Two-step method for rapid propagation of cinnamon buds

Abstract

This invention discloses a two-step method for rapid propagation of cinnamon buds, comprising the following steps: Step 1: Collecting cinnamon buds as explants, sterilizing and preventing browning of the explants, and then inoculating them into an induction medium for induction culture to obtain clustered buds. Culture is continued until the proliferation coefficient of the clustered buds reaches 5 or higher. Step 2: Cutting off healthy single buds with a height >2cm, transferring them to a rooting induction medium, and continuing culture to obtain rooted tissue culture seedlings with a bud height of 3-6cm and a root length of 1-2cm. These seedlings are then hardened off and transplanted. The clustered buds produced by this invention are of high quality and abundant quantity, with stable genetic traits and a proliferation coefficient of 5 or higher. This invention is simple to operate, has a short culture cycle, and low production costs, resulting in significant economic, ecological, and social benefits.

Description

Technical Field

[0001] This invention belongs to the technical field of plant tissue culture methods in agricultural biotechnology, and relates to tissue culture technology for perennial woody plants, especially a two-step method for rapid propagation of cinnamon buds. Background Technology

[0002] Currently, cinnamon is mainly propagated by seed. However, because cinnamon seeds are recalcitrant, seedlings are typically sown immediately after harvesting. Traditional seed propagation suffers from drawbacks such as high seedling costs, inconsistent seedling quality, and underutilization of the potential of superior varieties, resulting in a lack of high-quality cinnamon varieties in the industry. For perennial forest tree species, tissue culture technology provides an effective way to rapidly propagate a large number of superior clones. In 2018, Zhang Shishen disclosed a rapid tissue culture propagation method for cinnamon (CN109392717A) using tender shoots as explants. This method requires three steps: induction, proliferation, and rooting, which is time-consuming, costly, and the proliferation coefficient is unknown. Recently, South China Agricultural University disclosed a method for establishing a sterile culture system using seeds as explants in the related genus *Cinnamomum camphora* (CN115707378A), but it failed to induce clustered buds. Therefore, there is currently limited research on rapid propagation techniques for cinnamon through tissue culture, especially the technology using young shoots as explants, which severely restricts the breeding and propagation of superior cinnamon clones. Establishing an efficient rapid propagation technology system for cinnamon through tissue culture is of great significance for the breeding and propagation of superior cinnamon varieties. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] One objective of this invention is to provide a two-step seedling method for the rapid propagation of cinnamon buds, which enables simultaneous induction and proliferation of clustered buds during the induction stage, eliminating the need for subgeneration and seedling cultivation, thus achieving two-step seedling production, shortening the cultivation cycle, increasing the proliferation coefficient, and reducing production costs.

[0005] To achieve these objectives and other advantages of the present invention, a two-step method for rapid propagation of cinnamon buds is provided, comprising the following steps:

[0006] Step 1: Collect cinnamon buds as explants, sterilize and prevent browning of the explants, and then inoculate them into an induction medium for induction culture to obtain clustered buds. Continue to culture until the proliferation coefficient of the clustered buds reaches 5 or more.

[0007] Step 2: Cut off healthy single buds with a bud height >2cm, transfer them to a rooting medium, and continue culturing to obtain rooted tissue culture seedlings with a bud height of 3-6cm and a root length of 1-2cm. Then, harden off the seedlings and transplant them.

[0008] Preferably, the induction medium is prepared by adding the following raw material components to a modified WPM medium: 6-BA 4.0–6.0 mg / L, KT 0.5–1.0 mg / L, GA3 0.2–0.6 mg / L, NAA 0.2–0.5 mg / L, and activated carbon (AC).

[0009] 0.1%–0.2%, riboflavin 0.8–1.5 mg / L, vitamin C (VC) 5.0–10.0 mg / L, sucrose 25–30 g / L, agar 5.0–7.0 g / L, pH 6.0.

