A method for inducing differentiation and culturing callus of Rhizoma Citri Reticulatae

Abstract

The present invention belongs to the technical field of plant tissue culture, and specifically relates to an induced differentiation of callus of Amaranth and a culture method thereof. The induced differentiation method of Amaranth callus described in the present invention comprises the following steps: S1: washing and disinfecting Amaranth seedling leaves, cutting them, and obtaining Amaranth explants; S2: inoculating the Amaranth explants into an induction medium and culturing them to obtain callus tissue; S3: inoculating the callus tissue into a differentiation medium and culturing them to obtain Amaranth adventitious buds; S4: inoculating the Amaranth adventitious buds into a proliferation medium and culturing them to obtain Amaranth cluster buds; S5: inoculating the Amaranth cluster buds into a rooting medium and culturing them to obtain Amaranth seedlings. The method of the present invention effectively improves the reproductive efficiency and economic benefits of Amaranth, can be applied to large-scale, factory-scale cultivation of Amaranth, and promotes the development and utilization of Amaranth resources.

Description

Technical Field

[0001] The invention belongs to the technical field of plant tissue culture, and in particular relates to an induced differentiation of scutellaria callus and a culture method thereof. Background Art

[0002] Ilex asprella (Hook. et Am.) Champ. ex Benth, also known as Ilex asprella, star tree, wild liquorice, and mountain holly root, is the dried root and stem of the holly family. It is a key ingredient in Lingnan herbal teas. Currently, Ilex asprella is a major ingredient in traditional Chinese medicines for colds. Its chemical components, primarily triterpenes, phenolic acids, polysaccharides, and volatile oils, exhibit a wide range of pharmacological effects, including anti-inflammatory, antipyretic, analgesic, anti-pathogenic, anti-tumor, anti-complement, anti-ulcer, and anti-Alzheimer's disease activities, making it of great medicinal and economic value.

[0003] The sedge is distributed in many provinces of my country. It prefers a warm and humid climate and is not very particular about soil. It can grow on fertile or barren land. It often grows under sparse forests or in shrubs on hillsides. In recent years, the market demand for sedge has been increasing. Although a small number of sedges have been cultivated, they still cannot meet the market demand.

[0004] Callus induction and differentiation are characterized by a short cycle, high growth rate, and year-round production, making them an effective method for rapid propagation of plant seedlings. Callus induction and differentiation are independent of location and season, allowing for continuous and rapid propagation. They are also simple to perform and require minimal time, resulting in high-quality mature seedlings with well-developed root systems, significantly reducing production costs and improving productivity. To achieve large-scale cultivation of jasmine, a method for inducing and differentiating jasmine callus and cultivating it is crucial to meet the growing market demand. Summary of the Invention

[0005] The primary purpose of the present invention is to provide an induced differentiation of callus tissue of Atractylodes lanceolata and a culture method thereof, so as to realize the large-scale culture and planting of Atractylodes lanceolata.

[0006] To this end, the present invention provides the following technical solutions.

[0007] One aspect of the present invention provides a method for inducing differentiation of callus of Rhizoma Citri Reticulatae, comprising the following steps:

[0008] S1: Wash and disinfect the leaves of the safflower seedlings, then cut them to obtain safflower explants;

[0009] S2: Inoculate the explants of P. chebula into the induction medium and culture them at 25-30°C and 2000-3000 lux for 7-14 days to obtain callus tissue;

[0010] S3: The callus tissue was inoculated into a differentiation medium and cultured at 25-30°C and a light intensity of 2000-3000 lux for 10-20 days to obtain adventitious buds of the plant.

[0011] S4: inoculating the adventitious buds of the Chinese yew into a proliferation medium, and culturing them at 25-30°C and a light intensity of 2000-3000 lux for 15-25 days to obtain clusters of Chinese yew buds;

[0012] S5: Inoculate the buds of the Chinese yew tree into a rooting medium, and culture them at 25-30°C and a light intensity of 2000-3000 lux for 15-25 days to obtain Chinese yew tree seedlings.

[0013] In a preferred embodiment of the present invention, in step S2, the induction medium is an MS medium containing 0.01-0.03 mg / L inducer, 1-3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 25-32 g / L sucrose, and 4-8 g / L agar.

[0014] In a preferred embodiment of the present invention, the inducer is a compound of a small molecule inducer and polydopamine.

[0015] In a preferred embodiment of the present invention, the preparation method of the inducer comprises the following steps:

[0016] dissolving dopamine in an alkaline solution with a pH of 8 to 9 to obtain a dopamine solution with a concentration of 2 to 8 g / L, and stirring the resulting dopamine solution at 40 to 60° C. for 12 to 24 hours to obtain a polydopamine solution;

[0017] A cross-linking agent and a small molecule inducer are added to the polydopamine solution, stirred at 30-40° C. for 1-3 hours, and then cooled to room temperature and allowed to stand for 2-3 hours to obtain the inducer.

[0018] In a preferred embodiment of the present invention, the polydopamine solution, the cross-linking agent and the small molecule inducer are added in an amount according to a volume-to-mass ratio of 100 mL: 0.5-1 g: 1-3 mL.

[0019] In a preferred embodiment of the present invention, the cross-linking agent is selected from dicumyl peroxide or benzoyl peroxide.

[0020] In a preferred embodiment of the present invention, the small molecule inducer is selected from any one of diphenylamine, 2-methyldiphenylamine, 3-methyldiphenylamine, 4-methoxy-4'-methyldiphenylamine, 3-methoxydiphenylamine, and 2,4,4'-trimethyldiphenylamine.

