-benzylaminopurine, indolebutyric acid, chitosan, polyvinylpyrrolidone, and WPM medium, adjust the pH value to between 5.8 and 6.0, and sterilize in an autoclave at 121° C. for 20 minutes. After sterilization, set aside for use; Step 2, select tender shoots for processing: select tender shoots from the top of cold-resistant apple rootstocks and clean them with distilled water; Step 3, sprout disinfection and inoculation: disinfect the sprouts obtained in step 2, and then inoculate them into a tissue culture bottle containing the culture medium prepared in step 1; Step 4: Calculate the survival rate: Place the tissue culture bottle in the culture room and observe whether it survives; Step 5: The induced clustered buds were transferred into tissue culture bottles containing subculture proliferation medium and rooting medium respectively for culture, and the proliferation coefficient and rooting rate of the clustered buds were calculated.

2. A method for rapid propagation of cold-resistant apple rootstock tissue culture according to claim 1, characterized in that, In step 1, the MS medium includes the following components: NH4NO3, CaCl2·2H2O, MgSO4·7H2O, KH2PO4, MnSO4·4H2O, ZnSO4·7H2O, H3BO3, NaMoO4·2H2O, CuSO4·5H2O, CoCl2·6H2O, KI, niacin, vitamin B1, glycine, vitamin B6, inositol, FeSO4·7H2O, Na2-EDTA, sucrose, agar and distilled water.

3. A cold-resistant apple rootstock tissue culture rapid propagation culture method according to claim 2, characterized in that, The concentrations of the components of MS culture medium are: NH4NO30.3-0.5g / L, CaCl2·2H2O 0.067-0.078g / L, MgSO4·7H2O 0.46-0.78g / L, KH2PO40.43-0.56g / L, MnSO4·4H2O 20.5-22.5mg / L, ZnSO4·7H2O9.4-9.6mg / L, H3BO35.9mg / L, NaMoO4·2H2O 0.34-0.45mg / L, CuSO4·5H2O 0.029-0.032mg / L, CoCl2·6H2O0.025mg / L, KI 0.03-0.04mg / L, niacin 0.48-0.56mg / L, vitamin B11.4-1.5mg / L, glycine 2.5-3.5mg / L, vitamin B6 0.6mg / L, inositol 100mg / L, FeSO4·7H2O27.85mg / L, Na2-EDTA 35.25mg / L, sucrose 20-30g / L, agar 6-7g / L.

4. A method for rapid propagation of cold-resistant apple rootstock tissue culture according to claim 1, characterized in that: In step 1, the concentration of 6-benzylaminopurine is 0.4-1 mg / L, and the concentration of indolebutyric acid is 0.1-1 mg / L.

5. A method for rapid propagation of cold-resistant apple rootstock tissue culture according to claim 1, characterized in that: In step 2, the apple cold-resistant rootstock buds are selected from plants that are healthy and free of diseases and insect pests.

6. A method for rapid propagation of cold-resistant apple rootstock tissue culture according to claim 1, characterized in that: In step 3, the sprouts are disinfected in a clean bench, first disinfected with 75% alcohol for 30 seconds, rinsed with sterile water three times, then disinfected with 0.1% mercuric chloride for 6 minutes, rinsed with sterile water three times, and then the disinfected sprouts are placed on sterilized filter paper to absorb moisture on the surface of the sprouts. The disinfected sprouts are inoculated in tissue culture bottles, with one sprout inoculated in each bottle.

7. A method for rapid propagation of cold-resistant apple rootstock tissue culture according to claim 1, characterized in that: In step 4, the survival rate was calculated after 7-14 days of culture.

8. A method for rapid propagation of cold-resistant apple rootstocks through tissue culture according to claim 1, characterized in that: In step 5, the subculture proliferation medium uses WPM medium as the basic medium, adds 6-benzylaminoadenine, indolebutyric acid, gibberellin, humic acid, sucrose, and agar, and adjusts the pH value to 5.6-5.

8.

9. A method for rapid propagation of cold-resistant apple rootstocks through tissue culture according to claim 1, characterized in that: In step 5, the rooting medium uses 1 / 2 MS medium as the basic medium, and is supplemented with indolebutyric acid, naphthaleneacetic acid, activated carbon, seaweed extract, sucrose, and agar, and the pH value is adjusted to 5.6-5.

8.

10. The method for rapid propagation of cold-resistant apple rootstocks through tissue culture according to claim 1, wherein: In step 5, one value-added bud is inoculated into each tissue culture bottle, and the culture conditions are a temperature of 25±2° C., a humidity of 50-70%, and a culture period of 20-35 days.

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