Tissue culture and rapid propagation method for preventing calcium deficiency dwarf rootstock of apples

Abstract

The invention relates to the technical field of plant tissue culture, in particular to an apple calcium deficiency prevention dwarf rootstock tissue culture rapid propagation method which comprises the following preparation steps: selecting top tender shoots of apple calcium deficiency prevention dwarf rootstocks, cleaning and disinfecting the top tender shoots, and inoculating the top tender shoots into a differentiation induction culture medium for culture to obtain cluster buds; and inoculating the cluster buds into a proliferation subculture medium, carrying out proliferation culture to obtain proliferation buds, inoculating the proliferation buds into a rooting medium, and inducing rooting. According to the method, the components of all the culture media are optimized, breeding is carried out for apple calcium-deficiency-preventing dwarf rootstock tissue culture, on the premise that the seedling raising time is shortened, the terminal bud differentiation induction survival rate reaches 80% or above, the bud seedling multiplication coefficient is 5, the rooting rate reaches 95% or above, rapid and mass breeding of the good germplasm of the apple calcium-deficiency-preventing dwarf rootstock is achieved, and the survival rate is increased. And a powerful technical support is provided for genetic improvement and industrial development of the apple calcium-deficiency-preventing dwarfing rootstock.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for rapid tissue culture propagation of apple rootstocks to prevent calcium deficiency dwarfing. Background Art

[0002] Apple rootstocks are a crucial component of apple cultivation. They serve as the rootstock of the apple tree, also known as the rootstock. They provide support, absorb nutrients, regulate water, and transfer nutrients throughout the tree's growth. They also influence the tree's disease resistance, cold tolerance, and adaptability. Therefore, the quality of the rootstock is a crucial factor influencing apple tree cultivation. Improper selection can reduce not only the quality of the tree but also its fruit yield.

[0003] As a high-quality apple rootstock, calcium-deficiency-resistant apple dwarfing rootstocks enjoy high market demand, and their breeding and promotion are crucial to the development of the entire industry. However, while numerous studies have examined the asexual propagation of calcium-deficiency-resistant apple dwarfing rootstocks, methods such as grafting and cuttings remain ineffective in rapidly producing large numbers of high-quality plants. Therefore, establishing an efficient and stable rapid propagation system for calcium-deficiency-resistant apple dwarfing rootstocks using high-quality plants as material would be crucial for the genetic improvement of calcium-deficiency-resistant apple dwarfing rootstocks and the development and application of new varieties. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for rapid tissue culture propagation of apple dwarfing rootstocks for preventing calcium deficiency, so as to achieve rapid and large-scale propagation of excellent germplasm of apple dwarfing rootstocks for preventing calcium deficiency, and thus provide strong technical support for the genetic improvement and industrial development of apple dwarfing rootstocks for preventing calcium deficiency.

[0005] To achieve the above object, the present invention provides a method for tissue culture and rapid propagation of apple rootstocks to prevent calcium deficiency dwarfing, comprising the following preparation steps:

[0006] S1. Selecting the top tender shoots of apple calcium deficiency dwarfing rootstocks, cleaning and disinfecting the top tender shoots, and then inoculating them into a differentiation induction medium for cultivation to obtain clustered shoots;

[0007] S2. Inoculate the clustered buds into a proliferation subculture medium for proliferation culture. After obtaining the proliferated buds, inoculate the proliferated buds into a rooting medium to induce rooting.

[0008] In the present invention, the top tender shoots in S1 are tender shoots of plants that are healthy and free of diseases and insect pests.

[0009] In the present invention, the disinfection process in S1 includes: disinfecting and washing the top tender shoots with an ethanol solution, then disinfecting and washing with a mercuric chloride solution, and then placing the top tender shoots on a sterilized filter paper to absorb moisture from the surface of the tender shoots.

[0010] In the present invention, the mass concentration of the ethanol solution is 70%-80%, and the disinfection time of the ethanol solution is 25-35 seconds; the mass concentration of the mercuric chloride solution is 0.08%-0.12%, and the disinfection time of the mercuric chloride solution is 5-7 minutes.

[0011] In the present invention, the differentiation induction medium in S1 includes WPM medium + 6-benzylaminopurine 0.12-1 mg / L + indolebutyric acid 0.11-1 mg / L + sucrose 20-30 g / L + agar 6-7 g / L.

