Tissue culture and rapid propagation method for saline-alkaline tolerant cherry rootstock

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a cherry saline-alkaline tolerant rootstock tissue culture and rapid propagation method which comprises the following preparation steps: selecting top tender shoots of cherry saline-alkaline tolerant 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. By optimizing the components of each culture medium and breeding the saline-alkaline tolerant cherry rootstock plants, excellent nursery stocks can be quickly obtained, the seedling culture time is effectively shortened, and the seedling culture cost is reduced.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant tissue culture, in particular to a cherry salt-tolerant rootstock tissue culture rapid propagation method. BACKGROUND

[0002] Cherry rootstock is an important part of cherry cultivation, which is the rootstock of cherry tree, also known as rootstock, and is needed in the process of cherry tree cultivation. It plays a supporting, absorbing, regulating water and nutrient transfer role in the growth process of cherry tree. Therefore, the quality and selection of cherry rootstock is an important factor affecting the growth, fruit yield and quality of cherry tree. At the same time, it also affects the disease resistance, cold resistance and adaptability of cherry tree. If not properly selected, it will not only reduce the quality and yield of the fruit tree, but also cause the quality of the fruit on the tree to be poor.

[0003] Cherry salt-tolerant rootstock is a good cherry rootstock in China, and the market demand is large. Its breeding and popularization play a crucial role in the development of the entire industry. Therefore, the research on cherry salt-tolerant rootstock urgently needs a more efficient and rapid method for breeding, and asexual reproduction is one of the important ways to solve this problem. At present, there have been many studies on asexual reproduction of cherry salt-tolerant rootstock, but methods such as grafting and cutting still cannot achieve the expansion of a large number of excellent plants in a short time. Therefore, if the cherry salt-tolerant rootstock excellent plant is used as the material, a high-efficiency and stable cherry salt-tolerant rootstock rapid propagation system is established, which has important significance for the development and application of genetic improvement and new variety breeding of cherry salt-tolerant rootstock. SUMMARY

[0004] The purpose of the present application is to provide a cherry salt-tolerant rootstock tissue culture rapid propagation method, which realizes the rapid and large-scale propagation of cherry salt-tolerant rootstock excellent germplasm, and further provides strong technical support for the genetic improvement and industrial development of cherry salt-tolerant rootstock.

[0005] In order to achieve the above-mentioned purpose, the present application provides a cherry salt-tolerant rootstock tissue culture rapid propagation method, which comprises the following preparation steps:

[0006] S1, selecting the top buds of cherry salt-tolerant rootstock, washing and disinfecting the top buds, and inoculating them in a differentiation induction medium for culture to obtain a cluster of buds;

[0007] S2, inoculating the cluster of buds in a proliferation subculture medium for proliferation culture to obtain proliferation buds, and inoculating the proliferation buds in a rooting culture medium for root induction.

[0008] In the present application, the top buds of cherry salt-tolerant rootstock in S1 are the buds of healthy and pest-free plants.

[0009] In the present application, the disinfection process in S1 comprises: washing the top sprouts after disinfecting with an ethanol solution, disinfecting with a mercuric chloride solution, washing, and then placing the top sprouts on sterilized filter paper to absorb surface moisture.

[0010] In the present application, the concentration of the ethanol solution is 70%-80%, the ethanol solution disinfection time is 25-45s, the concentration of the mercuric chloride solution is 0.1%-0.15%, and the mercuric chloride solution disinfection time is 5-7min.

[0011] In the present application, the differentiation induction medium in S1 comprises: MS medium + 6-benzylaminopurine 0.13-1mg / L + indolebutyric acid 0.17-1mg / L + naphthaleneacetic acid 0.11-0.5mg / L, sucrose 20-30g / L + agar 6-7g / L.

[0012] In the present application, the components of the differentiation induction medium are preferably: NH4NO30.4g / L, CaCl2·2H2O 0.056g / L, MgSO4·7H2O 0.75g / L, KH2PO40.59g / L, MnSO4·4H2O 23.5mg / L, ZnSO4·7H2O 9.12mg / L, H3BO35.56mg / L, NaMoO4·2H2O 0.53mg / L, CuSO4·5H2O 0.042mg / L, CoCl2·6H2O 0.035mg / L, KI 0.032mg / L, nicotinic acid 0.57mg / L, vitamin B1 1.23mg / L, glycine 3.35mg / L, vitamin B60.65mg / L, myo-inositol 100mg / L, FeSO4·7H2O 27.65mg / L, Na2-EDTA 35.25mg / L, 6-benzylaminopurine 0.13-1mg / L, indolebutyric acid 0.17-1mg / L, naphthaleneacetic acid 0.11-0.5mg / L, sucrose 25g / L, agar 6g / L.