[0010] Preferably, the modified WPM medium formulation includes: KNO3 200 mg / L, Ca(NO3)2·4H2O 250 mg / L, (NH4)2SO4 750 mg / L, MgSO4·7H2O 400 mg / L, CaCl2·2H2O 150 mg / L, KH2PO4 300 mg / L, Na2MoO4·2H2O 0.25 mg / L, MnSO4·H2O 22.4 mg / L, ZnSO4·7H2O 15.0 mg / L, CuSO4·5H2O 0.25 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA 37.3 mg / L, inositol 100 mg / L, vitamin B1 1.0 mg / L, niacin 0.5 mg / L, and vitamin B6. 0.5 mg / L, glycine 2.0 mg / L.

[0011] Preferably, the rooting medium is prepared by adding the following raw material components to 1 / 2 MS medium: IBA 0.8-1.5 mg / L, NAA 0.3-0.8 mg / L, AC 0.1%-0.15%, sucrose 25-30 g / L, agar 5.0-7.0 g / L, and pH 6.0.

[0012] Preferably, the conditions for induction culture are: temperature 24-28℃, light intensity 2000 Lx, and light duration 10 h / day; the conditions for rooting culture are: temperature 24-28℃, light intensity 1000 Lx, and light duration 10 h / day.

[0013] Preferably, the sterilization and anti-browning treatment of explants specifically includes the following steps:

[0014] S1. Immediately after harvesting, cinnamon buds should be placed in a 4℃ refrigerator for 24 hours for cold treatment.

[0015] S2. Take out the cinnamon buds that have been refrigerated in step S1, soak them in a 0.05% detergent solution for 15 minutes, rinse them under running water for 30 minutes, and then soak them in a 2% NaClO solution for 10 minutes on a clean bench.

[0016] S3. Take out the cinnamon buds treated in step S2, soak them in the filtered and sterilized anti-browning combination solution for 5 minutes, and then inoculate them into the induction medium.

[0017] Preferably, the formulation of the anti-browning combination solution includes: 50-200 mg / L cysteine ​​salt, 3-5 g / L polyvinylpyrrolidone, 50-100 ml / L cinnamon aqueous extract, and 1.0-3.0 g / L agar.

[0018] Preferably, the cinnamon water extract is obtained by pulverizing cinnamon medicinal material through a 50-mesh sieve, mixing it with purified water at a mass ratio of 1:5 to 10, soaking for 3 hours, heating to boiling for 30 minutes, and then filtering.

[0019] Preferably, the specific method for hardening off and transplanting seedlings is as follows: the rooted tissue culture seedlings are hardened off indoors for 3-5 days with the cover off, the roots of the rooted tissue culture seedlings are washed clean to obtain transplanting seedlings; the transplanting seedlings are soaked in disinfectant for 30 seconds and then transplanted to the seedbed. The humidity of the seedbed is maintained at 80%-90% for 10 days before transplanting, and at 60%-80% for 10 days after transplanting. The temperature is controlled at 22-30℃ during the transplanting process. From the time the transplanting seedlings grow new leaves, compound fertilizer solution is applied every 10-15 days.

[0020] Preferably, the disinfectant solution is formulated with 1-1.5 g / L potassium permanganate and 0.5-1 g / L carbendazim.

[0021] The substrate in the seedbed is a mixture of perlite and seedling soil in a volume ratio of 1:2, with a pH of 4.5–6.5.

[0022] Disinfect the substrate with a 0.2%–0.3% potassium permanganate solution 12–24 hours before transplanting;

[0023] The mass fraction of the compound fertilizer solution is 0.1% to 0.3%, and the mass ratio of N:P:K in the compound fertilizer solution is 1:1:1.

[0024] The present invention has at least the following beneficial effects:

[0025] First, the complete method provided by this invention is simple to operate, has low production cost, high propagation coefficient, and high transplant survival rate, making it easy to apply in large-scale industrial production. It can obtain a large number of high-quality transplanted seedlings quickly and continuously, and is highly practical.

