Method for reducing browning rate of zanthoxylum bungeanum in tissue culture

Abstract

The application discloses a method for reducing the browning rate of Zanthoxylum bungeanum in tissue culture, and belongs to the technical field of Zanthoxylum bungeanum tissue culture. The method is characterized by adopting polyvinylpyrrolidone and modified chitosan as raw materials to prepare a prevention and control agent, coating the tissue culture material with the prevention and control agent, inoculating the coated tissue culture material into a culture medium, and then carrying out tissue culture, so that the phenolic substances released by the tissue culture material and the quinone products formed by oxidation are adsorbed and fixed and covalently combined, the damage to the tissue culture material is reduced, the browning rate of Zanthoxylum bungeanum in tissue culture is effectively reduced, the propagation efficiency of Zanthoxylum bungeanum in tissue culture is ensured, and the method has a good application prospect in the field of Zanthoxylum bungeanum tissue culture.

Description

Technical Field

[0001] This invention relates to the field of Sichuan pepper tissue culture technology, and in particular to a method for reducing the browning rate of Sichuan pepper tissue culture. Background Technology

[0002] Sichuan pepper (Zanthoxylum bungeanum Maxim.) is a deciduous small tree or perennial plant belonging to the Rutaceae family and the Zanthoxylum genus. As an important economic crop, Sichuan pepper possesses high nutritional, economic, and ecological value. With the increasing market demand for Sichuan pepper, traditional propagation methods such as cuttings and sowing are no longer sufficient to meet the needs of large-scale production. Plant tissue culture technology, due to its advantages of rapid propagation and stable genetic traits, has shown great potential for application in Sichuan pepper propagation.

[0003] Currently, many problems still hinder the development of Sichuan pepper tissue culture. Among these, browning is a particularly serious issue. The phenolic substances produced during browning are toxic to the tissue culture material, affecting normal cell metabolism and growth, inhibiting bud differentiation, and reducing the overall reproductive efficiency of the tissue culture process. The conventional method for inhibiting browning is to add activated carbon or polyvinylpyrrolidone (PVP) to the culture medium. Through physical adsorption or complexation, these methods capture phenolic substances released from the explants into the medium, reducing the contact between phenolic substances and polyphenol oxidase, thereby inhibiting the oxidative browning reaction of phenolic substances and ultimately reducing the browning rate. However, both methods have significant limitations in practical applications. Activated carbon exhibits non-selective adsorption of substances in the culture medium, adsorbing plant growth regulators and other nutrients along with phenols, thus affecting the normal growth and differentiation of the tissue culture material. Compared to activated carbon, PPV is more specific in its adsorption of phenolic substances; however, the concentration of PPV is difficult to control precisely—high concentrations slow down the proliferation rate of differentiated buds, while low concentrations have little inhibitory effect on browning.

[0004] Therefore, there is an urgent need to find a method to reduce the browning rate of Sichuan pepper tissue culture and to solve the problem that it is difficult to simultaneously inhibit browning and ensure the normal growth of tissue culture materials when adding polyvinylpyrrolidone to the tissue culture medium in a conventional way. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide a method for reducing the browning rate of Sichuan pepper tissue culture, solving the problem that conventional methods of adding polyvinylpyrrolidone to tissue culture media are difficult to simultaneously inhibit browning and ensure the normal growth of tissue culture materials.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] A method for reducing the browning rate of Sichuan pepper tissue culture, the method comprising the following steps:

[0008] (1) Selection and treatment of explants: collect leaves or stems of healthy, pollution-free subcultured pepper seedlings as explants, cut the leaves into leaf pieces with a side length of 1-2 cm or cut the stems into stem segments with a length of 1-2 cm, and then coat them with a control agent.

