Tissue culture method for improving germination rate of zanthoxylum

Abstract

This invention belongs to the field of plant tissue culture technology, specifically relating to a tissue culture method for improving the germination rate of Sichuan pepper (Zanthoxylum bungeanum). The method includes the following steps: Sichuan pepper seeds are placed statically at 0℃~10℃ for 3~7 days, then bathed in water at 30℃~42℃ for 1~3 days. After the water bath, the seeds are disinfected and the seed coats are broken, leaving the seed coats partially peeled. The broken seeds are then scattered on MS medium containing vermiculite, covered with a 0.5cm~1.5cm thick layer of vermiculite, and cultured at 26℃~30℃. The method provided by this invention significantly increases the germination rate of Sichuan pepper seeds from the conventional 10%~20% to 40%, shortens the culture cycle from seed pretreatment to seedling formation to 30d~35d, and is applicable to seed treatment of various Sichuan pepper varieties.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a tissue culture method for improving the germination rate of Sichuan pepper. Background Technology

[0002] In the current context of the development of Sichuan pepper planting and breeding technology, the germination rate of Sichuan pepper seeds has always been a key factor restricting its large-scale planting and the selection of high-quality varieties. Sichuan pepper seeds, due to their hard seed coat and high oil content, have poor water permeability and are difficult to germinate; untreated seeds have a germination rate of almost zero. Furthermore, traditional seedling raising methods, such as direct sowing or simple soaking, often fail to meet the modern Sichuan pepper industry's demand for efficient and high-survival-rate seedling raising. Current methods for treating Sichuan pepper seeds mainly include physical shell breaking, chemical soaking, and alternating wet and dry methods, but these methods are generally cumbersome and result in low seed germination rates. Summary of the Invention

[0003] To address the aforementioned problems, this invention provides a tissue culture method for improving the germination rate of Sichuan pepper seeds. By combining physical shell breaking and chemical treatment, the germination rate of Sichuan pepper seeds is significantly increased from the conventional 10%~20% to 40%, and the culture cycle of Sichuan pepper tissue culture seedlings is also shortened.

[0004] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0005] The first aspect of this invention provides a tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0006] Place the pepper seeds at 0℃~10℃ for 3~7 days to break seed dormancy, and then bathe them in water at 30℃~42℃ for 1~3 days to allow the pepper seeds to absorb water and activate enzyme activity.

[0007] After being disinfected and cracked by water bath, the seeds of Sichuan pepper are in a semi-peeled state, which protects the radicle and shortens the germination time.

[0008] After the seeds were cracked, they were scattered on MS medium containing vermiculite, and then covered with a 0.5cm to 1.5cm thick layer of vermiculite. The seeds were then cultured at 26℃ to 30℃.

[0009] Furthermore, the culture conditions consist of alternating 12 hours of light and 12 hours of darkness.

[0010] Furthermore, the light intensity for the light culture is 2000 Lx to 2400 Lx.

[0011] Furthermore, the disinfection steps specifically include: soaking in 75% alcohol for 1 to 3 minutes, soaking in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 10 to 30 minutes, and rinsing with sterile water.

[0012] Further, soak in 75% alcohol for 2 minutes, soak in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes, and rinse with sterile water.

[0013] Furthermore, the volume ratio of vermiculite to MS culture medium is 1.5:2~4.

[0014] Furthermore, the method also includes inoculating the cultured pepper seed embryo root length into a subculture medium for subculture when the cultured seed embryo root length is 1.0cm~2.0cm.

[0015] Furthermore, the subculture temperature is 23℃~27℃, the light intensity is 2000Lx~2400Lx, and the light duration is 12h / d.

[0016] Furthermore, the subculture medium is based on MS medium, and each liter of subculture medium contains 25g~45g sucrose, 4g~8g agarose, 0.3g~0.6mg 6-benzylaminopurine and 0.2g~0.4mg indolebutyric acid.

[0017] Furthermore, the pepper seeds are mature pepper seeds with degreased surfaces.

[0018] Furthermore, dish soap is used to degrease the surface of the pepper seeds.