[0026] Secondly, this invention enables simultaneous induction and proliferation culture of cinnamon buds during the bud induction stage, eliminating the need for subculture and seedling strengthening. This allows for rapid propagation of sterile cinnamon seedlings in a two-step process, significantly shortening the cycle and reducing production costs.

[0027] Third, the induction culture medium formula of the present invention can achieve a proliferation coefficient of more than 5, which can achieve good economic benefits.

[0028] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0029] Figure 1 This is a flowchart of the cultivation process of the present invention;

[0030] Figure 2 The results of the anti-browning treatment on the induction of clustered buds were shown. Among them, A was treated with soaking in the anti-browning solution; B was not treated with soaking in the anti-browning solution.

[0031] Figure 3 The results show the effect of culture medium on the induction of shoot clusters. In this paper, A represents the modified WPM basal medium, and B represents the WPM basal medium. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to the accompanying drawings, so that those skilled in the art can implement it based on the description.

[0033] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0034] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0035] like Figure 1 As shown, this invention provides a two-step method for rapid propagation of cinnamon buds, comprising the following steps:

[0036] Step 1: Collect cinnamon buds as explants, sterilize and prevent browning of the explants, and then inoculate them into an induction medium for induction culture to obtain clustered buds. Continue to culture until the proliferation coefficient of the clustered buds reaches 5 or more.

[0037] Step 2: Cut off healthy single buds with a bud height >2cm, transfer them to rooting medium, and continue culturing to obtain rooted tissue culture seedlings with a bud height of 3-6cm and a root length of 1-2cm. Then, harden off the seedlings and transplant them.

[0038] In another technical solution, the induction medium is prepared by adding the following raw material components to a modified WPM medium: 6-BA 4.0-6.0 mg / L, KT 0.5-1.0 mg / L, GA3 0.2-0.6 mg / L, NAA 0.2-0.5 mg / L, activated carbon (AC) 0.1%-0.2%, riboflavin 0.8-1.5 mg / L, vitamin C (VC) 5.0-10.0 mg / L, sucrose 25-30 g / L, agar 5.0-7.0 g / L, and pH 6.0.

[0039] In another technical solution, the modified WPM culture medium formulation includes: KNO3 200 mg / L, Ca(NO3)2·4H2O 250 mg / L, (NH4)2SO4 750 mg / L, MgSO4·7H2O 400 mg / L, CaCl2·2H2O 150 mg / L, KH2PO4 300 mg / L, Na2MoO4·2H2O 0.25 mg / L, MnSO4·H2O 22.4 mg / L, ZnSO4·7H2O 15.0 mg / L, CuSO4·5H2O 0.25 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA 37.3 mg / L, inositol 100 mg / L, vitamin B1 1.0 mg / L, niacin 0.5 mg / L, and vitamin B6. 0.5 mg / L, glycine 2.0 mg / L.

[0040] In another technical solution, the rooting medium is prepared by adding the following raw material components to 1 / 2MS medium: IBA 0.8-1.5 mg / L, NAA 0.3-0.8 mg / L, AC 0.1%-0.15%, sucrose 25-30 g / L, agar 5.0-7.0 g / L, and pH 6.0.

[0041] In another technical solution, the conditions for induction culture are: temperature 24-8℃, light intensity 2000Lx, and light duration 10h / day; the conditions for rooting culture are: temperature 24-28℃, light intensity 1000Lx, and light duration 10h / day.

[0042] In another technical solution, the sterilization and anti-browning treatment of explants specifically includes the following steps:

[0043] S1. Immediately after harvesting, cinnamon buds should be placed in a 4℃ refrigerator for 24 hours for cold treatment.

[0044] S2. Take out the cinnamon buds that have been refrigerated in step S1, soak them in a 0.05% detergent solution for 15 minutes, rinse them under running water for 30 minutes, and then soak them in a 2% NaClO solution for 10 minutes on a clean bench.

[0045] S3. Take out the cinnamon buds treated in step S2, soak them in the filtered and sterilized anti-browning combination solution for 5 minutes, and then inoculate them into the induction medium.

