Tissue culture and rapid propagation method for Shannong crisp pears

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a Shannong crisp pear tissue culture and rapid propagation method which comprises the following preparation steps: S1, selecting top tender shoots of Shannong crisp pears, 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; s2, transferring the buds into a proliferation culture medium for proliferation culture, then transferring the buds into a rooting culture medium, and inducing rooting. By optimizing the components of each culture medium, the Shannong crisp pear plants are bred, 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 invention relates to the technical field of plant tissue culture, in particular to a method for rapid tissue culture of Shannong pear. Background Art

[0002] The Shannong Crispy Pear (Shannong Crispy Pear) is a perennial deciduous tree of the genus Pyrus in the Rosaceae family. A popular fruit, Shannong Crispy Pear is renowned for its smooth surface, large, plump, juicy, crisp texture, small, residue-free core, and unique flavor of honey and rock sugar. Its high yield, early maturity, rich nutrition, and pollution-free environment make it a highly sought-after fruit. Crispy pears contain sugar, minerals, organic acids, and multiple vitamins, making them a rare and desirable fruit. Besides being eaten fresh, they can also be processed into pear wine, pear paste, pear candy, and canned goods, making them a valuable raw material for the food industry. Pear juice and pear paste have medicinal properties such as clearing heat, phlegm, cough, and lung moistening, making them popular supplements for daily use and promising development prospects.

[0003] The breeding and promotion of Shannong Pear is crucial to the development of the entire industry. Therefore, a more efficient and rapid breeding method is urgently needed for the research of Shannong Pear, and asexual reproduction is one of the important ways to solve this problem.

[0004] While much research has been conducted on the asexual propagation of the Shannong Pear, methods such as grafting and cuttings remain incapable of rapidly producing large numbers of high-quality plants. Plant tissue culture can be used as a rapid method for inducing sterile seedlings. Therefore, establishing an efficient and stable rapid propagation system for the Shannong Pear using high-quality plants would be crucial for the genetic improvement of the pear and the development and application of new varieties. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for rapid tissue culture propagation of Shannong pear, so as to achieve rapid and large-scale propagation of high-quality Shannong pear germplasm, and further provide strong technical support for the genetic improvement and industrial development of Shannong pear.

[0006] To achieve the above object, the present invention provides a method for rapid propagation of Shannong pear through tissue culture, comprising the following preparation steps:

[0007] S1. Selecting the top tender shoots of Shannongsu pear, washing and disinfecting the top tender shoots, and then inoculating them into a differentiation induction medium for cultivation to obtain clustered shoots;

[0008] S2. The buds are transferred to a proliferation medium for proliferation culture, and then transferred to a rooting medium to induce rooting.

[0009] In the present invention, the top tender shoots of the Shannong Pear described in S1 are tender shoots of plants that are healthy and free of diseases and insect pests.

[0010] In the present invention, the disinfection process in S1 includes: disinfecting the top tender shoots with ethanol and then washing them, then disinfecting and washing them with mercuric chloride, and then placing the top tender shoots on sterilized filter paper to absorb surface moisture.

[0011] In the present invention, the concentration of the ethanol is 70%-80%, the disinfection time of the ethanol is 25-45 seconds, the concentration of the mercuric chloride is 0.1%-0.15%, and the disinfection time of the mercuric chloride is 5-7 minutes.

[0012] In the present invention, the differentiation induction medium in S1 includes: WPM medium + 0.1-1 mg / L 6-benzylaminopurine + 0.1-1 mg / L indolebutyric acid IBA + 0.1-1 mg / L gibberellin GA3 + 20-30 g / L sucrose + 6-7 g / L agar.

[0013] In the present invention, the components of the differentiation induction medium in S1 are preferably: NH4NO3 0.3 g / L, CaCl2·2H2O 0.072 g / L, MgSO4·7H2O 0.42 g / L, KH2PO4 0.23 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 0.47 mg / L, vitamin B1 1.2 mg / L, glycine 2.5 mg / L, vitamin B6 0.6 mg / L, inositol 100 mg / L, FeSO4·7H2O 27.85mg / L, Na2-EDTA 35.25mg / L, 6-benzylaminopurine 0.1-1mg / L, indolebutyric acid IBA 0.1-1mg / L, gibberellin GA30.1-1mg / L, sucrose 25g / L, agar 6g / L.

[0014] In the present invention, the differentiation induction culture medium needs to be sterilized before use. The sterilization process includes: placing the differentiation induction culture medium in a high pressure sterilizer for sterilization at a temperature of 120-122° C. for 18-22 minutes.

[0015] 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.