[0019] Furthermore, the varieties of Sichuan pepper include Fengxian Dahongpao, Yaojiao, Cijiao, Xinong thornless Sichuan pepper, or Hancheng Dahongpao.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] This invention provides a tissue culture method to improve the germination rate of Sichuan pepper, comprising the following steps: Sichuan pepper seeds are placed at 0℃~10℃ for 3~7 days, then bathed in water at 30℃~42℃ for 1~3 days, then the seeds after water bath are disinfected and the shells are broken, so that the seed shells are in a semi-peeled state, then the broken seeds are scattered on MS medium containing vermiculite, and then covered with a layer of vermiculite 0.5cm~1.5cm thick, and cultured at 26℃~30℃. (1) The method provided by this invention can significantly improve the germination rate of Sichuan pepper seeds: the germination rate of Sichuan pepper seeds is significantly increased from the conventional 10%~20% to 40%, which not only improves the germination efficiency of seeds, but also provides a good physiological basis for the formation of subsequent tissue culture seedlings. (2) Shortened cultivation cycle: In traditional methods, the time from pretreatment of pepper seeds to formation of tissue culture seedlings usually takes 40 to 60 days, while this invention shortens this cycle to 30 to 35 days, significantly improving production efficiency. This provides strong support for the large-scale tissue culture seedling production of pepper. (3) Simple operation and strong adaptability: The operation process of this method is simple and suitable for large-scale industrial production. The required materials and equipment are all conventional materials, and it is applicable to the seed treatment of various pepper varieties such as Fengxian Dahongpao, Yaojiao, Cijiao, Xinong Wuzhenhuajiao, and Hancheng Dahongpao, and has good versatility. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0023] Figure 1 Image showing the cultivation of Sichuan pepper seeds on a sterile vermiculite substrate.

[0024] Figure 2 Image showing the cultivation of Sichuan pepper seeds in a sterile nutrient soil substrate. Detailed Implementation

[0025] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0026] In the current context of the development of Sichuan pepper planting and breeding technology, the germination rate of Sichuan pepper seeds has always been a key factor restricting its large-scale planting and the selection of high-quality varieties. Sichuan pepper seeds, due to their hard seed coat and high oil content, have poor water permeability and are difficult to germinate; untreated seeds have a germination rate of almost zero. Furthermore, traditional seedling raising methods, such as direct sowing or simple soaking, often fail to meet the modern Sichuan pepper industry's demand for efficient and high-survival-rate seedling raising. Current methods for treating Sichuan pepper seeds mainly include physical shell breaking, chemical soaking, and alternating wet and dry methods, but these methods are generally cumbersome and result in low seed germination rates.

[0027] This invention provides a tissue culture method for improving the germination rate of Sichuan pepper seeds, comprising the following steps: Sichuan pepper seeds are placed statically at 0℃~10℃ for 3~7 days, then bathed in water at 30℃~42℃ for 1~3 days. After water bathing, the seeds are disinfected and the seed coats are broken, leaving the seed coats partially peeled. The broken seeds are then scattered on MS medium containing vermiculite, covered with a 0.5cm~1.5cm thick layer of vermiculite, and cultured at 26℃~30℃. The method provided by this invention significantly increases the germination rate of Sichuan pepper seeds from the conventional 10%~20% to 40%, shortens the culture cycle from seed pretreatment to tissue culture seedling formation to 30d~35d, and is applicable to seed treatment of various Sichuan pepper varieties.

[0028] Example 1: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0029] (1) Seed collection, screening and treatment

[0030] Collect mature, plump, and disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds to ensure seed quality, then rub the oil film on the surface of the seeds with dish soap and rinse them clean with water. After degreasing the surface of the pepper seeds, store them in a 4℃ refrigerator for 3 days, and finally heat shock them in a 37℃ water bath for 1 day.

[0031] (2) Surface disinfection treatment

[0032] After treatment, rinse the seeds under running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes. Next, soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween with shaking for 15 minutes. Finally, rinse them 3 times with sterile water to remove surface microorganisms and prevent diseases from occurring during subsequent cultivation.

[0033] (3) Physical shell breaking treatment

[0034] After sterilization, place the seeds in a sterilized petri dish and use sterilized and cooled scissors and tweezers to break the shell, so that the pepper seed shell is partially peeled off, in order to enhance the water permeability of the seeds and promote the softening of the endosperm and germination.

[0035] (4) Preparation of sterile vermiculite matrix (MS medium containing vermiculite)

[0036] Preparation of MS medium: Dissolve 1g of 1 / 2 MS in 1L of ultrapure water and adjust the pH to 6.0.

[0037] Preparation of sterile vermiculite matrix: Fill the culture flask with vermiculite to 1 / 3 volume, then pour in the prepared MS medium to cover the vermiculite, then sterilize at 121℃ for 20 min, and cool before use.