[0046] In another technical solution, the formulation of the anti-browning combination solution includes: 50-200 mg / L cysteine ​​salt, 3-5 g / L polyvinylpyrrolidone, 50-100 ml / L cinnamon aqueous extract, and 1.0-3.0 g / L agar.

[0047] In another technical solution, the cinnamon water extract is obtained by pulverizing cinnamon medicinal materials through a 50-mesh sieve, mixing them with purified water at a mass ratio of 1:5 to 10, soaking for 3 hours, heating to boiling for 30 minutes, and then filtering.

[0048] In another technical solution, the specific method for hardening off and transplanting seedlings is as follows: the rooted tissue culture seedlings are hardened off indoors for 3-5 days with the cover off, the roots of the rooted tissue culture seedlings are washed clean to obtain transplanting seedlings; the transplanting seedlings are soaked in disinfectant for 30 seconds and then transplanted to the seedbed. The humidity of the seedbed is maintained at 80%-90% for 10 days before transplanting, and at 60%-80% for 10 days after transplanting. The temperature is controlled at 22-30℃ during the transplanting process. From the time the transplanting seedlings grow new leaves, compound fertilizer solution is applied every 10-15 days.

[0049] In another technical solution, the disinfectant formulation includes 1-1.5 g / L potassium permanganate and 0.5-1 g / L carbendazim.

[0050] The substrate in the seedbed is a mixture of perlite and seedling soil in a volume ratio of 1:2, with a pH of 4.5–6.5.

[0051] Disinfect the substrate with a 0.2%–0.3% potassium permanganate solution 12–24 hours before transplanting;

[0052] The mass fraction of the compound fertilizer solution is 0.1% to 0.3%, and the mass ratio of N:P:K in the compound fertilizer solution is 1:1:1.

[0053] The formulas for calculating the induction rate, multiplication factor, rooting rate, and transplant survival rate of this invention are as follows:

[0054] Induction rate (%) = (Number of buds induced to form clusters / Number of inoculated buds) × 100%;

[0055] Multiplication factor = Number of effective buds with a height greater than 1.0 cm / Number of inoculated buds;

[0056] Rooting rate (%) = (Number of rooted buds / Number of inoculated buds) × 100%;

[0057] Transplant survival rate (%) = (number of surviving tissue culture seedlings / number of transplanted tissue culture seedlings) × 100%.

[0058] <Example 1>

[0059] A two-step method for rapid propagation of cinnamon buds, comprising the following steps:

[0060] Step 1: Collect cinnamon buds as explants. After sterilizing and preventing browning of the explants, inoculate them into an induction medium and induce them for 25 days to obtain clustered buds. Continue to culture for 30 days until the proliferation coefficient of the clustered buds reaches 5 or more.

[0061] Step 2: Cut off healthy single buds with a bud height >2cm, transfer them to rooting medium, and continue culturing to obtain rooted tissue culture seedlings with a bud height of 3-6cm and a root length of 1-2cm. Then, harden off the seedlings and transplant them.

[0062] The induction medium was prepared by adding the following raw material components to a modified WPM medium: 6-BA 5.0 mg / L, KT 1.0 mg / L, GA3 0.5 mg / L, NAA 0.2 mg / L, activated carbon (AC) 0.2%, riboflavin 1.0 mg / L, vitamin C (VC) 8.0 mg / L, sucrose 25 g / L, agar 5.5 g / L, and pH 6.0.

[0063] The modified WPM medium formulation includes: KNO3 200 mg / L, Ca(NO3)2·4H2O 250 mg / L, (NH4)2SO4 750 mg / L, MgSO4·7H2O 400 mg / L, CaCl2·2H2O 150 mg / L, KH2PO4 300 mg / L, Na2MoO4·2H2O 0.25 mg / L, MnSO4·H2O 22.4 mg / L, ZnSO4·7H2O 15.0 mg / L, CuSO4·5H2O 0.25 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA 37.3 mg / L, inositol 100 mg / L, and vitamin B1.