[0016] In the present invention, the inoculation method in S1 is: one top bud is inoculated into each differentiation induction culture medium.

[0017] In the present invention, the proliferation medium in S2 is a 1 / 2MS medium, and the components of the 1 / 2MS medium include: 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, Na2MoO4·2H2O0.25mg / L, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O 27.8mg / L, Na2-EDTA 37.3 mg / L, inositol 100 mg / L, glycine 2 mg / L vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, niacin 0.5 mg / L.

[0018] In the present invention, the proliferation culture medium needs to be sterilized before use. The sterilization process includes: placing the proliferation culture medium in a high pressure sterilizer at a temperature of 120-122°C and sterilizing for 18-22 minutes.

[0019] In the present invention, the temperature of proliferation culture in S2 is 18-25° C., the humidity is 40%-50%, and the culture period is 30-35 days.

[0020] In the present invention, the components of the S2 rooting medium include: WPM medium + 0.1-1 mg / L 6-benzylaminopurine + 0.1-1 mg / L indolebutyric acid IBA + 0.1-1 mg / L + sucrose 20-30 g / L + agar 6-7 g / L.

[0021] In the present invention, the components of the rooting medium in S2 are preferably:

[0022] NH4NO30.3 g / L, CaCl2·2H2O 0.072g / L, MgSO4·7H2O 0.42g / L, KH2PO40.23 g / L, MnSO4·4H2O 20.5mg / L, ZnSO4·7H2O 9.4mg / L, H3BO35.9 mg / L, NaMoO4·2H2O 0.34mg / L, CuSO4·5H2O 0.029mg / L, CoCl2·6H2O 0.025mg / L, KI 0.03mg / L, niacin 0.47mg / L, vitamin B11.2mg / L, glycine 2.5mg / L, vitamin B6 0.6 mg / L, inositol 100mg / L, FeSO4·7H2O 27.85mg / L, Na2-EDTA35.25 mg / L, 6-benzylaminopurine 0.05-1mg / L, indolebutyric acid IBA 0.3-1mg / L, sucrose 25g / L, agar 6g / L.

[0023] In the present invention, the rooting culture medium needs to be sterilized before use. The sterilization process includes: placing the rooting culture medium in a high pressure sterilizer at a temperature of 120-122° C. and sterilizing it for 18-22 minutes.

[0024] In the present invention, the temperature for inducing rooting is 18-25° C., the humidity is 50%-70%, and the number of days for inducing rooting is 20-35 days.

[0025] The present invention has the following beneficial effects:

[0026] The present invention provides a method for rapid tissue culture propagation of a Shannong Pear, comprising the following steps: S1. Selecting the top tender shoots of a Shannong Pear, cleaning and disinfecting the shoots, and then inoculating them in a differentiation induction medium for cultivation to obtain clustered shoots; S2. Transferring the sprouts to a proliferation medium for proliferation culture, and then transferring them to a rooting medium for rooting induction. By optimizing the components of each culture medium for propagation of Shannong Pear plants, the present invention can rapidly obtain high-quality seedlings, effectively shortening seedling cultivation time and reducing seedling cultivation costs.

[0027] After being cultured in the differentiation induction medium of the present invention, the survival rate of apical bud differentiation induction is as high as over 80%, after being cultured in the proliferation medium, the seedling proliferation coefficient is 3-5, and after being cultured in the rooting medium, the rooting rate can reach over 95%, with excellent reproduction effect, thus realizing the rapid and large-scale reproduction of the excellent germplasm of Shannong Pear.

[0028] The present invention further defines a differentiation induction culture medium comprising: WPM culture medium + 0.1-1 mg / L 6-benzylaminopurine + 0.1-1 mg / L indolebutyric acid (IBA) + 0.1-1 mg / L gibberellin GA3 + 20-30 g / L sucrose + 6-7 g / L agar. The present invention adds gibberellin (GA3) and indolebutyric acid (IBA) to the WPM culture medium. Gibberellin is an important plant growth regulator. In the tissue culture of Shannong pear, its main function is to promote the differentiation and elongation of tender shoots, break dormancy, accelerate cell division and growth, help improve the survival rate of terminal bud differentiation, and lay a good foundation for subsequent proliferation and rooting.

[0029] Indolebutyric acid (IBA) is a commonly used auxin hormone that, in tissue culture, is primarily responsible for promoting cell elongation and division, as well as root induction and development. In Shannong Pear cultivation, IBA synergizes with gibberellins and 6-benzylaminopurine (6-BA) to promote bud differentiation during the differentiation induction phase. Adjusting its concentration during the rooting phase significantly increases the rooting rate (reaching over 95%), ensuring the formation of a complete root system in tissue culture seedlings and improving plant quality.