[0038] (5) Seed culture

[0039] Spread the shelled seeds evenly on the surface of the sterile vermiculite substrate, avoiding adhesion to the walls, and then cover with a 1cm thick layer of sterilized vermiculite (see [link to product]). Figure 1 The tissue culture medium was placed in a culture chamber and cultured at a temperature of 28℃, a light intensity of 2200 lx, and a photoperiod of 12 h / d. MS medium was added promptly to prevent it from drying out. When the radicle reached a length of 1.5 cm, the radicle was separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle emerging facing up. The radicles were then transplanted into a subculture medium using forceps to obtain tissue culture seedlings, achieving rapid growth and differentiation. The subculture medium was based on MS medium, with each liter containing 30 g sucrose, 6 g agarose, 0.4 mg 6-benzylaminopurine, and 0.25 mg indolebutyric acid.

[0040] The subculture conditions were: temperature 23℃~27℃, light intensity 2200lx, and light duration 12h / d.

[0041] Example 2: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0042] (1) Seed collection, screening and treatment: Collect mature, plump, disease-free pepper seeds, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a refrigerator at 2℃ for 5 days, they are finally placed in a water bath at 30℃ for 3 days of heat shock.

[0043] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 1 minute, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 30 minutes with shaking, and finally rinse them with sterile water 3 times.

[0044] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0045] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterile vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 30℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.0cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0046] Example 3: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0047] (1) Seed collection, screening and treatment: Collect mature, plump, disease-free pepper seeds, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a 0℃ refrigerator for 3 days, they are finally placed in a 40℃ water bath for 2 days of heat shock.

[0048] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0049] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0050] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 0.5 cm thick layer of sterilized vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 27°C, a light intensity of 2300 lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.8 cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0051] Example 4: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0052] (1) Seed collection, screening and treatment: Collect mature, plump, disease-free and thornless pepper seeds from Xinnong, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in an 8℃ refrigerator for 7 days, they are finally placed in a 42℃ water bath for 1 day of heat shock.

[0053] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 20 minutes with shaking, and finally rinse them with sterile water 3 times.

[0054] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0055] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1.5cm thick layer of sterilized vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 26℃, a light intensity of 2400lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 2.0cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0056] Example 5: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0057] (1) Seed collection, screening and treatment: Collect mature, plump, disease-free Hancheng Dahongpao seeds, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a 10℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0058] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 3 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 10 minutes with shaking, and finally rinse them with sterile water 3 times.

[0059] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0060] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterile vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 26℃~30℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0061] Comparative Example 1: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0062] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Fengxian Dahongpao seeds, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a 4℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0063] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0064] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0065] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterile vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0066] Comparative Example 2: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0067] (1) Seed collection, screening and treatment: Collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a 4℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0068] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0069] (3) Seed culture: The sterilized seeds were evenly sprinkled on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterilized vermiculite. The seeds were then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium was added in time to prevent the seeds from drying out. When the radicle length reached 1.5cm, the radicle was separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds were then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0070] Comparative Example 3: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0071] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a refrigerator at 4℃ for 7 days, they are finally placed in a water bath at 37℃ for 3 days of heat shock.

[0072] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0073] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0074] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterile vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0075] Comparative Example 4: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0076] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a refrigerator at 4℃ for 7 days, they are finally placed in a water bath at 37℃ for 3 days of heat shock.

[0077] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0078] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0079] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterile vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0080] Comparative Example 5: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0081] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a refrigerator at 4℃ for 7 days, they are finally placed in a water bath at 37℃ for 3 days of heat shock.

[0082] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0083] (3) Seed culture: The sterilized seeds were evenly sprinkled on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterilized vermiculite. The seeds were then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium was added in time to prevent the seeds from drying out. When the radicle length reached 1.5cm, the radicle was separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds were then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0084] Comparative Example 6: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0085] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0086] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0087] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0088] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterile vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0089] Comparative Example 7: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0090] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0091] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0092] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0093] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterile vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0094] Comparative Example 8: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0095] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0096] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0097] (3) Seed culture: The sterilized seeds were evenly sprinkled on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterilized vermiculite. The seeds were then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium was added in time to prevent the seeds from drying out. When the radicle length reached 1.5cm, the radicle was separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds were then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0098] Comparative Example 9: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0099] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then wash the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 7 days, and finally placed in a 37℃ water bath for 3 days of heat shock.

[0100] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0101] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0102] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 1cm thick layer of sterile vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0103] Comparative Example 10: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0104] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then wash the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 7 days, and finally placed in a 37℃ water bath for 3 days of heat shock.