[0012] In the present invention, the components of the differentiation induction medium are preferably: NH4NO3 0.54 g / L, CaCl2·2H2O 0.065 g / L, MgSO4·7H2O 0.85 g / L, KH2PO4 0.74 g / L, MnSO4·4H2O 25.6 mg / L, ZnSO4·7H2O 10.5 mg / L, H3BO3 7.8 mg / L, NaMoO4·2H2O 0.58 mg / L, CuSO4·5H2O 0.049 mg / L, CoCl2·6H2O 0.072 mg / L, KI 0.07 mg / L, niacin 0.68 mg / L, vitamin B1 1.58 mg / L, glycine 5.23 mg / L, vitamin B6 0.87 mg / L, inositol 100 mg / L, FeSO4·7H2O 28.75 mg / L, Na2-EDTA33.25 mg / L, 6-benzylaminopurine0.12-1 mg / L, indolebutyric acid0.11-1 mg / L, sucrose25 g / L, agar6 g / L.

[0013] In the present invention, the culture temperature in S1 is 23-27° C., the culture humidity is 50%-70%, the culture time is 7-14 days, and the culture pH is 5.8-6.0.

[0014] In the present invention, the proliferation subculture medium in S2 is 1 / 2MS medium.

[0015] In the present invention, the components of 1 / 2MS culture medium are preferably: NH4NO3825mg / L, KNO3950mg / L, KH2PO485mg / L, MgSO4·7H2O 185mg / L, CaCl2·2H2O220mg / L, KI 0.83mg / L, H3BO36.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O 27.8mg / L, Na2-EDTA37.3mg / L, inositol 100mg / L, glycine 2mg / L, vitamin B10.1 mg / L, vitamin B6 0.5 mg / L, niacin 0.5 mg / L.

[0016] In the present invention, the proliferation culture temperature in S2 is 18-25° C., the humidity is 40%-50%, and the number of days is 30-35 days.

[0017] In the present invention, the S2 mesorooting medium includes WPM medium+6-benzylaminopurine 0.12-1 mg / L+indolebutyric acid 0.11-1 mg / L+sucrose 20-30 g / L+agar 6-7 g / L.

[0018] In the present invention, the components of the rooting medium are preferably: NH4NO3 0.25 g / L, CaCl2·2H2O 0.075 g / L, MgSO4·7H2O 0.65 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 23.15 mg / L, ZnSO4·7H2O 12.24 mg / L, H3BO3 6.08 mg / L, NaMoO4·2H2O 0.33 mg / L, CuSO4·5H2O 0.027 mg / L, CoCl2·6H2O 0.028 mg / L, KI 0.05 mg / L, niacin 0.46 mg / L, vitamin B1 1.5 mg / L, glycine 3.5 mg / L, vitamin B6 0.55 mg / L, inositol 100 mg / L, FeSO4·7H2O 26.85 mg / L, Na2-EDTA35.25 mg / L, 6-benzylaminopurine0.12-1 mg / L, indolebutyric acid0.11-1 mg / L, sucrose25 g / L, agar6 g / L.

[0019] In the present invention, the rooting medium is inoculated in the following manner: one proliferation bud is inoculated into each rooting medium.

[0020] In the present invention, the rooting induction temperature in S2 is 23-27° C., the humidity is 50%-70%, and the time is 20-35 days.

[0021] In the present invention, the differentiation induction medium, proliferation subculture medium, and rooting medium all need to be sterilized before use. The sterilization step includes placing the culture medium in an autoclave and sterilizing it at 120-122°C for 18-22 minutes.

[0022] The present invention has the following beneficial effects:

[0023] The present invention provides a method for rapid tissue culture propagation of apple calcium-deficient dwarfing rootstocks, comprising the following steps: selecting top tender buds of the apple calcium-deficient dwarfing rootstock, cleaning and disinfecting the tender buds, and then inoculating them into a differentiation induction medium for cultivation to obtain clustered buds; inoculating the clustered buds into a proliferation subculture medium for proliferation culture to obtain proliferating buds; and inoculating the proliferating buds into a rooting medium for rooting induction. The present invention optimizes the composition of the culture medium based on the characteristics of the apple calcium-deficient dwarfing rootstock. While shortening the seedling raising time, the survival rate of terminal bud differentiation induction is as high as over 80%, the seedling proliferation coefficient is 5, and the rooting rate reaches over 95%. This method achieves rapid and large-scale propagation of high-quality germplasm of the apple calcium-deficient dwarfing rootstock, providing strong technical support for the genetic improvement and industrial development of the apple calcium-deficient dwarfing rootstock.