[0030] The combined application of gibberellins and indolebutyric acid not only addresses the issue of low shoot differentiation efficiency but also ensures success in the subsequent rooting phase. These two agents, working synergistically with other nutrients in the WPM medium, form a highly efficient culture system, achieving a shoot proliferation coefficient of 3-5. This enables the rapid and extensive propagation of high-quality Shannong Pear germplasm, ultimately providing technical support for its genetic improvement and industrial expansion.

[0031] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a diagram showing the growth of the top tender buds of Shannong Pear after culture in a differentiation induction medium in Example 1;

[0033] Figure 2 This is a diagram showing the growth of the sprouts in Example 1 after culture in a proliferation medium;

[0034] Figure 3 This is a diagram of the root morphology of Example 1 after 25 days of root induction. DETAILED DESCRIPTION

[0035] 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.

[0036] Example 1

[0037] Prepare differentiation induction medium, the components of which include:

[0038] NH4NO30.3 g / L, CaCl2·2H2O 0.072g / L, MgSO4·7H2O 0.42g / L, KH2PO40.23 g / L, MnSO4·4H2O 20.5mg / L, ZnSO4·7H2O 9.4mg / L, H3BO35.9 mg / L, NaMoO4·2H2O 0.34mg / L, CuSO4·5H2O 0.029mg / L, CoCl2·6H2O 0.025mg / L, KI 0.03mg / L, niacin 0.47mg / L, vitamin B11.2mg / L, glycine 2.5mg / L, vitamin B6 0.6 mg / L, inositol 100mg / L, FeSO4·7H2O 27.85mg / L, Na2-EDTA35.25 mg / L, 6-benzylaminopurine 1mg / L, indolebutyric acid IBA 1mg / L, gibberellin GA31 mg / L, sucrose 25g / L, agar 6g / L.

[0039] Prepare proliferation medium, the components of which include:

[0040] 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, CuSO4·5H2O 0.025mg / L, CoCl2·6H2O 0.025mg / L, FeSO4·7H2O27.8mg / L, Na2-EDTA 37.3mg / L, inositol 100mg / L, glycine 2mg / L, vitamin B1 0.1 mg / L, vitamin B6 0.5 mg / L, niacin 0.5mg / L.

[0041] Prepare rooting medium. The components of rooting medium include:

[0042] NH4NO30.3 g / L, CaCl2·2H2O 0.072g / L, MgSO4·7H2O 0.42g / L, KH2PO40.23 g / L, MnSO4·4H2O 20.5mg / L, ZnSO4·7H2O 9.4mg / L, H3BO35.9 mg / L, NaMoO4·2H2O 0.34mg / L, CuSO4·5H2O 0.029mg / L, CoCl2·6H2O 0.025mg / L, KI 0.03mg / L, niacin 0.47mg / L, vitamin B11.2mg / L, glycine 2.5mg / L, vitamin B6 0.6 mg / L, inositol 100mg / L, FeSO4·7H2O27.85 mg / L, Na2-EDTA35.25 mg / L, 6-benzylaminopurine 1mg / L, indolebutyric acid IBA 1mg / L, sucrose 25g / L, agar 6g / L.

[0043] After the configuration is completed, the above culture medium is placed in a high pressure sterilizer at a temperature of 121°C and the differentiation induction medium is sterilized for 20 minutes.

[0044] S1. Select robust, fruit-bearing, pest-free top shoots. Rinse them thoroughly with distilled water. Then, disinfect them in a laminar flow hood with 75% alcohol for 30 seconds, rinse three times with sterile water, and then disinfect them with 0.1% mercuric chloride for 6 minutes and rinse three times with sterile water. Place the disinfected top shoots on sterile filter paper to dry them. Inoculate them in differentiation induction medium for 7 days to obtain buds. The survival rate was 85%. Culture conditions were: 25°C, 50% humidity, and pH 6.8. One top shoot was inoculated into each differentiation induction medium.

[0045] S2. The shoots were transferred to a proliferation medium and cultured for 35 days. The proliferation coefficient of the shoots was 5. The culture conditions were: 25°C, 40% humidity. The shoots were then transferred to a rooting medium and induced to root for 25 days. The rooting rate was 98%. The rooting conditions were: 25°C, 50% humidity.

[0046] 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 Shannong Pear were inoculated into the induction culture medium, they survived well under the culture conditions, with leaves stretching and normal color, which confirmed the effectiveness of the differentiation induction culture medium of the present invention.