[0105] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0106] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0107] (4) Seed culture: The seeds that have been broken open are evenly scattered on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 3cm thick layer of sterilized vermiculite. The seeds are then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium is added in time to prevent the seeds from drying out. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds are then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0108] Comparative Example 11: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0109] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then wash the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 7 days, and finally placed in a 37℃ water bath for 3 days of heat shock.

[0110] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0111] (3) Seed culture: The sterilized seeds were evenly sprinkled on the surface of the sterile vermiculite substrate prepared in Example 1, and then covered with a 0.5cm thick layer of sterilized vermiculite. The seeds were then placed in a tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. MS medium was added in time to prevent the seeds from drying out. When the radicle length reached 1.5cm, the radicle was separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seeds were then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0112] Comparative Example 12: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0113] (1) Seed collection, screening and treatment: Collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a 4℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0114] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0115] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0116] (4) Seed culture: Spread the shelled seeds evenly on the nutrient soil (purchased from Siweibo Biotechnology Horticulture Taobao store; name: organic nutrient soil), and then cover them with a 1cm layer of nutrient soil (see See Figure 2The tissue culture seedlings were placed in a tissue culture room and cultured at a temperature of 28°C, a light intensity of 2200 lx, and a light duration of 12 h / d. When the radicle length reached 1.5 cm, the radicle was separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seedlings were then transplanted to a subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0117] Comparative Example 13: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0118] (1) Seed collection, screening and treatment: Collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a 4℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0119] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0120] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0121] (4) Seed culture: The seeds that have been cracked are evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds are then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seedlings are then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0122] Comparative Example 14: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0123] (1) Seed collection, screening and treatment: Collect mature, plump seeds free from pests and diseases, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a 4℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0124] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0125] (3) Seed culture: After disinfection, the seeds were evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds were then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle length reached 1.5cm, the radicle was separated from the seed coat. The radicle connected to the seed coat was placed with the seed coat facing down and the first radicle growing was placed with the seed coat facing up. The seedlings were then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0126] Comparative Example 15: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0127] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a refrigerator at 4℃ for 7 days, they are finally placed in a water bath at 37℃ for 3 days of heat shock.

[0128] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0129] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0130] (4) Seed culture: The seeds that have been cracked are evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds are then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seedlings are then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0131] Comparative Example 16: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0132] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a refrigerator at 4℃ for 7 days, they are finally placed in a water bath at 37℃ for 3 days of heat shock.

[0133] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0134] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0135] (4) Seed culture: The seeds that have been cracked are evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds are then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seedlings are then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0136] Comparative Example 17: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0137] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a refrigerator at 4℃ for 7 days, they are finally placed in a water bath at 37℃ for 3 days of heat shock.

[0138] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0139] (3) Seed culture: After disinfection, the seeds were evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds were then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle length reached 1.5cm, the radicle was separated from the seed coat. The radicle connected to the seed coat was placed with the seed coat facing down and the first radicle growing was placed with the seed coat facing up. The seedlings were then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0140] Comparative Example 18: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0141] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0142] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0143] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0144] (4) Seed culture: The seeds that have been cracked are evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds are then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seedlings are then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0145] Comparative Example 19: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0146] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0147] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0148] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0149] (4) Seed culture: The seeds that have been cracked are evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds are then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seedlings are then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0150] Comparative Example 20: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0151] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then rub the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 3 days, they are finally placed in a 37℃ water bath for 1 day of heat shock.

[0152] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0153] (3) Seed culture: After disinfection, the seeds were evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds were then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle length reached 1.5cm, the radicle was separated from the seed coat. The radicle connected to the seed coat was placed with the seed coat facing down and the first radicle growing was placed with the seed coat facing up. The seedlings were then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0154] Comparative Example 21: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0155] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then wash the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 7 days, and finally placed in a 37℃ water bath for 3 days of heat shock.

[0156] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0157] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shell so that the pepper seed shell is in a semi-peeled state.

[0158] (4) Seed culture: The seeds that have been cracked are evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds are then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seedlings are then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0159] Comparative Example 22: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0160] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then wash the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 7 days, and finally placed in a 37℃ water bath for 3 days of heat shock.

[0161] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0162] (3) Physical shell breaking treatment: Place the disinfected seeds into a sterilized petri dish, and use sterilized and cooled scissors and tweezers to break the shells so that the pepper seed shells are completely peeled off.