[0013] In the present application, the culture temperature in S1 is 23-27℃, the culture humidity is 50%-70%, the culture time is 7-14d, and the culture pH is 5.8-6.0.

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

[0015] In the present application, the components of the 1 / 2 culture medium are preferably: NH4NO3825 mg / L, KNO3950 mg / L, KH2PO485 mg / L, MgSO4·7H2O 185 mg / L, CaCl2·2H2O 220 mg / L, KI 0.83 mg / L, H3BO36.2 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-EDTA 37.3 mg / L, myo-inositol 100 mg / L, glycine 2 mg / L, vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0016] In the present application, the temperature of the proliferation culture in S2 is 18-25℃, the humidity is 40%-50%, and the days of the proliferation culture are 30-35d.

[0017] In the present application, the rooting culture medium in S2 comprises: MS culture medium + 6-benzylaminopurine 0.08-1 mg / L + indole-3-butyric acid 0.2-1 mg / L + naphthaleneacetic acid 0.1-0.5 mg / L, sucrose 20-30 g / L + agar 6-7 g / L.

[0018] In the present application, the components of the rooting culture medium are preferably: NH4NO30.6 g / L, CaCl2·2H2O 0.068 g / L, MgSO4·7H2O 0.56 g / L, KH2PO40.46 g / L, MnSO4·4H2O 23.5 mg / L, ZnSO4·7H2O 11.4 mg / L, H3BO35.45 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.037 mg / L, CoCl2·6H2O 0.035 mg / L, KI 0.04 mg / L, nicotinic acid 0.47 mg / L, vitamin B1 1.04 mg / L, glycine 3.5 mg / L, vitamin B6 0.7 mg / L, myo-inositol 100 mg / L, FeSO4·7H2O 27.75 mg / L, Na2-EDTA 35.65 mg / L, 6-benzylaminopurine 0.08-1 mg / L, indole-3-butyric acid 0.2-1 mg / L, naphthaleneacetic acid 0.1-0.5 mg / L, sucrose 25 g / L, agar 6 g / L.

[0019] In the present application, the temperature for inducing rooting is 18-25℃, the humidity is 50%-70%, and the days for inducing rooting are 20-35d.

[0020] In the present application, the above-mentioned differentiation induction medium, proliferation subculture medium and rooting medium all need to be sterilized before use. The sterilization process includes: placing the above-mentioned medium in a high-pressure sterilization pot, and sterilizing at a temperature of 120-122 DEG C for 19-21 min.

[0021] The present application has the following beneficial effects:

[0022] The present application provides a method for tissue culture and rapid propagation of cherry salt-tolerant rootstock, comprising the following preparation steps: selecting the top tender buds of cherry salt-tolerant rootstock, washing and disinfecting the top tender buds, and then inoculating them into a differentiation induction medium for culture to obtain a cluster of buds; inoculating the cluster of buds into a proliferation subculture medium for proliferation culture to obtain proliferated buds, and inoculating the proliferated buds into a rooting medium for root induction.

[0023] After the differentiation induction medium of the present application is used for culture, the survival rate of the top bud differentiation induction is as high as 80% or more, after the proliferation subculture medium is used for culture, the bud seedling proliferation coefficient is 4-5, and after the rooting medium is used for culture, the rooting rate can reach 95%, the propagation effect is excellent, and the rapid and large-scale propagation of the cherry salt-tolerant rootstock is realized.

[0024] The present application further limits the differentiation induction medium to include: MS medium + 6-benzylaminopurine 0.13-1 mg / L + indole-3-butyric acid 0.17-1 mg / L + naphthalene acetic acid 0.11-0.5 mg / L, sucrose 20-30 g / L + agar 6-7 g / L. The rooting medium includes: MS medium + 6-benzylaminopurine 0.08-1 mg / L + indole-3-butyric acid 0.2-1 mg / L + naphthalene acetic acid 0.1-0.5 mg / L, sucrose 20-30 g / L + agar 6-7 g / L. The present application adds 6-benzylaminopurine, indole-3-butyric acid and naphthalene acetic acid to the MS medium, which act together as plant growth regulators on the stages of cell differentiation, bud proliferation and root development. By adjusting the endogenous hormone balance, the tissue culture efficiency is significantly improved.