[0009] (2) Induction culture: The explants after the coating treatment are inoculated into the induction culture medium and induced to form callus tissue;

[0010] (3) Differentiation culture: When the callus tissue grows to 0.8-1.5cm, it is coated with a control agent and then transferred to a differentiation culture medium to form adventitious shoots;

[0011] (4) Subculture: When the adventitious shoots grow to a height of 1.5-2.5 cm, they are coated with a control agent and then transferred to a subculture medium to form proliferating seedlings.

[0012] (5) Rooting culture: After the propagation seedlings grow to a height of 2-3 cm, they are transferred to the rooting culture medium to obtain rooted seedlings.

[0013] Furthermore, the control agent comprises the following raw materials:

[0014] Polyvinylpyrrolidone, sodium alginate, chitosan, thioglycolic acid, EDC·HCl, N-hydroxysuccinimide, sodium hexametaphosphate, maltodextrin, calcium chloride.

[0015] Furthermore, the preparation method of the control agent is as follows:

[0016] A: Dissolve sodium alginate in water to prepare a 1wt% sodium alginate solution, add polyvinylpyrrolidone and stir to dissolve, to obtain control agent A;

[0017] B: Add chitosan to 1wt% acetic acid solution and stir to dissolve. Then add EDC·HCl and N-hydroxysuccinimide, stir for 15-30 min, add mercaptoacetic acid, adjust pH to 5-5.5 and stir to react for 4-6 h. After the reaction is complete, adjust pH to 8, then centrifuge to collect the precipitate and wash it twice with ethanol. Dry it under vacuum at 40℃ for 12-24 h to obtain modified chitosan.

[0018] C: Sodium hexametaphosphate and maltodextrin were dissolved in 5 times their weight of water to prepare sodium hexametaphosphate solution and maltodextrin solution, respectively. The sodium hexametaphosphate solution and maltodextrin solution were mixed and stirred continuously at 500-800 r / min for 10-30 min to obtain a mixture. Then, the mixture was spray-dried at an inlet air temperature of 160-180℃, an outlet air temperature of 80-90℃, and a feed rate of 3-5 mL / min to obtain composite microparticles.

[0019] D: Prepare a 0.5wt% modified chitosan solution by dissolving modified chitosan in water, then add composite microparticles and calcium chloride and mix evenly to obtain agent B.

[0020] Furthermore, in step A, the mass ratio of sodium alginate solution to polyvinylpyrrolidone is 1000:(0.1-0.2).

[0021] Furthermore, in step B, the mass ratio of chitosan, EDC·HCl, N-hydroxysuccinimide, and mercaptoacetic acid is 10:(20-25):(8-12):(8-10).

[0022] Furthermore, in step C, the mass ratio of sodium hexametaphosphate solution to maltodextrin solution is 1:(4-5).

[0023] Furthermore, in step D, the mass ratio of modified chitosan solution, composite microparticles, and calcium chloride is 1000:(8-10):(3-5).

[0024] Furthermore, the coating process is specifically as follows:

[0025] First, soak the product in agent A for 3-5 minutes, then remove it and air dry for 20-30 minutes. Next, soak the product in agent B for 3-5 minutes, then remove it and air dry for 1-2 hours.

[0026] Furthermore, the components of the induction medium, differentiation medium, subculture medium, and rooting medium are as follows:

[0027] Induction medium: MS + 0.5–0.7 mg / L 6-BA + 0.2–0.4 mg / L 2,4-D + 20–40 g / L sucrose + 5–7 g / L agar; pH of the induction medium is 5.8–6.0.

[0028] Differentiation medium: MS + 0.03–0.05 mg / L TDZ + 0.1–0.3 mg / L IBA + 20–40 g / L sucrose + 5–7 g / L agar; pH of the differentiation medium is 5.8–6.0.

[0029] Subculture medium: 1 / 2 MS + 0.5-1.5 mg / L 6-BA + 0.2-0.6 mg / L IBA + 7.0-7.5 g / L agar + 25-30 g / L sucrose, wherein the pH of the subculture medium is 5.8-6.0;

[0030] Rooting medium: 1 / 2 MS + 0.5-0.7 mg / L NAA + 0.3-0.4 mg / L IBA + 7.0-7.5 g / L agar + 25-30 g / L sucrose, wherein the pH of the rooting medium is 5.8-6.0.