[0064] 1.0 mg / L, niacin 0.5 mg / L, vitamin B6 0.5 mg / L, glycine 2.0 mg / L;

[0065] The rooting medium was prepared by adding the following raw material components to 1 / 2 MS medium: IBA 1.0 mg / L, NAA 0.5 mg / L, AC 0.15%, sucrose 25 g / L, agar 6.0 g / L, pH 6.0;

[0066] The specific steps for sterilizing and preventing browning of explants include:

[0067] S1. Immediately after harvesting, cinnamon buds should be placed in a 4℃ refrigerator for 24 hours for cold treatment.

[0068] S2. Take out the cinnamon buds that have been refrigerated in step S1, soak them in a 0.05% detergent solution for 15 minutes, rinse them under running water for 30 minutes, and then soak them in a 2% NaClO solution for 10 minutes on a clean bench.

[0069] S3. Take out the cinnamon buds treated in step S2, soak them in the filtered and sterilized anti-browning combination solution for 5 minutes, and then inoculate them into the induction medium.

[0070] The specific method for hardening off and transplanting seedlings is as follows: Harden off the rooted tissue culture seedlings indoors for 3 days with the cover off, wash the roots of the rooted tissue culture seedlings to obtain transplanting seedlings; soak the transplanting seedlings in disinfectant solution for 30 seconds and then transplant them to the seedbed. Keep the humidity of the seedbed at 80% to 90% for 10 days before transplanting, and keep the humidity of the seedbed at 60% to 80% for 10 days after transplanting. Control the temperature at 25℃ during the transplanting process. Once the transplanting seedlings grow new leaves, apply compound fertilizer solution every 10 days.

[0071] The formulation of the anti-browning combination solution includes: 100 mg / L cysteine ​​salt, 4 g / L polyvinylpyrrolidone, 80 ml / L cinnamon aqueous extract, and 2.0 g / L agar;

[0072] The disinfectant solution is formulated with 1.0 g / L potassium permanganate and 1.0 g / L carbendazim.

[0073] The substrate in the seedbed is a mixture of perlite and seedling soil in a volume ratio of 1:2, with a pH of 6.5.

[0074] Disinfect the substrate with a 0.2% potassium permanganate solution 12 hours before transplanting;

[0075] The compound fertilizer solution has a mass fraction of 0.1% and a mass ratio of N:P:K of 1:1:1.

[0076] The method provided in this embodiment can induce and proliferate cinnamon bud clusters, with a bud cluster induction rate of 100% and a proliferation multiple of 5.86 times. It can also achieve two-step seedling formation, with a rooting rate of 100% and a transplant survival rate of 88.2%.

[0077] <Example 2>

[0078] A two-step method for rapid propagation of cinnamon buds is identical to the method provided in Example 1, except for the formulation of the induction and rooting media. In this example, the induction media is prepared by adding the following components to a modified WPM medium: 6-BA 4.0 mg / L, KT 0.5 mg / L, GA3 0.2 mg / L, NAA 0.2 mg / L, activated carbon (AC) 0.1%, riboflavin 1.5 mg / L, vitamin C 5.0 mg / L, sucrose 25 g / L, agar 5.0 g / L, and pH 6.0. The rooting media is prepared by adding the following components to a 1 / 2 MS medium: IBA 0.8 mg / L, NAA 0.3 mg / L, AC 0.1%, sucrose 25 g / L, agar 5.0 g / L, and pH 6.0.

[0079] The method in this embodiment can induce and proliferate cinnamon bud clusters, with a 100% bud induction rate and a proliferation multiple of 5.63 times. It can also achieve two-step seedling formation, with a 100% rooting rate and a transplant survival rate of 87.1%.