[0025] 6-benzylaminopurine, as a cytokinin, dominates the differentiation of tender buds, promotes cell division and bud primordium formation, drives the differentiation of tender buds and the proliferation of cluster of buds, and makes them form multiple bud points quickly. Indole-3-butyric acid, as an auxin, dominates root development, induces root development and vascular bundle formation, and improves the rooting rate and root quality. Naphthalene acetic acid, as an auxiliary auxin, assists in root formation and bud differentiation, enhances the synergistic effect of hormones, and stabilizes the level of auxin. The three work together to realize the optimization of the whole process of "differentiation-proliferation-rooting".

[0026] The technical solutions of the present application are described in further detail below with reference to the accompanying drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a growth condition diagram of cherry salt-tolerant rootstock top bud differentiation induction medium after culture in Example 1;

[0028] Figure 2 is a growth condition diagram of the chrysanthemum after culture in the proliferation subculture medium in Example 1;

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

[0030] The present application is further described below with reference to the accompanying drawings and examples. Unless otherwise defined, the technical terms or scientific terms used in the present application should be understood as the usual meanings understood by persons having ordinary skills in the art to which the present application belongs. The features mentioned above or the features mentioned in the specific examples can be combined arbitrarily, and these specific examples are only used to illustrate the present application and are not used to limit the scope of the present application.

[0031] Example 1

[0032] The differentiation induction medium is configured, and the components of the differentiation induction medium include:

[0033] NH4NO30.4 g / L, CaCl2·2H2O 0.056 g / L, MgSO4·7H2O 0.75 g / L, KH2PO40.59 g / L, MnSO4·4H2O 23.5 mg / L, ZnSO4·7H2O 9.12 mg / L, H3BO35.56 mg / L, NaMoO4·2H2O 0.53 mg / L, CuSO4·5H2O 0.042 mg / L, CoCl2·6H2O 0.035 mg / L, KI 0.032 mg / L, nicotinic acid 0.57 mg / L, vitamin B1 1.23 mg / L, glycine 3.35 mg / L, vitamin B60.65 mg / L, myo-inositol 100 mg / L, FeSO4·7H2O 27.65 mg / L, Na2-EDTA 35.25 mg / L, 6-benzylaminopurine 1 mg / L, indolebutyric acid 1 mg / L, naphthaleneacetic acid 0.5 mg / L, sucrose 25 g / L, agar 6 g / L.

[0034] The proliferation subculture medium is configured, and the components of the proliferation subculture medium include:

[0035] NH4NO3825mg / L, KNO3950mg / L, KH2PO485mg / L, MgSO4·7H2O 185mg / L, CaCl2·2H2O 220mg / L, KI 0.83mg / L, H3BO36.2mg / 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 37.3mg / L, myo-inositol 100mg / L, glycine 2mg / L, vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5mg / L.

[0036] The rooting medium is configured, and components of the rooting medium include:

[0037] NH4NO30.6 g / L, CaCl2·2H2O 0.068g / L, MgSO4·7H2O 0.56g / L, KH2PO40.46 g / L, MnSO4·4H2O 23.5mg / L, ZnSO4·7H2O 11.4mg / L, H3BO35.45mg / L, NaMoO4·2H2O 0.54mg / L, CuSO4·5H2O 0.037mg / L, CoCl2·6H2O 0.035mg / L, KI 0.04mg / L, nicotinic acid 0.47mg / L, vitamin B1 1.04mg / L, glycine 3.5mg / L, vitamin B6 0.7 mg / L, myo-inositol 100mg / L, FeSO4·7H2O 27.75mg / L, Na2-EDTA 35.65mg / L, 6-benzylaminopurine 1mg / L, indole-3-butyric acid 1mg / L, naphthalene acetic acid 0.5mg / L, sucrose 25g / L, agar 6g / L.

[0038] After the configuration is completed, the above-mentioned differentiation medium, proliferation subculture medium and rooting medium are respectively placed in a high-pressure sterilization pot, and sterilized at a temperature of 121°C for 20min.