[0024] The present invention further defines that the components of the differentiation induction medium and the rooting medium each include: WPM medium + 6-benzylaminopurine 0.12-1 mg / L + indolebutyric acid 0.11-1 mg / L + sucrose 20-30 g / L + agar 6-7 g / L. Among them, 6-benzylaminopurine (6-BA) as a cytokinin can activate cell division and differentiation, promote the germination of top tender shoots and the formation of clustered buds, so that the differentiation induction survival rate of the top tender shoots is as high as more than 80%, and the seedling proliferation coefficient reaches 5 times, realizing rapid asexual reproduction. Indolebutyric acid (IBA) as an auxin promotes root primordium differentiation and root system development, synergistically acts with 6-BA, balances cytokinins, and prevents seedlings from growing too tall; high concentration (1 mg / L) directly induces rooting and shortens the seedling cultivation period.

[0025] The present invention adds indolebutyric acid and 6-benzylaminopurine to the WPM culture medium, realizes "bud-root synchronous development", breaks through the limitation of a single hormone, promotes the successful tissue culture seedlings of apple dwarfing rootstocks for preventing calcium deficiency, and realizes the rapid propagation of apple dwarfing rootstocks for preventing calcium deficiency.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a graph showing the growth of the top tender shoots of apple rootstocks for preventing calcium deficiency dwarfing in Example 1 after being cultured in a differentiation induction medium;

[0028] Figure 2 This is a diagram showing the growth of the clustered buds in Example 1 after culture in a proliferation subculture medium;

[0029] Figure 3 This is a diagram of the root morphology of Example 1 after 25 days of root induction. DETAILED DESCRIPTION

[0030] The present invention is further described below with reference to the accompanying drawings and examples. Unless otherwise defined, technical or scientific terms used herein shall have the same meanings as those commonly understood by persons of ordinary skill in the art to which the present invention pertains. The above-mentioned features or features described in the specific examples of the present invention may be combined in any manner. These specific examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0031] Example 1

[0032] Prepare differentiation induction medium, the components of which include:

[0033] NH4NO30.54 g / L, CaCl2·2H2O 0.065g / L, MgSO4·7H2O 0.85g / L, KH2PO40.74 g / L, MnSO4·4H2O 25.6mg / L, ZnSO4·7H2O 10.5mg / L, H3BO37.8 mg / L, NaMoO4·2H2O 0.58mg / L, CuSO4·5H2O 0.049mg / L, CoCl2·6H2O 0.072mg / L, KI 0.07mg / L, niacin 0.68mg / L, vitamin B11.58mg / L, glycine 5.23mg / L, vitamin B6 0.87 mg / L, inositol 100mg / L, FeSO4·7H2O 28.75mg / L, Na2-EDTA 33.25 mg / L, 6-benzylaminopurine 1 mg / L, indolebutyric acid 1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0034] Prepare proliferation subculture medium (1 / 2MS medium), the components of which include: NH4NO3 825 mg / L, KNO3 950 mg / L, KH2PO4 85 mg / L, MgSO4·7H2O 185 mg / L, CaCl2·2H2O 220 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA37.3 mg / L, inositol100 mg / L, glycine2 mg / Lvitamin B10.1 mg / L, vitamin B60.5 mg / L, niacin0.5 mg / L.

[0035] Prepare rooting medium, the components of which include: NH4NO3 0.25 g / L, CaCl2·2H2O 0.075 g / L, MgSO4·7H2O 0.65 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 23.15 mg / L, ZnSO4·7H2O 12.24 mg / L, H3BO3 6.08 mg / L, NaMoO4·2H2O 0.33 mg / L, CuSO4·5H2O 0.027 mg / L, CoCl2·6H2O0.028 mg / L, KI 0.05 mg / L, niacin 0.46 mg / L, vitamin B1 1.5 mg / L, glycine 3.5 mg / L, vitamin B6 0.55 mg / L, inositol 100 mg / L, FeSO4·7H2O 26.85 mg / L, Na2-EDTA 35.25 mg / L, 6-benzylaminopurine 1 mg / L, indolebutyric acid 1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0036] Before use, the above-mentioned differentiation induction medium, proliferation subculture medium, and rooting medium were placed in a high pressure sterilizer and sterilized at 121° C. for 20 minutes.