[0047] After S2 was cultured in the proliferation medium from the bud, the proliferation situation was as follows Figure 2 As shown. Figure 1 and Figure 2It can be seen that the growth state of the clustered buds is that multiple healthy new buds are differentiated from the single original tender buds, and the increase in the number of buds is obviously seen, which confirms the promoting effect of the proliferation culture medium on the rapid reproduction of Shannong Pear buds, and shows that the proliferation culture medium of the present invention can effectively achieve the quantitative expansion of excellent germplasm.

[0048] 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.

[0049] Comparative Example 1

[0050] The sterilization process of the culture medium is consistent with that of Example 1.

[0051] S1. Select robust, fruit-bearing, pest-free top shoots. Rinse the shoots thoroughly with distilled water. Then, disinfect them in a laminar flow hood with 75% alcohol for 30 seconds, rinse three times with sterile water, and then disinfect them with 0.1% mercuric chloride for 6 minutes and rinse three times with sterile water. Place the disinfected top shoots on sterile filter paper to dry them. Inoculate them in WPM medium for 7 days to obtain buds. The survival rate was 70%. Culture conditions were: 25°C, 50% humidity, and pH 6.8. One top shoot was inoculated into each differentiation induction medium.

[0052] S2: The shoots were transferred to 1 / 2 MS medium and cultured for 35 days. The shoot proliferation coefficient was 1. Culture conditions were: 25°C, 40% humidity. The shoots were then transferred to WPM medium and rooting was induced for 25 days. Rooting was difficult. Rooting induction conditions were: 25°C, 50% humidity.

[0053] Comparative Example 2

[0054] Prepare differentiation induction medium, the components of which include:

[0055] NH4NO30.3 g / L, CaCl2·2H2O 0.072g / L, MgSO4·7H2O 0.42g / L, KH2PO40.23 g / L, MnSO4·4H2O 20.5mg / L, ZnSO4·7H2O 9.4mg / L, H3BO35.9 mg / L, NaMoO4·2H2O 0.34mg / L, CuSO4·5H2O 0.029mg / L, CoCl2·6H2O 0.025mg / L, KI 0.03mg / L, niacin 0.47mg / L, vitamin B11.2mg / L, glycine 2.5mg / L, vitamin B6 0.6 mg / L, inositol 100mg / L, FeSO4·7H2O 27.85mg / L, Na2-EDTA35.25 mg / L, 6-benzylaminopurine 1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0056] Prepare rooting medium. The components of rooting medium include:

[0057] NH4NO30.3 g / L, CaCl2·2H2O 0.072g / L, MgSO4·7H2O 0.42g / L, KH2PO40.23 g / L, MnSO4·4H2O 20.5mg / L, ZnSO4·7H2O 9.4mg / L, H3BO35.9 mg / L, NaMoO4·2H2O 0.34mg / L, CuSO4·5H2O 0.029mg / L, CoCl2·6H2O 0.025mg / L, KI 0.03mg / L, niacin 0.47mg / L, vitamin B11.2mg / L, glycine 2.5mg / L, vitamin B6 0.6 mg / L, inositol 100mg / L, FeSO4·7H2O27.85 mg / L, Na2-EDTA35.25 mg / L, 6-benzylaminopurine 1 mg / L, sucrose 25 g / L, agar 6 g / L.

[0058] After the configuration is completed, the above culture medium and 1 / 2 MS culture medium are placed in a high pressure sterilizer at a temperature of 121°C and sterilized for 20 minutes.

[0059] S1. Select robust, fruit-bearing, pest-free top shoots. Rinse the shoots thoroughly with distilled water. Then, disinfect them in a laminar flow hood with 75% alcohol for 30 seconds, rinse three times with sterile water, and then disinfect them with 0.1% mercuric chloride for 6 minutes and rinse three times with sterile water. Place the disinfected top shoots on sterile filter paper to dry them. Inoculate them in WPS medium for 7 days to obtain buds. The survival rate was 60%. Culture conditions were: 25°C, 50% humidity, and pH 6.8. One top shoot was inoculated into each differentiation induction medium.

[0060] S2: The shoots were transferred to a proliferation medium and cultured for 35 days. The proliferation coefficient of the shoots was 2. The culture conditions were: 25°C, 40% humidity. The shoots were then transferred to a rooting medium and induced to root for 25 days. However, rooting was difficult. The rooting induction conditions were: 25°C, 50% humidity.

[0061] Comparative Example 3

[0062] The sterilization conditions of the culture medium were the same as those in Example 1.