[0031] Furthermore, the conditions for the induction culture, differentiation culture, subculture culture, and rooting culture are as follows:

[0032] The induction culture temperature was 23–27℃, the light intensity was 1500–2000 lux, the light duration was 12–14 h / d, and the induction culture time was 10–45 days.

[0033] The differentiation culture temperature was 23–27℃, the light intensity was 2000–2500 lux, the light duration was 14–16 h / d, and the differentiation culture time was 15–55 days.

[0034] The subculture temperature was 23–27℃, the light intensity was 2000–3000 lux, the light duration was 12–16 h / d, and the subculture time was 25–40 days.

[0035] The rooting culture temperature is 23–27℃, the light intensity is 1500–2000 lux, the light duration is 12 h / d, and the rooting culture time is 20–25 days.

[0036] During the process of cutting and inoculating tissue culture materials into the culture medium, phenolic substances are released from the wounds in the early stages of tissue culture. These released phenolic substances are then further oxidized to form quinones, which damage the tissue culture materials and cause browning. This invention prepares a control agent to coat the tissue culture materials before transferring them to the culture medium for tissue culture. By using the control agent to chelate phenolic substances, the formation of quinones is reduced, the toxicity of quinones is decreased, and the contact between quinones and tissue culture materials is inhibited, effectively reducing the browning rate and ensuring the normal growth of the tissue culture materials, thereby effectively ensuring the propagation efficiency of Sichuan pepper tissue culture.

[0037] Specifically, this invention prepares control agent A by adding polyvinylpyrrolidone to sodium alginate solution, and prepares control agent B by adding calcium chloride after modifying chitosan. Then, control agents A and B are used sequentially to coat the tissue culture material. The sodium alginate in control agent A crosslinks with the calcium chloride in control agent B to form calcium alginate, achieving the purpose of forming a double film layer on the surface of the tissue culture material. In control agent A, polyvinylpyrrolidone first complexes with phenolic substances released from the wounds of the tissue culture material in the early stage, inhibiting their conversion into quinone substances. Then, in control agent B, chitosan covalently binds with quinone substances that are not complexed with polyvinylpyrrolidone, as well as those formed by desorption after complexation with polyvinylpyrrolidone, thereby reducing their toxicity to the tissue culture material.

[0038] This invention also adds maltodextrin-coated sodium hexametaphosphate composite microparticles to the control agent B. In the high-humidity culture flask of Sichuan pepper tissue culture, the composite microparticles in the control agent B slowly release sodium hexametaphosphate. Then, the sodium hexametaphosphate competes with calcium ions in calcium alginate, causing the destruction of the film layer formed by the control agent A. As a result, the control agent film layer will gradually be lost and detached, and then polyvinylpyrrolidone carrying phenolic substances and chitosan carrying quinone substances will detach from the tissue culture material, thereby achieving the purpose of reducing the contact between quinone substances and tissue culture material.

[0039] Beneficial effects:

[0040] This invention prepares control agent A and control agent B to coat tissue culture materials, forming a double-layer structure on the surface of the materials. The membrane layer complexes the phenolic substances released by the tissue culture materials, reduces the toxicity of quinone substances, and reduces the contact between quinone substances and the tissue culture materials, thereby effectively inhibiting the browning of Sichuan pepper tissue culture and ensuring good growth of the materials. This achieves efficient Sichuan pepper tissue culture and is of great significance for promoting the development of the Sichuan pepper industry. Attached Figure Description

[0041] Figure 1 Image showing the induction culture process of Sichuan pepper tissue culture according to the present invention;

[0042] Figure 2 Image showing rooting culture in the tissue culture of Sichuan pepper according to the present invention. Detailed Implementation

[0043] The present invention will now be described in detail with reference to specific embodiments and accompanying drawings:

[0044] Example 1: Preparation of Control Agent

[0045] A: Dissolve sodium alginate in water to prepare 1000g of 1wt% sodium alginate solution, add 0.15g of polyvinylpyrrolidone and stir to dissolve, to obtain control agent A;

[0046] B: Add 10g of chitosan to 100g of 1wt% acetic acid solution and stir to dissolve. Then add 22g of EDC·HCl and 10g of N-hydroxysuccinimide. Stir for 15-30min and then add 9g of mercaptoacetic acid. Adjust the pH to 5.2 and stir to react for 5h. After the reaction is complete, adjust the pH to 8. Then centrifuge to collect the precipitate and wash it twice with ethanol. Dry it under vacuum at 40℃ for 18h to obtain modified chitosan.

[0047] C: Sodium hexametaphosphate and maltodextrin were dissolved in 5 times their mass of water to prepare sodium hexametaphosphate solution and maltodextrin solution, respectively. They were mixed at a mass ratio of sodium hexametaphosphate solution to maltodextrin solution = 1:4 and stirred continuously at 600 r / min for 20 min to obtain a mixture. Then, the mixture was spray-dried at an inlet air temperature of 170℃, an outlet air temperature of 85℃, and a feed rate of 4 mL / min to obtain composite microparticles.

[0048] D: Dissolve the modified chitosan in water to prepare 1000g of 0.5wt% modified chitosan solution, then add 9g of composite microparticles and 4g of calcium chloride and mix evenly to obtain agent B.

[0049] Example 2: Preparation of Control Agent II

[0050] A: Dissolve sodium alginate in water to prepare 1000g of 1wt% sodium alginate solution, add 0.1g of polyvinylpyrrolidone and stir to dissolve, to obtain control agent A;

[0051] B: Add 10g of chitosan to 100g of 1wt% acetic acid solution and stir to dissolve. Then add 20g of EDC·HCl and 8g of N-hydroxysuccinimide. Stir for 15min and then add 8g of mercaptoacetic acid. Adjust the pH to 5 and stir to react for 4h. After the reaction is complete, adjust the pH to 8, then centrifuge to collect the precipitate and wash it twice with ethanol. Dry it under vacuum at 40℃ for 12h to obtain modified chitosan.

[0052] C: Sodium hexametaphosphate and maltodextrin were dissolved in 5 times their mass of water to prepare sodium hexametaphosphate solution and maltodextrin solution, respectively. They were mixed at a mass ratio of sodium hexametaphosphate solution to maltodextrin solution = 1:4 and stirred continuously at 500 r / min for 30 min to obtain a mixture. Then, the mixture was spray-dried at an inlet air temperature of 160℃, an outlet air temperature of 80℃, and a feed rate of 3 mL / min to obtain composite microparticles.

[0053] D: Dissolve the modified chitosan in water to prepare 1000g of 0.5wt% modified chitosan solution, then add 8g of composite microparticles and 3g of calcium chloride and mix evenly to obtain agent B.

[0054] Example 3: Preparation of Control Agent

[0055] A: Dissolve sodium alginate in water to prepare 1000g of 1wt% sodium alginate solution, add 0.2g of polyvinylpyrrolidone and stir to dissolve, to obtain control agent A;

[0056] B: Add 10g of chitosan to 100g of 1wt% acetic acid solution and stir to dissolve. Then add 25g of EDC·HCl and 12g of N-hydroxysuccinimide. Stir for 15-30min and then add 10g of mercaptoacetic acid. Adjust the pH to 5.5 and stir to react for 6h. After the reaction is complete, adjust the pH to 8. Then centrifuge to collect the precipitate and wash it twice with ethanol. Dry it under vacuum at 40℃ for 24h to obtain modified chitosan.