[0039] S1, select the top tender shoots which grow healthily, bear fruits abundantly and have no diseases and pests, wash the tender shoots with distilled water for three times, then sterilize the tender shoots with 75% volume concentration of ethanol solution for 30s in the super-clean bench, wash with sterile water for three times, sterilize the tender shoots with 0.1% mass concentration of mercury chloride solution for 6min, wash with sterile water for three times, place the sterilized top tender shoots on sterilized filter paper to absorb the water on the surface of the tender shoots, complete the sterilization process, then inoculate the tender shoots in the differentiation induction medium and culture for 7d to obtain the clustered shoots, and the survival rate is 85%. The culture conditions are as follows: 25°C, humidity 50%, pH 5.8, and inoculate one top tender shoot in each differentiation induction medium.

[0040] S2, inoculate the clustered shoots in the proliferation subculture medium, proliferate and culture for 35d, and the proliferation coefficient of the tender shoots is 5. The culture conditions are as follows: 25°C, humidity 40%, pH 5.8, and inoculate one clustered shoot in each proliferation subculture medium. Then, transfer the clustered shoots into the rooting medium, induce rooting for 25d, and the rooting rate is 98%. The conditions for inducing rooting are as follows: 25°C, humidity 50%, pH 5.8.

[0041] S1, the top tender shoots after being cultured in the differentiation induction medium are as shown in Figure 1 From Figure 1 it can be seen that the leaves of the top tender shoots are unfolded and the color is healthy after the culture, which indicates that the differentiation induction medium can effectively maintain the activity of the top tender shoots.

[0042] S2, the proliferation after the clustered shoots are placed in the proliferation subculture medium and cultured is as shown in Figure 2 From Figure 1 and Figure 2 it can be seen that a plurality of healthy new shoots are differentiated around the single original tender shoot, and the number of shoots is significantly increased.

[0043] S2, the situation after the clustered shoots are induced to root in the rooting medium is as shown in Figure 3 From Figure 3 it can be seen that only after 25d of culture, the base of the shoots has formed a large number of roots with moderate length, which are thick and have normal color.

[0044] Example 2

[0045] The differentiation induction medium is configured, and the components of the differentiation induction medium include:

[0046] NH4NO30.4 g / L, CaCl2*2H2O 0.056 g / L, MgSO4*7H2O 0.75 g / L, KH2PO40.59 g / L, MnSO4*4H2O 23.5 mg / L, ZnSO4*7H2O 9.12 mg / L, H3BO35.56 mg / L, NaMoO4*2H2O 0.53 mg / L, CuSO4*5H2O 0.042 mg / L, CoCl2*6H2O 0.035 mg / L, KI 0.032 mg / L, nicotinic acid 0.57 mg / L, vitamin B1 1.23 mg / L, glycine 3.35 mg / L, vitamin B6 0.65 mg / L, myo-inositol 100 mg / L, FeSO4*7H2O 27.65 mg / L, Na2-EDTA 35.25 mg / L, 6-benzylaminopurine 0.13 mg / L, indolebutyric acid 0.17 mg / L, naphthalene acetic acid 0.11 mg / L, sucrose 25 g / L, agar 6 g / L.

[0047] A proliferation subculture medium is configured, the components of the proliferation subculture medium including:

[0048] NH4NO3825 mg / L, KNO3950 mg / L, KH2PO485 mg / L, MgSO4*7H2O 185 mg / L, CaCl2*2H2O 220 mg / L, KI 0.83 mg / L, H3BO36.2 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-EDTA 37.3 mg / L, myo-inositol 100 mg / L, glycine 2 mg / L, vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0049] A rooting medium is configured, the components of the rooting medium including:

[0050] NH4NO30.6 g / L, CaCl2·2H2O 0.068 g / L, MgSO4·7H2O 0.56 g / L, KH2PO40.46 g / L, MnSO4·4H2O 23.5 mg / L, ZnSO4·7H2O 11.4 mg / L, H3BO35.45 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.037 mg / L, CoCl2·6H2O 0.035 mg / L, KI 0.04 mg / L, nicotinic acid 0.47 mg / L, vitamin B1 1.04 mg / L, glycine 3.5 mg / L, vitamin B60.7 mg / L, myo-inositol 100 mg / L, FeSO4·7H2O 27.75 mg / L, Na2-EDTA 35.65 mg / L, 6-benzylaminopurine 0.08 mg / L, indole-3-butyric acid 0.2 mg / L, naphthalene acetic acid 0.1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0051] After the configuration is completed, the above differentiation medium, proliferation subculture medium, and rooting medium are respectively placed in a high-pressure sterilization pot and sterilized at a temperature of 121°C for 20 min.