[0080] <Example 3>

[0081] A two-step method for rapid propagation of cinnamon buds is identical to the method provided in Example 1, except for the formulation of the induction and rooting media. In this example, the induction media is prepared by adding the following components to a modified WPM medium: 6.0 mg / L 6-BA, 1.0 mg / L KT, 0.6 mg / L GA3, 0.5 mg / L NAA, 0.2% AC, 1.5 mg / L riboflavin, 10.0 mg / L vitamin C, 30 g / L sucrose, 7.0 g / L agar, with a pH of 6.0. The rooting media is prepared by adding the following components to 1 / 2 MS medium: 1.5 mg / L IBA, 0.8 mg / L NAA, 0.15% AC, 30 g / L sucrose, 7.0 g / L agar, with a pH of 6.0.

[0082] The method in this embodiment can induce and proliferate cinnamon bud clusters, with a bud cluster induction rate of 100% and a proliferation multiple of 5.02 times. It can also achieve two-step seedling formation, with a rooting rate of 100% and a transplant survival rate of 85.7%.

[0083] <Comparative Example 1>

[0084] A two-step method for rapid propagation of cinnamon buds is the same as the method provided in Example 1, except for the formulation of the rooting medium. The rooting medium formulation of this example is 1 / 2 MS medium with the following raw material components added: IBA 0.5 mg / L, AC 0.1%, sucrose 25 g / L, agar 5.5 g / L, pH 6.0.

[0085] Although the method in this embodiment can achieve rooting of aseptic cinnamon buds, the rooting rate is 67.5%, the roots are relatively thin and long, and the transplant survival rate is 45.3%.

[0086] <Comparative Example 2>

[0087] A two-step method for rapid propagation of cinnamon buds is the same as the method provided in Example 1, except that the rooting medium is formulated. The induction medium in this example is prepared by adding the following raw materials to MS medium: 6-BA 5.0 mg / L, NAA 0.2 mg / L, AC 0.2%, sucrose 25 g / L, agar 5.5 g / L, pH 6.0.

[0088] This comparative method cannot induce cinnamon cluster buds.

[0089] <Comparative Example 3>

[0090] A two-step method for rapid propagation of cinnamon buds is the same as the method provided in Example 1, except that the rooting medium is formulated. The induction medium in this example is prepared by adding the following raw materials to MS medium: GA3 0.5 mg / L, NAA 0.2 mg / L, AC 0.2%, sucrose 25 g / L, agar 5.5 g / L, pH 6.0.

[0091] This comparative method cannot induce cinnamon cluster buds.

[0092] <Comparative Example 4>

[0093] A two-step method for rapid propagation of cinnamon buds is the same as the method provided in Example 1, except that no anti-browning treatment is performed. That is, the cinnamon buds are disinfected immediately after collection and then placed in an induction culture medium for induction culture.

[0094] The results are as follows Figure 2 As shown in the figure. The method in this comparative example could not achieve a good effect in preventing browning. All the young shoots were severely browned after sterilization, and no clustered shoots could be induced in the induction medium.

[0095] <Comparative Example 5>

[0096] A two-step method for rapid propagation of cinnamon buds is the same as the method provided in Example 1, except that WPM basic medium is used instead of modified WPM basic medium.

[0097] The results are as follows Figure 3 As shown in the figure, although the method of this comparative example can induce and proliferate cinnamon bud clusters, the induction rate of bud clusters decreased from 100% to 67.3%, the multiplication factor decreased from 5.86 times to 2.37 times, and the leaves of the bud clusters were prone to yellowing and falling off, which seriously affected the next step of rooting culture.