[0037] S1. Select the top shoots of healthy, disease-free, and insect-free apple dwarfing rootstocks. Wash the top shoots three times with sterile distilled water. Place the top shoots in a clean bench and disinfect them with 75% ethanol for 30 seconds, rinse them three times with sterile water, and then disinfect them with 0.1% mercuric chloride solution for 6 minutes. Rinse them three times with sterile water. Place the top shoots on sterile filter paper to dry them. Then, inoculate them into differentiation induction medium and culture them for 7 days. Clustered shoots were obtained, and the survival rate was 85%. Culture conditions were: 25°C, 50% humidity, and pH 5.8. One top shoot was inoculated into each differentiation induction medium.

[0038] S2. Inoculate the clustered buds into a proliferation subculture medium for 35 days, achieving a proliferation coefficient of 5. Culture conditions: 25°C, 40% humidity, with one clustered bud inoculated into each proliferation subculture medium. Then, inoculate into a rooting medium and induce rooting for 25 days, achieving a rooting rate of 95%. Rooting induction conditions: 25°C, 50% humidity, with one clustered bud inoculated into each rooting medium.

[0039] After S1 was cultured in differentiation induction medium, the top buds were as follows Figure 1 As shown. Figure 1 It can be seen that after the top shoots of the apple calcium deficiency dwarfing rootstock were inoculated into the differentiation induction medium, they survived well under the culture conditions, with leaves stretching and normal color, which confirmed the effectiveness of the differentiation induction medium of the present invention.

[0040] After the clustered buds of S2 were placed in the proliferation subculture medium, the proliferation situation was as follows: Figure 2 As shown. Figure 1 and Figure 2 It can be seen that the growth state of the clustered buds is that multiple healthy new buds are differentiated from the single original tender bud, and the increase in the number of buds is obviously seen, which confirms the role of the proliferation and subculture medium in promoting the rapid reproduction of apple dwarf rootstocks to prevent calcium deficiency, and shows that the proliferation and subculture medium of the present invention can effectively achieve the quantitative expansion of excellent germplasm.

[0041] S2 in rooting medium, the situation after rooting induction is as follows Figure 3 As shown. Figure 3 It can be seen that after only 25 days of cultivation, the root morphology has grown at the base of the bud, and the number, length, thickness and root color of the roots are all at normal levels, indicating that the rooting medium of the present invention can successfully induce the bud to form a complete root system, laying the foundation for the survival of tissue culture seedlings after transplantation.

[0042] Comparative Example 1

[0043] Prepare differentiation induction medium, the components of which include:

[0044] NH4NO30.54 g / L, CaCl2·2H2O 0.065g / L, MgSO4·7H2O 0.85g / L, KH2PO40.74 g / L, MnSO4·4H2O 25.6mg / L, ZnSO4·7H2O 10.5mg / L, H3BO37.8 mg / L, NaMoO4·2H2O 0.58mg / L, CuSO4·5H2O 0.049mg / L, CoCl2·6H2O 0.072mg / L, KI 0.07mg / L, niacin 0.68mg / L, vitamin B11.58mg / L, glycine 5.23mg / L, vitamin B6 0.87 mg / L, inositol 100mg / L, FeSO4·7H2O 28.75 mg / L, Na2-EDTA33.25 mg / L.

[0045] Prepare proliferation subculture medium (1 / 2MS medium), the components of which include: NH4NO3 825 mg / L, KNO3 950 mg / L, KH2PO4 85 mg / L, MgSO4·7H2O 185 mg / L, CaCl2·2H2O 220 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA37.3 mg / L, inositol100 mg / L, glycine2 mg / Lvitamin B10.1 mg / L, vitamin B60.5 mg / L, niacin0.5 mg / L.

[0046] Prepare rooting medium, the components of which include: NH4NO3 0.25 g / L, CaCl2·2H2O 0.075 g / L, MgSO4·7H2O 0.65 g / L, KH2PO4 0.46 g / L, MnSO4·4H2O 23.15 mg / L, ZnSO4·7H2O 12.24 mg / L, H3BO3 6.08 mg / L, NaMoO4·2H2O 0.33 mg / L, CuSO4·5H2O 0.027 mg / L, CoCl2·6H2O0.028 mg / L, KI 0.05 mg / L, niacin 0.46 mg / L, vitamin B1 1.5 mg / L, glycine 3.5 mg / L, vitamin B6 0.55 mg / L, inositol 100 mg / L, FeSO4·7H2O 26.85 mg / L, Na2-EDTA35.25 mg / L.

[0047] Before use, the above-mentioned differentiation induction medium, proliferation subculture medium, and rooting medium were placed in a high pressure sterilizer and sterilized at 121° C. for 20 minutes.