[0163] (4) Seed culture: The seeds that have been cracked are evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds are then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle reaches 1.5cm in length, the radicle is separated from the seed coat, with the radicle connected to the seed coat facing down and the first radicle growing facing up. The seedlings are then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0164] Comparative Example 23: A tissue culture method for improving the germination rate of Sichuan pepper, comprising the following steps:

[0165] (1) Seed collection, screening and treatment: collect mature, plump, disease-free Dahongpao seeds from Fengxian County, soak them in water to remove impurities and shriveled seeds, then wash the oil film on the surface of the seeds with detergent and rinse them with clean water. After the surface of the degreased pepper seeds are placed in a -20℃ refrigerator for 7 days, and finally placed in a 37℃ water bath for 3 days of heat shock.

[0166] (2) Surface disinfection treatment: Rinse the treated seeds with running water for 4 hours, then place them in a sterile operating table and soak them in 75% alcohol for 2 minutes, then soak them in a mixed solution containing 3% sodium hypochlorite and 0.1% Tween for 15 minutes with shaking, and finally rinse them with sterile water 3 times.

[0167] (3) Seed culture: After disinfection, the seeds were evenly scattered on the nutrient soil, and then covered with a 1cm thick layer of nutrient soil. The seeds were then placed in the tissue culture room for culture at a temperature of 28℃, a light intensity of 2200lx, and a light duration of 12 h / d. When the radicle length reached 1.5cm, the radicle was separated from the seed coat. The radicle connected to the seed coat was placed with the seed coat facing down and the first radicle growing was placed with the seed coat facing up. The seedlings were then transplanted to the subculture medium with tweezers and cultured according to the subculture conditions in Example 1 to obtain tissue culture seedlings.

[0168] Examples 1 through 5 have similar effects. For ease of subsequent discussion and reference, the experimental results of Example 1 will be used as an example. The following are the experimental results of Example 1.

[0169] Sichuan pepper seeds were cultured according to the tissue culture methods of Example 1 and Comparative Examples 1 to 23. Each group had 50 seeds per bottle. The seed culture was observed in the tissue culture room: seed germination was observed every 5 days for the first 15 days, and then every other day thereafter, with the number of germinated seeds recorded. Germination was considered complete when the radicle broke through the seed coat. The germination rate was calculated using the following formula: Germination rate (%) = (Number of germinated seeds / Total number of seeds tested) × 100%, and the culture period was recorded.

[0170] Table 1. Statistics on Seed Germination Rate and Culture Cycle

[0171]

[0172] Note: / indicates that the corresponding method does not perform culture cycle statistics.

[0173] Table 1 shows the effects of different Sichuan pepper tissue culture methods on seed germination rate and culture cycle. As can be seen from Table 1, the Sichuan pepper tissue culture method provided by this invention enables seeds to have a high germination rate (40%) and shortens the culture cycle (30d~35d), significantly improving production efficiency and providing strong support for the large-scale tissue culture seedling production of Sichuan pepper.

[0174] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.

[0175] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A tissue culture method for improving the germination rate of Sichuan pepper, characterized in that, Includes the following steps: The seeds of Sichuan pepper were left to stand at 0℃~10℃ for 3 days, and then bathed in water at 30℃~42℃ for 1 day. After water bath, the seeds are disinfected and the shells are broken, so that the pepper seed shells are in a semi-peeled state. After the seeds have broken open, they are scattered on MS medium containing vermiculite, and then covered with a 0.5cm to 1.5cm thick layer of vermiculite. The seeds are then cultured at 26℃ to 30℃ with alternating light and dark conditions for 12 hours. When the radicle length of the cultured Sichuan pepper seeds is 1.0cm~2.0cm, they are inoculated into a subculture medium for subculture. The varieties of Sichuan pepper include Fengxian Dahongpao, Yaojiao, Cijiao, Xinong thornless Sichuan pepper or Hancheng Dahongpao. The volume ratio of vermiculite to MS medium is 1.5:(2~4).

2. The tissue culture method according to claim 1, characterized in that, The light intensity for the light culture was 2000 Lx to 2400 Lx.

3. The tissue culture method according to claim 1, characterized in that, The disinfection steps are as follows: soaking in 75% (v / v) alcohol for 1 min to 3 min, soaking in a mixed solution containing 3% (w / v) sodium hypochlorite and 0.1% (w / v) Tween for 10 min to 30 min, and rinsing with sterile water.

4. The tissue culture method according to claim 1, characterized in that, The subculture temperature was 23℃~27℃, the light intensity was 2000Lx~2400Lx, and the light duration was 12h / d.

5. The tissue culture method according to claim 1, characterized in that, The pepper seeds are mature pepper seeds with their surface degreased.

6. The tissue culture method according to claim 5, characterized in that, Use dish soap to degrease the surface of the pepper seeds.

Images