[0052] S1, select the top sprouts that are healthy, fruitful, and free of pests and diseases, wash the sprouts with distilled water three times, then sterilize the sprouts with an ethanol solution with a volume concentration of 75% for 30 s in a clean bench, wash with sterile water three times, sterilize the sprouts with a mercury chloride solution with a mass concentration of 0.1% for 6 min, wash with sterile water three times, place the sterilized top sprouts on sterilized filter paper to absorb the water on the surface of the sprouts, complete the sterilization process, then inoculate the sprouts in the differentiation induction medium and culture for 7 d to obtain the sprouts, and the survival rate is 82%. The culture conditions are: 25°C, humidity 50%, pH 5.8, and one top sprout is inoculated in each differentiation induction medium.

[0053] S2, inoculate the sprouts in the proliferation subculture medium, proliferate and subculture for 35 d, and the proliferation coefficient of the sprouts is 4. The culture conditions are: 25°C, humidity 40%, pH 5.8, and one sprout is inoculated in each proliferation subculture medium. Then, transfer the sprouts to the rooting medium, induce rooting for 25 d, and the rooting rate is 95%. The conditions for inducing rooting are: 25°C, humidity 50%, and pH 5.8.

[0054] Comparative Example 1

[0055] The differentiation induction medium is configured, and the components of the differentiation induction medium include:

[0056] NH4NO30.4 g / L, CaCl2*2H2O 0.056 g / L, MgSO4*7H2O 0.75 g / L, KH2PO40.59 g / L, MnSO4*4H2O 23.5 mg / L, ZnSO4*7H2O 9.12 mg / L, H3BO35.56 mg / L, NaMoO4*2H2O 0.53 mg / L, CuSO4*5H2O 0.042 mg / L, CoCl2*6H2O 0.035 mg / L, KI 0.032 mg / L, nicotinic acid 0.57 mg / L, vitamin B1 1.23 mg / L, glycine 3.35 mg / L, vitamin B6 0.65 mg / L, myo-inositol 100 mg / L, FeSO4*7H2O 27.65 mg / L, Na2-EDTA 35.25 mg / L, naphthalene acetic acid 0.5 mg / L, sucrose 25 g / L, agar 6 g / L.

[0057] A proliferation subculture medium was configured, the components of the proliferation subculture medium including:

[0058] NH4NO3825 mg / L, KNO3950 mg / L, KH2PO485 mg / L, MgSO4*7H2O 185 mg / L, CaCl2*2H2O 220 mg / L, KI 0.83 mg / L, H3BO36.2 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-EDTA 37.3 mg / L, myo-inositol 100 mg / L, glycine 2 mg / L, vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0059] A rooting medium was configured, the components of the rooting medium including:

[0060] NH4NO30.6 g / L, CaCl2·2H2O 0.068 g / L, MgSO4·7H2O 0.56 g / L, KH2PO40.46 g / L, MnSO4·4H2O 23.5 mg / L, ZnSO4·7H2O 11.4 mg / L, H3BO35.45 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.037 mg / L, CoCl2·6H2O 0.035 mg / L, KI 0.04 mg / L, nicotinic acid 0.47 mg / L, vitamin B1 1.04 mg / L, glycine 3.5 mg / L, vitamin B60.7 mg / L, myo-inositol 100 mg / L, FeSO4·7H2O 27.75 mg / L, Na2-EDTA 35.65 mg / L, naphthalene acetic acid 0.5 mg / L, sucrose 25 g / L, agar 6 g / L.

[0061] After the configuration is completed, the above differentiation medium, proliferation subculture medium, and rooting medium are respectively placed in an autoclave, and sterilized at a temperature of 121°C for 20 min.