[0057] C: Sodium hexametaphosphate and maltodextrin were dissolved in 5 times their mass of water to prepare sodium hexametaphosphate solution and maltodextrin solution, respectively. They were mixed at a mass ratio of sodium hexametaphosphate solution to maltodextrin solution = 1:5 and stirred continuously at 800 r / min for 10 min to obtain a mixture. Then, the mixture was spray-dried at an inlet air temperature of 180℃, an outlet air temperature of 90℃, and a feed rate of 5 mL / min to obtain composite microparticles.

[0058] D: Dissolve the modified chitosan in water to prepare 1000g of 0.5wt% modified chitosan solution, then add 10g of composite microparticles and 5g of calcium chloride and mix evenly to obtain agent B.

[0059] Comparative Example 1: Preparation of Control Agent

[0060] Compared with Example 1, the only difference is that the original step B was omitted in the preparation of the control agent in Comparative Example 1, that is, the chitosan was not modified and conventional chitosan was used instead. The remaining steps are the same as in Example 1, as shown below.

[0061] A: Same as Example 1;

[0062] B: Step C in Example 1 is the same;

[0063] C: Dissolve chitosan in water to prepare 1000g of 0.5wt% chitosan solution, then add 9g of composite microparticles and 4g of calcium chloride and mix evenly to obtain agent B.

[0064] Comparative Example 2: Preparation of Control Agent

[0065] Compared with Example 1, the only difference in Comparative Example 2 is that step C was omitted in the preparation of the control agent, i.e., instead of using maltodextrin to coat sodium hexametaphosphate, sodium hexametaphosphate was added directly. The remaining steps are the same as in Example 1, as detailed below:

[0066] A-B: Same as in Example 1;

[0067] C: Dissolve modified chitosan in water to prepare 1000g of 0.5wt% modified chitosan solution, then add 0.2g of sodium hexametaphosphate and 4g of calcium chloride and mix well to obtain agent B.

[0068] Comparative Example 3: Preparation of Control Agent

[0069] Compared with Example 1, the only difference is that polyvinylpyrrolidone was not added in step A of the preparation of the control agent in Comparative Example 3, while the other steps were the same as in Example 1.

[0070] Comparative Example 4: Preparation of Control Agent

[0071] Compared with Example 1, the only difference is that in Comparative Example 4, sodium alginate was used instead of modified chitosan in the preparation of agent B. All other steps are the same as in Example 1, as detailed below:

[0072] A: Same as Example 1;

[0073] B: Same as step C in Example 1;

[0074] C: Dissolve sodium alginate in water to prepare 1000g of 0.5wt% sodium alginate solution, then add 9g of composite microparticles and 4g of calcium chloride and mix evenly to obtain control agent B.

[0075] Comparative Example 5: Preparation of Control Agent

[0076] Compared with Example 1, the only difference is that in Comparative Example 5, no composite microparticles were added in step D during the preparation of the control agent; all other steps were the same as in Example 1.

[0077] Comparative Example 6: Preparation of Control Agent

[0078] Compared with Example 1, the only difference is that the concentration of sodium alginate solution in step A of the preparation of the control agent in Comparative Example 6 is 2 wt%, while the rest of the steps are the same as in Example 1.

[0079] Comparative Example 7: Preparation of Control Agent

[0080] Compared with Example 1, the only difference is that the concentration of the modified chitosan solution in step D of the preparation of the control agent in Comparative Example 7 is 1 wt%, while the rest of the steps are the same as in Example 1.

[0081] Example 4: Sichuan pepper tissue culture method

[0082] The culture medium components used in this embodiment are as follows:

[0083] Induction medium: MS + 0.6 mg / L 6-BA + 0.3 mg / L 2,4-D + 30 g / L sucrose + 6 g / L agar; pH of the induction medium was 5.8.

[0084] Differentiation medium: MS + 0.034 mg / L TDZ + 0.2 mg / L IBA + 30 g / L sucrose + 6 g / L agar; pH of the differentiation medium was 5.8.