[0048] S1. Select top shoots from healthy, disease- and insect-free apple dwarfing rootstocks. Wash the top shoots three times with sterile distilled water. Place the top shoots in a clean bench and disinfect them with 75% ethanol for 30 seconds, rinse them three times with sterile water, and then disinfect them with 0.1% mercuric chloride solution for 6 minutes. Rinse them three times with sterile water. Place the top shoots on sterile filter paper to dry them. Then, inoculate them in differentiation-inducing medium and culture them for 7 days. Clustered shoots were obtained, with a survival rate of 32%. Culture conditions were: 25°C, 50% humidity, and pH 5.8. One top shoot was inoculated into each differentiation-inducing medium.

[0049] S2. Inoculate the clustered buds into a proliferation subculture medium for 35 days, achieving a proliferation coefficient of 2. Culture conditions were: 25°C, 40% humidity, with one clustered bud inoculated into each proliferation subculture medium. Then, inoculate the buds into a rooting medium and induce rooting for 25 days. Rooting was difficult. Rooting induction conditions were: 25°C, 50% humidity, with one clustered bud inoculated into each rooting medium.

[0050] Comparative Example 2

[0051] Prepare differentiation induction medium, the components of which include:

[0052] NH4NO3 1650mg / L, KNO3 1900mg / L, KH2PO4 170mg / L, MgSO4·7H2O 370mg / L, CaCl2·2H2O 440mg / L, KI 0.83mg / L, H3BO 36.2mg / L, MnSO4·4H2O 22.3mg / L, ZnSO4·7H2O 8.6mg / L, Na2MoO4·2H2O 0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O 27.8mg / L, Na2-EDTA 37.3 mg / L, inositol 100mg / L, glycine 2mg / L, vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, niacin 0.5mg / L, 6-benzylaminopurine 1mg / L, indolebutyric acid 1mg / L.

[0053] Prepare proliferation subculture medium (1 / 2MS medium), the components of which include: NH4NO3 825 mg / L, KNO3 950 mg / L, KH2PO4 85 mg / L, MgSO4·7H2O 185 mg / L, CaCl2·2H2O 220 mg / L, KI 0.83 mg / L, H3BO3 6.2 mg / L, MnSO4·4H2O 22.3 mg / L, ZnSO4·7H2O 8.6 mg / L, Na2MoO4·2H2O 0.25 mg / L, CuSO4·5H2O 0.025 mg / L, CoCl2·6H2O 0.025 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA37.3 mg / L, inositol100 mg / L, glycine2 mg / Lvitamin B10.1 mg / L, vitamin B60.5 mg / L, niacin0.5 mg / L.

[0054] Prepare rooting medium. The components of the rooting medium include: 1650 mg / L NH4NO3, 1900 mg / L KNO3, 170 mg / L KH2PO4, 370 mg / L MgSO4·7H2O, 440 mg / L CaCl2·2H2O, 0.83 mg / L KI, 36.2 mg / L H3BO, 22.3 mg / L MnSO4·4H2O, 8.6 mg / L ZnSO4·7H2O, 0.25 mg / L Na2MoO4·2H2O, 0.025 mg / L CuSO4·5H2O, 0.025 mg / L CoCl2·6H2O, 27.8 mg / L FeSO4·7H2O, 37.3 mg / L Na2-EDTA, 100 mg / L inositol, 2 mg / L glycine, and 0.1 mg vitamin B1. mg / L, vitamin B6 0.5 mg / L, niacin 0.5 mg / L, 6-benzylaminopurine 1 mg / L, indolebutyric acid 1 mg / L.

[0055] Before use, the above-mentioned differentiation induction medium, proliferation subculture medium, and rooting medium were placed in a high pressure sterilizer and sterilized at 121° C. for 20 minutes.

[0056] S1. Select the top shoots of healthy, disease- and insect-free apple dwarfing rootstocks. Wash the top shoots three times with sterile distilled water. Place the top shoots in a clean bench and disinfect them with 75% ethanol for 30 seconds, rinse them three times with sterile water, and then disinfect them with 0.1% mercuric chloride solution for 6 minutes. Rinse them three times with sterile water. Place the top shoots on sterile filter paper to dry the surface moisture. Then, inoculate them into differentiation induction medium and culture them for 7 days to obtain clustered shoots. The survival rate was 60%. Culture conditions were: 25°C, 50% humidity, pH 5.8. One top shoot was inoculated into each differentiation induction medium.