[0062] S1, select the top sprouts that are healthy, fruitful, and free of pests and diseases, wash the sprouts with distilled water three times, then sterilize the sprouts with an ethanol solution with a volume concentration of 75% for 30 s in a clean bench, rinse with sterile water three times, sterilize the sprouts with a mercury chloride solution with a mass concentration of 0.1% for 6 min, rinse with sterile water three times, place the sterilized top sprouts on sterilized filter paper to absorb the water on the surface of the sprouts, complete the sterilization process, then inoculate the sprouts in the differentiation induction medium and culture for 7 d to obtain the sprouts, and the survival rate is 30%. The culture conditions are: 25°C, humidity 50%, pH 5.8, and one top sprout is inoculated in each differentiation induction medium.

[0063] S2, inoculate the sprouts in the proliferation subculture medium, and proliferate the sprouts for 35 d, and the proliferation coefficient of the sprouts is 1. The culture conditions are: 25°C, humidity 40%, pH 5.8, and one sprout is inoculated in each proliferation subculture medium. Then, transfer the sprouts to the rooting medium, and induce the sprouts to root for 25 d, and the rooting rate is 20%. The conditions for inducing the sprouts to root are: 25°C, humidity 50%, and pH 5.8.

[0064] Comparative Example 2

[0065] The differentiation induction medium is configured, and the components of the differentiation induction medium include:

[0066] NH4NO30.4 g / L, CaCl2 2H2O 0.056 g / L, MgSO4 7H2O 0.75 g / L, KH2PO40.59 g / L, MnSO4 4H2O 23.5 mg / L, ZnSO4 7H2O 9.12 mg / L, H3BO3 5.56 mg / L, NaMoO4 2H2O 0.53 mg / L, CuSO4 5H2O 0.042 mg / L, CoCl2 6H2O 0.035 mg / L, KI 0.032 mg / L, nicotinic acid 0.57 mg / L, vitamin B1 1.23 mg / L, glycine 3.35 mg / L, vitamin B6 0.65 mg / L, myo-inositol 100 mg / L, FeSO4 7H2O 27.65 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.

[0067] A proliferation subculture medium was configured, the components of the proliferation subculture medium including:

[0068] NH4NO3825 mg / L, KNO3950 mg / L, KH2PO485 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-EDTA 37.3 mg / L, myo-inositol 100 mg / L, glycine 2 mg / L, vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L.

[0069] A rooting medium was configured, the components of the rooting medium including:

[0070] NH4NO30.6 g / L, CaCl2·2H2O 0.068 g / L, MgSO4·7H2O 0.56 g / L, KH2PO40.46 g / L, MnSO4·4H2O 23.5 mg / L, ZnSO4·7H2O 11.4 mg / L, H3BO35.45 mg / L, NaMoO4·2H2O 0.54 mg / L, CuSO4·5H2O 0.037 mg / L, CoCl2·6H2O 0.035 mg / L, KI 0.04 mg / L, nicotinic acid 0.47 mg / L, vitamin B1 1.04 mg / L, glycine 3.5 mg / L, vitamin B60.7 mg / L, myo-inositol 100 mg / L, FeSO4·7H2O 27.75 mg / L, Na2-EDTA 35.65 mg / L, 6-benzylaminopurine 1 mg / L, indolebutyric acid 1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0071] After the configuration is completed, the above differentiation medium, proliferation subculture medium, and rooting medium are respectively placed in a high-pressure sterilization pot and sterilized at a temperature of 121°C for 20 min.

[0072] S1, select the top sprouts that are healthy, fruitful, and free of pests and diseases, wash the sprouts with distilled water three times, then sterilize the sprouts with an ethanol solution with a volume concentration of 75% for 30 s in a clean bench, rinse with sterile water three times, sterilize the sprouts with a mercury chloride solution with a mass concentration of 0.1% for 6 min, rinse with sterile water three times, place the sterilized top sprouts on sterilized filter paper to absorb the water on the surface of the sprouts, complete the sterilization process, then inoculate the sprouts in the differentiation induction medium and culture for 7 d to obtain the cluster sprouts, and the survival rate is 70%. The culture conditions are as follows: 25°C, humidity 50%, pH 5.8, and one top sprout is inoculated in each differentiation induction medium.