[0063] S1. Select healthy, fruitful, pest-free top shoots. Wash them thoroughly with distilled water. Then, disinfect them in a laminar flow hood with 75% alcohol for 30 seconds, rinse three times with sterile water, and then disinfect them with 0.1% mercuric chloride for 6 minutes and rinse three times with sterile water. Place the disinfected top shoots on sterile filter paper to dry them. Inoculate them in MS medium for 7 days to obtain buds. The survival rate was 70%. Culture conditions were: 25°C, 50% humidity, and pH 6.8. One top shoot was inoculated per MS medium.

[0064] S2: Shoots were transferred to 1 / 2 MS medium and cultured for 35 days. The proliferation coefficient of the shoots was 2.5. Culture conditions were: 25°C, 40% humidity. They were then transferred to MS medium and rooted for 25 days. The rooting rate was 35%. Rooting induction conditions were: 25°C, 50% humidity.

[0065] Comparative Example 1, compared to Example 1, used WPM medium for culture. The results of survival rate, proliferation coefficient, and rooting rate showed that due to the lack of hormone regulation in the basal medium, the explants had difficulty adapting and were less resistant to stress, resulting in a low survival rate of only 45%. During the proliferation culture process, due to the lack of mitogenic and differentiation hormones, proliferation was limited, and the final proliferation rate was only 1. During the rooting process, without the induction of auxins such as IBA, rooting was extremely difficult.

[0066] Compared with the culture medium of Example 1, gibberellins and indolebutyric acid were not added in Comparative Example 2, but 6-benzylaminopurine basic hormone was added to maintain partial physiology, and the survival rate was higher than that of Comparative Example 1. However, due to the lack of gibberellins and indolebutyric acid, it was difficult to break dormancy and balance growth. Therefore, the survival rate and proliferation coefficient were lower than those of Example 1, and subsequent rooting was difficult.

[0067] In Comparative Example 3, compared with Example 1, after the basal culture medium was replaced with MS culture medium, the differentiation of Shannong Pear buds was inhibited due to the high concentration of nitrogen and inorganic salts in the MS culture medium, and the MS culture medium had a low matching degree with the requirements of Shannong Pear, with a rooting rate of only 35%.

[0068] 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 Shannong pear tissue culture, characterized in that: The method comprises the following preparation steps: S1. Selecting the top tender shoots of Shannongsu pear, washing and disinfecting the top tender shoots, and then inoculating them into a differentiation induction medium for cultivation to obtain clustered shoots; S2. The buds are transferred to a proliferation medium for proliferation culture, and then transferred to a rooting medium to induce rooting.

2. The method for rapid propagation of Shannong Pear tissue culture according to claim 1, wherein The top tender shoots of the Shannong Pear described in S1 are tender shoots from plants that are healthy and free of diseases and insect pests.

3. The method for rapid propagation of Shannong Pear tissue culture according to claim 1, wherein: The disinfection process described in S1 includes: disinfecting the top tender shoots with ethanol and then washing them, then disinfecting them with mercuric chloride and washing them, and then placing the top tender shoots on sterilized filter paper to absorb surface moisture.

4. The method for rapid propagation of Shannong Pear tissue culture according to claim 3, wherein: The concentration of the ethanol is 70%-80%, and the disinfection time of the ethanol is 25-45 seconds. The concentration of the mercuric chloride is 0.1%-0.15%, and the disinfection time of the mercuric chloride is 5-7 minutes.

5. The method for rapid propagation of Shannong Pear tissue culture according to claim 1, characterized in that: The differentiation induction medium described in S1 includes: WPM medium + 0.1-1 mg / L 6-benzylaminopurine + 0.1-1 mg / L indolebutyric acid IBA + 0.1-1 mg / L gibberellin GA3 + 20-30 g / L sucrose + 6-7 g / L agar.

6. The method for rapid propagation of Shannong Pear tissue culture according to claim 1, characterized in that: 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 rapid propagation of Shannong Pear tissue culture according to claim 1, characterized in that: The temperature of proliferation culture in S2 is 18-25°C, the humidity is 40%-50%, and the number of days of proliferation culture is 30-35 days.

8. The method for rapid propagation of Shannong Pear tissue culture according to claim 1, characterized in that: The components of the S2 rooting medium include: WPM medium + 0.1-1 mg / L 6-benzylaminopurine + 0.1-1 mg / L indolebutyric acid IBA + 0.1-1 mg / L + sucrose 20-30 g / L + agar 6-7 g / L.

9. The method for rapid propagation of Shannong Pear tissue culture according to claim 1, characterized in that: The temperature for inducing rooting is 18-25℃, the humidity is 50%-70%, and the number of days for inducing rooting is 20-35 days.

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