[0057] S2. Inoculate the clustered buds into a proliferation subculture medium for 35 days, achieving a proliferation coefficient of 2. Culture conditions: 25°C, 40% humidity, with one clustered bud inoculated into each proliferation subculture medium. Then, inoculate into a rooting medium and induce rooting for 25 days, achieving a rooting rate of 75%. Rooting induction conditions: 25°C, 50% humidity, with one clustered bud inoculated into each rooting medium.

[0058] Compared to Example 1, Comparative Example 1 omitted 6-benzylaminopurine, indolebutyric acid, sucrose, and agar from the differentiation induction medium and rooting medium. Its survival rate, proliferation coefficient, and rooting rate were lower than those in Example 1. This is because the absence of 6-benzylaminopurine prevents effective activation of cell division and promotion of shoot differentiation, leading to decreased survival rate and proliferation coefficient. Furthermore, the absence of indolebutyric acid hinders root induction, resulting in incomplete root development and difficulty in rooting.

[0059] Compared to Example 1, in Comparative Example 2, the basal medium for the differentiation induction medium and rooting medium was changed to MS medium. The salt ion concentration in MS medium was significantly higher than that in WPM medium. Therefore, despite the addition of two hormones, 6-benzylaminopurine and indolebutyric acid, the higher salt ion concentration disrupted the ion-hormone balance. This resulted in a significant decrease in the survival rate, proliferation coefficient, and rooting rate in Comparative Example 2.

[0060] 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 preventing calcium deficiency in apple dwarfing rootstocks by tissue culture and rapid propagation, characterized in that: The method comprises the following preparation steps: S1. Selecting the top tender shoots of apple calcium deficiency dwarfing rootstocks, cleaning and disinfecting the top tender shoots, and then inoculating them into a differentiation induction medium for cultivation to obtain clustered shoots; S2. Inoculate the clustered buds into a proliferation subculture medium for proliferation culture. After obtaining the proliferated buds, inoculate the proliferated buds into a rooting medium to induce rooting.

2. A method for preventing calcium deficiency dwarfing rootstock tissue culture and rapid propagation of apple according to claim 1, characterized in that, The top shoots in S1 are the shoots of healthy and pest-free plants.

3. A method for preventing calcium deficiency dwarfing rootstock tissue culture and rapid propagation of apple according to claim 1, characterized in that, The disinfection process in S1 includes: disinfecting and washing the top tender shoots with an ethanol solution, then disinfecting and washing with a mercuric chloride solution, and then placing the top tender shoots on a sterilized filter paper to absorb moisture from the surface of the tender shoots.

4. A method for preventing calcium deficiency dwarfing rootstock tissue culture and rapid propagation of apple according to claim 3, characterized in that, The mass concentration of the ethanol solution is 70%-80%, and the disinfection time of the ethanol solution is 25-35 seconds; the mass concentration of the mercuric chloride solution is 0.08%-0.12%, and the disinfection time of the mercuric chloride solution is 5-7 minutes.

5. The method for preventing calcium deficiency dwarfing rootstock tissue culture and rapid propagation of apple according to claim 1, wherein: The differentiation induction medium in S1 includes WPM medium + 6-benzylaminopurine 0.12-1 mg / L + indolebutyric acid 0.11-1 mg / L + sucrose 20-30 g / L + agar 6-7 g / L.

6. The method for preventing calcium deficiency dwarfing rootstock tissue culture and rapid propagation of apple according to claim 1, wherein: The culture temperature in S1 is 23-27° C., the culture humidity is 50%-70%, the culture time is 7-14 days, and the culture pH is 5.8-6.

0.

7. The method for preventing calcium deficiency dwarfing rootstock tissue culture and rapid propagation of apple according to claim 1, wherein: The proliferation subculture medium in S2 is 1 / 2MS medium.

8. The method for preventing calcium deficiency dwarfing rootstock tissue culture and rapid propagation of apple according to claim 1, wherein: The temperature of proliferation culture in S2 is 18-25°C, the humidity is 40%-50%, and the number of days is 30-35 days.

9. The method for preventing calcium deficiency dwarfing rootstock tissue culture and rapid propagation of apple according to claim 1, wherein: The S2 rooting medium includes WPM medium + 6-benzylaminopurine 0.12-1 mg / L + indolebutyric acid 0.11-1 mg / L + sucrose 20-30 g / L + agar 6-7 g / L.

10. The method for preventing calcium deficiency in apple dwarfing rootstocks through tissue culture and rapid propagation according to claim 1, wherein: The rooting induction temperature in S2 is 23-27°C, the humidity is 50%-70%, and the time is 20-35 days.

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