[0085] Subculture medium: 1 / 2 MS + 1 mg / L 6-BA + 0.4 mg / L IBA + 7.0 g / L agar + 25 g / L sucrose, the pH of the subculture medium is 5.8;

[0086] Rooting medium: 1 / 2 MS + 0.6 mg / L NAA + 0.3 mg / L IBA + 7.0 g / L agar + 25 g / L sucrose, the pH of the rooting medium is 5.8;

[0087] (1) Selection and treatment of explants: collect the stems of healthy, pollution-free subcultured pepper seedlings as explants, cut the stems into 1.5cm long segments, and then coat them with the control agent of Example 2. Specifically, first soak them in control agent A for 4 minutes, then take them out and air dry for 25 minutes, then soak them in control agent B for 4 minutes, then take them out and air dry for 1 hour.

[0088] (2) Induction culture: The explants after the coating treatment were inoculated into the induction culture medium and the induction culture temperature was 24℃, the light intensity was 1800 lux, the light time was 12h / d, and the induction culture was carried out for 28 days to form callus tissue;

[0089] (3) Differentiation culture: When the callus tissue grows to 1cm, it is cut off and first soaked in the control agent A of Example 2 for 4min, then taken out and dried for 25min. Then it is soaked in the control agent B of Example 2 for 4min, then taken out and dried for 1h before being transferred to the differentiation culture medium. The differentiation culture temperature is 24℃, the light intensity is 2200lux, the light time is 14h / d, and adventitious buds are formed after 30 days of differentiation.

[0090] (4) Subculture: When the adventitious shoots grow to a height of 2cm, cut them off and soak them in the control agent A of Example 2 for 4min, then take them out and air dry them for 25min. Then soak them in the control agent B of Example 2 for 4min, then take them out and air dry them for 1h before transferring them to the subculture medium. The subculture temperature is 24℃, the light intensity is 2200 lux, and the light time is 12h / d. After 30 days of subculture, proliferating seedlings are formed.

[0091] (5) Rooting culture: After the propagation seedlings grow to a height of 2.5cm, they are transferred to the rooting culture medium. The rooting culture temperature is 24℃, the light intensity is 1800 lux, the light time is 12h / d, and the rooting culture time is 22 days to obtain rooted seedlings.

[0092] Experiment: Sichuan pepper tissue culture experiment

[0093] The control agents prepared in Example 1 and Comparative Examples 1-7 were used in a tissue culture experiment on Sichuan pepper. The experiment was divided into 9 groups: experimental group 1, control groups 1-7, and blank control group. Experimental group 1 used the control agent prepared in Example 1 and the tissue culture method of Example 4; control groups 1-7 used the control agents prepared in Comparative Examples 1-7 and the tissue culture method of Example 4, respectively; the blank control group was not treated with the control agent, but 200 mg / L of polyvinylpyrrolidone was added directly to the culture medium, and the remaining steps were the same as those in Example 4.

[0094] Stems from healthy, vigorous subcultured Sichuan pepper seedlings with similar growth were collected as explants in each group. There were 30 explants (stem segments) in each group. After 20 days of induction culture, the browning rate of explants in each group was recorded. After 20 days of differentiation culture, the bud differentiation rate of each group was counted. The average data obtained from the three replicate experiments are shown in Table 1.

[0095] Table 1

[0096]

[0097] Based on the data analysis in Table 1, we can conclude that:

[0098] (1) In experimental group 1 of this invention, the browning rate of Sichuan pepper tissue culture was only 6.7%, and the differentiation rate reached 93.3%. In contrast, the browning rate of the blank control, when a higher concentration of polyvinylpyrrolidone was directly added, reached 11.1%, and the differentiation rate decreased to 83.3%. This indicates that preparing the control agent according to the method of this invention, coating the Sichuan pepper tissue culture material with a membrane, and then inoculating it into the culture medium for tissue culture can effectively reduce the browning rate of the tissue, ensure the good growth of the tissue culture seedlings, and achieve efficient tissue culture propagation of Sichuan pepper. However, directly increasing the concentration of polyvinylpyrrolidone in the culture medium to reduce the browning rate has a limited effect and will also affect the normal growth of the tissue culture material and reduce the differentiation rate.