[0098] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A two-step method for rapid propagation of cinnamon buds, characterized in that, Including the following steps: Step 1: Collect cinnamon buds as explants, sterilize and prevent browning of the explants, and then inoculate them into an induction medium for induction culture to obtain clustered buds. Continue to culture until the proliferation coefficient of the clustered buds reaches 5 or more. The specific steps for sterilizing and preventing browning of explants include: S1. Immediately after harvesting, cinnamon buds should be placed in a 4℃ refrigerator for 24 hours for cold treatment. S2. Take out the cinnamon buds that have been refrigerated in step S1, soak them in a 0.05% detergent solution for 15 minutes, rinse them under running water for 30 minutes, and then soak them in a 2% NaClO solution for 10 minutes on a clean bench. S3. Take out the cinnamon buds treated in step S2, soak them in the filtered and sterilized anti-browning combination solution for 5 minutes, and then inoculate them into the induction medium. The aforementioned anti-browning combination solution is composed of the following raw materials: cysteine ​​salt 50~200 mg / L, polyvinylpyrrolidone 3~5 g / L, cinnamon aqueous extract 50~100 ml / L, and agar 1.0~3.0 g / L; The cinnamon water extract is obtained by pulverizing cinnamon medicinal material through a 50-mesh sieve, mixing it with purified water at a mass ratio of 1:5~10, soaking it for 3 hours, heating it to a boil for 30 minutes, and then filtering it. The induction medium consists of the following ingredients: modified WPM medium, 6-BA 4.0~6.0 mg / L, KT 0.5~1.0 mg / L, GA3 0.2~0.6 mg / L, NAA 0.2~0.5 mg / L, activated carbon 0.1%~0.2%, riboflavin 0.8~1.5 mg / L, vitamin C 5.0~10.0 mg / L, sucrose 25~30 g / L, agar 5.0~7.0 g / L, and pH 6.

0. The modified WPM medium is composed of the following ingredients: KNO3 200 mg / L, Ca(NO3)2·4H2O 250 mg / L, (NH4)2SO4 750 mg / L, MgSO4·7H2O 400 mg / L, CaCl2·2H2O 150 mg / L, KH2PO4 300 mg / L, Na2MoO4·2H2O 0.25 mg / L, MnSO4·H2O 22.4 mg / L, ZnSO4·7H2O 15.0 mg / L, CuSO4·5H2O 0.25 mg / L, FeSO4·7H2O 27.8 mg / L, Na2-EDTA 37.3 mg / L, inositol 100 mg / L, vitamin B1 1.0 mg / L, nicotinic acid 0.5 mg / L, vitamin B6 0.5 mg / L, and glycine 2.0 mg / L. mg / L; The conditions for induction culture were: temperature 24~28℃, light intensity 2000 Lx, and light duration 10 h / day; the conditions for rooting culture were: temperature 24~28℃, light intensity 1000 Lx, and light duration 10 h / day. Step 2: Cut off healthy single buds with a bud height >2 cm, transfer them to rooting medium, and continue culturing to obtain rooted tissue culture seedlings with a bud height of 3-6 cm and a root length of 1-2 cm. Then, harden off the seedlings and transplant them. The rooting medium consists of the following ingredients: 1 / 2 MS medium, IBA 0.8~1.5 mg / L, NAA 0.3~0.8 mg / L, AC 0.1%~0.15%, sucrose 25~30 g / L, agar 5.0~7.0 g / L, and pH 6.0; The specific method for hardening off and transplanting seedlings is as follows: Harden off the rooted tissue culture seedlings indoors for 3-5 days with the cover off. Wash the roots of the rooted tissue culture seedlings to obtain transplanting seedlings. Soak the transplanting seedlings in disinfectant for 30 seconds and then transplant them to the seedbed. Keep the humidity of the seedbed at 80%-90% for 10 days before transplanting and at 60%-80% for 10 days after transplanting. Control the temperature at 22-30℃ during the transplanting process. Once the transplanted seedlings grow new leaves, apply compound fertilizer solution every 10-15 days. The disinfectant solution is formulated with potassium permanganate 1~1.5g / L and carbendazim 0.5~1g / L; The substrate in the seedbed is a mixture of perlite and seedling soil at a volume ratio of 1:2, with a pH of 4.5-6.

5. Disinfect the substrate with a 0.2%–0.3% potassium permanganate solution 12–24 hours before transplanting; The mass fraction of the compound fertilizer solution is 0.1%~0.3%, and the mass ratio of N:P:K in the compound fertilizer solution is 1:1:1.

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