[0073] S2, inoculate the cluster sprouts in the proliferation subculture medium, and proliferate and culture the sprouts for 35 d, and the proliferation coefficient of the sprouts is 3. The culture conditions are as follows: 25°C, humidity 40%, pH 5.8, and one cluster sprout is inoculated in each proliferation subculture medium. Then, the cluster sprouts are transferred to the rooting medium, and the cluster sprouts are induced to root for 25 d, and the rooting rate is 85%. The conditions for inducing the cluster sprouts to root are as follows: 25°C, humidity 50%, and pH 5.8.

[0074] The cultivation process of the comparative example 1 is basically the same as that of the example 1, except that 6-benzylaminopurine and indolebutyric acid are not added in the differentiation induction medium and the rooting medium. It is tested that the survival rate, the proliferation coefficient, and the rooting rate greatly decrease. This is because the cells cannot be effectively differentiated and the roots cannot be effectively developed after the 6-benzylaminopurine and the indolebutyric acid are removed, so that the rapid propagation system almost fails.

[0075] The cultivating process of Comparative Example 2 is basically the same as that of Example 1, except that no naphthaleneacetic acid is added in the differentiation induction cultivating medium and the rooting culture medium. It is found through testing that the survival rate, proliferation coefficient and rooting rate also decrease. This is because naphthaleneacetic acid plays the role of auxiliary auxin, which is used to stabilize rooting and reduce callus, and after the removal of naphthaleneacetic acid, the differentiation and rooting stability are weakened.

[0076] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, but not to limit it. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can still be modified or equivalently replaced, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present application.

Claims

1. A method for tissue culture rapid propagation of cherry salt-tolerant rootstock, characterized in that, The preparation steps include the following steps: S1, selecting top buds of cherry salt-tolerant rootstocks, washing and disinfecting the top buds, inoculating the top buds into a differentiation induction medium for culture to obtain a cluster bud; S2, inoculating the cluster bud into a proliferation subculture medium for proliferation culture to obtain a proliferation bud, and inoculating the proliferation bud into a rooting medium to induce rooting.

2. The method for tissue culture rapid propagation of cherry salt-tolerant rootstock according to claim 1, characterized in that, The top buds of the cherry salt-tolerant rootstocks in S1 are healthy and pest-free.

3. The method for tissue culture rapid propagation of cherry salt-tolerant rootstock according to claim 1, characterized in that, The disinfection process in S1 includes disinfecting the top buds with an ethanol solution, washing, disinfecting the top buds with a mercuric chloride solution, washing, and then placing the top buds on sterilized filter paper to absorb surface moisture.

4. The method for tissue culture rapid propagation of cherry salt-tolerant rootstock according to claim 3, characterized in that, The concentration of the ethanol solution is 70%-80%, the ethanol solution disinfection time is 25-45s, the concentration of the mercuric chloride solution is 0.1%-0.15%, and the mercuric chloride solution disinfection time is 5-7min.

5. The method of cherry salt-tolerant rootstock tissue culture rapid propagation according to claim 1, characterized in that, The differentiation induction medium in S1 includes MS medium+6-benzylaminopurine 0.13-1mg / L+indole-3-butyric acid 0.17-1mg / L+naphthaleneacetic acid 0.11-0.5mg / L+sucrose 20-30g / L+agar 6-7g / L.

6. The method for tissue culture rapid propagation of cherry salt-tolerant rootstock according to claim 1, characterized in that, The culture temperature in S1 is 23-27℃, the culture humidity is 50%-70%, the culture time is 7-14d, and the culture pH is 5.8-6.

0.

7. The method of cherry salt-tolerant rootstock tissue culture rapid propagation according to claim 1, characterized in that, The proliferation subculture medium in S2 is 1 / 2MS medium.

8. The method of tissue culture rapid propagation of cherry salt-tolerant rootstock according to claim 1, characterized in that, The proliferation culture temperature in S2 is 18-25℃, the humidity is 40%-50%, and the proliferation culture time is 30-35d.

9. The method of tissue culture rapid propagation of cherry salt-tolerant rootstock according to claim 1, characterized in that, The rooting medium in S2 includes MS medium+6-benzylaminopurine 0.08-1mg / L+indole-3-butyric acid 0.2-1mg / L+naphthaleneacetic acid 0.1-0.5mg / L+sucrose 20-30g / L+agar 6-7g / L.

10. The method of tissue culture rapid propagation of cherry salt-tolerant rootstock according to claim 1, characterized in that, The rooting temperature is 18-25℃, the humidity is 50%-70%, and the rooting time is 20-35d.

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