[0099] (2) In control group 1, chitosan was not modified during the preparation of the control agent, and the binding ability of chitosan to quinones was limited, resulting in an increase in the browning rate of the tissue culture. In control group 4, the control agent B was prepared using sodium alginate instead of modified chitosan, which lacked the binding ability of modified chitosan to quinones, causing toxicity to the tissue culture material, increasing the browning rate and decreasing the differentiation rate to some extent. In control group 3, polyvinylpyrrolidone was not added during the preparation of the control agent, which failed to effectively adsorb phenolic substances. A large amount of phenolic substances were transformed into toxic quinones. The effect of modified chitosan alone in reducing the browning rate by binding quinones was limited, and the browning rate increased significantly. The toxicity of quinones to the tissue culture material also caused a significant decrease in the differentiation rate.

[0100] (3) In control group 2, sodium hexametaphosphate was not coated with maltodextrin during the preparation of the control agent, but was added directly. This caused sodium hexametaphosphate to bind to calcium ions in control agent B in advance, which affected the cross-linking and film formation of control agent B on the surface of the tissue culture material. The swelling and shedding of the film in the early stage reduced the effect of the control agent. In control group 5, no composite microparticles were added during the preparation of the control agent, which failed to release sodium hexametaphosphate in time to destroy the film structure and cause the film to fall off from the surface of the tissue culture material. The contact between quinone substances and tissue culture material increased, which affected the normal growth of tissue culture material.

[0101] (4) The concentration of sodium alginate solution was too high when the control agent was prepared in control group 6, and the concentration of chitosan solution was too high when the control agent was prepared in control group 7. The membrane structure formed in control groups 7 and 8 was stable and difficult to detach from the surface of the tissue culture material, which increased the contact rate with quinone substances and thus affected the browning and differentiation of the tissue culture material.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A method for reducing the browning rate of Sichuan pepper tissue culture, characterized in that, The method includes the following steps: (1) Selection and treatment of explants: collect leaves or stems of healthy, pollution-free subcultured pepper seedlings as explants, cut the leaves into leaf pieces with a side length of 1-2 cm or cut the stems into stem segments with a length of 1-2 cm, and then coat them with a control agent. (2) Induction culture: The explants after the coating treatment are inoculated into the induction culture medium and induced to form callus tissue; (3) Differentiation culture: When the callus tissue grows to 0.8-1.5cm, it is coated with a control agent and then transferred to a differentiation culture medium to form adventitious shoots; (4) Subculture: When the adventitious shoots grow to a height of 1.5-2.5 cm, they are coated with a control agent and then transferred to a subculture medium to form proliferating seedlings. (5) Rooting culture: After the propagation seedlings grow to a height of 2-3 cm, they are transferred to the rooting culture medium to obtain rooted seedlings.

2. The method for reducing the browning rate of Sichuan pepper tissue culture according to claim 1, characterized in that, The control agent comprises the following raw materials: Polyvinylpyrrolidone, sodium alginate, chitosan, thioglycolic acid, EDC·HCl, N-hydroxysuccinimide, sodium hexametaphosphate, maltodextrin, calcium chloride.

3. The method for reducing the browning rate of Sichuan pepper tissue culture according to claim 2, characterized in that, The preparation method of the control agent is as follows: A: Dissolve sodium alginate in water to prepare a 1wt% sodium alginate solution, add polyvinylpyrrolidone and stir to dissolve, to obtain control agent A; B: Add chitosan to 1wt% acetic acid solution and stir to dissolve. Then add EDC·HCl and N-hydroxysuccinimide, stir for 15-30 min, add mercaptoacetic acid, adjust pH to 5-5.5 and stir to react for 4-6 h. After the reaction is complete, adjust pH to 8, then centrifuge to collect the precipitate and wash it twice with ethanol. Dry it under vacuum at 40℃ for 12-24 h to obtain modified chitosan. C: Sodium hexametaphosphate and maltodextrin were dissolved in 5 times their weight of water to prepare sodium hexametaphosphate solution and maltodextrin solution, respectively. The sodium hexametaphosphate solution and maltodextrin solution were mixed and stirred continuously at 500-800 r / min for 10-30 min to obtain a mixture. Then, the mixture was spray-dried at an inlet air temperature of 160-180℃, an outlet air temperature of 80-90℃, and a feed rate of 3-5 mL / min to obtain composite microparticles. D: Prepare a 0.5wt% modified chitosan solution by dissolving modified chitosan in water, then add composite microparticles and calcium chloride and mix evenly to obtain agent B.

4. The method for reducing the browning rate of Sichuan pepper tissue culture according to claim 3, characterized in that, In step A, the mass ratio of sodium alginate solution to polyvinylpyrrolidone is 1000:(0.1-0.2).

5. The method for reducing the browning rate of Sichuan pepper tissue culture according to claim 4, characterized in that, In step B, the mass ratio of chitosan, EDC·HCl, N-hydroxysuccinimide, and thioglycolic acid is 10:(20-25):(8-12):(8-10).

6. The method for reducing the browning rate of Sichuan pepper tissue culture according to claim 5, characterized in that, In step C, the mass ratio of sodium hexametaphosphate solution to maltodextrin solution is 1:(4-5).

7. The method for reducing the browning rate of Sichuan pepper tissue culture according to claim 6, characterized in that, In step D, the mass ratio of modified chitosan solution, composite microparticles, and calcium chloride is 1000:(8-10):(3-5).

8. The method for reducing the browning rate of Sichuan pepper tissue culture according to claim 1, characterized in that, The specific coating process is as follows: First, soak the product in agent A for 3-5 minutes, then remove it and air dry for 20-30 minutes. Next, soak the product in agent B for 3-5 minutes, then remove it and air dry for 1-2 hours.

9. A method for reducing the browning rate of Sichuan pepper tissue culture according to claim 8, characterized in that, The components of the induction medium, differentiation medium, subculture medium, and rooting medium are as follows: Induction medium: MS + 0.5–0.7 mg / L 6-BA + 0.2–0.4 mg / L 2,4-D + 20–40 g / L sucrose + 5–7 g / L agar; pH of the induction medium is 5.8–6.

0. Differentiation medium: MS + 0.03–0.05 mg / L TDZ + 0.1–0.3 mg / L IBA + 20–40 g / L sucrose + 5–7 g / L agar; pH of the differentiation medium is 5.8–6.

0. Subculture medium: 1 / 2 MS + 0.5-1.5 mg / L 6-BA + 0.2-0.6 mg / L IBA + 7.0-7.5 g / L agar + 25-30 g / L sucrose, wherein the pH of the subculture medium is 5.8-6.0; Rooting medium: 1 / 2 MS + 0.5-0.7 mg / L NAA + 0.3-0.4 mg / L IBA + 7.0-7.5 g / L agar + 25-30 g / L sucrose, wherein the pH of the rooting medium is 5.8-6.

0.

10. A method for reducing the browning rate of Sichuan pepper tissue culture according to claim 9, characterized in that, The conditions for induction culture, differentiation culture, subculture culture, and rooting culture are as follows: The induction culture temperature was 23–27℃, the light intensity was 1500–2000 lux, the light duration was 12–14 h / d, and the induction culture time was 10–45 days. The differentiation culture temperature was 23–27℃, the light intensity was 2000–2500 lux, the light duration was 14–16 h / d, and the differentiation culture time was 15–55 days. The subculture temperature was 23–27℃, the light intensity was 2000–3000 lux, the light duration was 12–16 h / d, and the subculture time was 25–40 days. The rooting culture temperature is 23–27℃, the light intensity is 1500–2000 lux, the light duration is 12 h / d, and the rooting culture time is 20–25 days.

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