[0021] In a preferred embodiment of the present invention, in step S3, the differentiation medium is an MS medium containing 0.03-0.1 mg / L hinokitiol, 0.2-0.5 mg / L N-ethyl-L-glutamine, 0.1-0.5 mg / L 2,4-D, 1-2 mg / L 5-hydroxy-IAA, 10-20 g / L sucrose, and 4-8 g / L agar.

[0022] In a preferred embodiment of the present invention, in step S4, the proliferation medium is MS medium containing 1.5-2.5 mg / L 6-BA, 0.02-0.08 mg / L NAA, and 1-3 g / L fucoidan.

[0023] In a preferred embodiment of the present invention, in step S5, the rooting medium is a 1 / 2MS medium containing 0.2-0.6 mg / L NAA, 1-2 mg / L IBA, 0.03-0.08 mg / L vitamin C, and 1-3 g / L fucoidan.

[0024] By means of the above technical solution, the present invention has at least the following advantages:

[0025] 1. The present invention adds a small amount of inducer to the induction medium. Experiments have shown that the inducer can promote the formation of callus tissue, increase the induction rate of callus tissue of Rhizoma Cibotii, and promote the subsequent differentiation, proliferation and rooting culture of callus tissue. The inducer of the present invention is a composite of a small molecule inducer and polydopamine. The small molecule inducer is a small molecule aniline organic compound. By combining with polydopamine, the properties of polydopamine can be utilized to organically combine multiple functional molecules or groups through PDA self-polymerization reaction, covalent modification, π-π stacking and hydrogen bonding, thereby achieving a synergistic effect between the active substances.

[0026] 2. The present invention adds hinokitiol and N-ethyl-L-glutamine to the differentiation medium, and the two components cooperate with each other to reduce the browning rate and the contamination rate. The differentiation medium of the present invention can promote the growth and reproduction of the callus of the hyacinthus annuus, is conducive to promoting the generation of the adventitious buds of the hyacinthus annuus, can significantly improve the differentiation ability of the cells, thereby improving the differentiation rate of the adventitious buds of the hyacinthus annuus, and realizing the high-frequency differentiation and regeneration of the callus.

[0027] 3. The method of the present invention effectively improves the reproductive efficiency and economic benefits of the Chinese hawthorn, can be applied to the large-scale, factory-based cultivation of the Chinese hawthorn, and promotes the development and utilization of the Chinese hawthorn resources.

[0028] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a detailed description of the preferred embodiments of the present invention. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0030] Unless otherwise specified, the percentage content involved in the present invention refers to mass percentage for solid-liquid mixing and solid-solid mixing, and refers to volume percentage for liquid-liquid mixing.

[0031] Unless otherwise specified, percentage concentrations referred to in the present invention refer to final concentrations, which refer to the percentage of an added component in the system after the addition of the component.

[0032] The temperature parameters in the present invention, unless otherwise specified, allow for either constant temperature treatment or treatment within a certain temperature range. The constant temperature treatment allows for temperature fluctuations within the precision range controlled by the instrument.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0034] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0035] Example 1

[0036] This embodiment provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0037] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0038] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 6 g / L. The obtained dopamine solution was stirred and reacted at 50°C for 18 hours to obtain a polydopamine solution; diisopropylbenzene peroxide and 2-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:0.75 g:2 mL, and the mixture was stirred at 35°C for 2 hours. The mixture was then cooled to room temperature and allowed to stand for 2.5 hours to obtain an inducer.

[0039] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration be 0.02 mg / L, 6-BA concentration be 2 mg / L, NAA concentration be 0.15 mg / L, sucrose concentration be 28.5 g / L, agar concentration be 6 g / L, sterilize at 121°C for 20 min to obtain induction medium.

[0040] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0041] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a hinokitiol concentration of 0.065 mg / L, a N-ethyl-L-glutamine concentration of 0.35 mg / L, a 2,4-D concentration of 0.3 mg / L, a 5-hydroxy-IAA concentration of 1.5 mg / L, a sucrose concentration of 15 g / L, and agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0042] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0043] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0044] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0045] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0046] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0047] Example 2

[0048] This embodiment provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0049] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0050] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 8 g / L. The obtained dopamine solution was stirred and reacted at 60°C for 12 hours to obtain a polydopamine solution. Benzoyl peroxide and 3-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:1 g:3 mL, and the mixture was stirred at 30°C for 3 hours. The mixture was then cooled to room temperature and allowed to stand for 2 hours to obtain an inducer.

[0051] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration 0.01 mg / L, 6-BA concentration 3 mg / L, NAA concentration 0.1 mg / L, sucrose concentration 32 g / L, and agar concentration 4 g / L. The medium was sterilized at 121°C for 20 min to obtain an induction medium.

[0052] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0053] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a hinokitiol concentration of 0.1 mg / L, N-ethyl-L-glutamine concentration of 0.2 mg / L, 2,4-D concentration of 0.5 mg / L, 5-hydroxy-IAA concentration of 1 mg / L, sucrose concentration of 20 g / L, and agar concentration of 4 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0054] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0055] S4: Add 6-BA, NAA, and fucoidan to MS medium to make the concentration of 6-BA 1.5 mg / L, NAA 0.08 mg / L, and fucoidan 1 g / L. Sterilize at 121°C for 20 min to obtain proliferation medium.

[0056] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0057] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.6 mg / L, IBA concentration 1 mg / L, vitamin C concentration 0.08 mg / L, and fucoidan concentration 1 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0058] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0059] Example 3

[0060] This embodiment provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0061] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0062] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 2 g / L. The obtained dopamine solution was stirred and reacted at 40°C for 24 hours to obtain a polydopamine solution. Diisopropyl peroxide and 4-methoxy-4'-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:0.5 g:1 mL, and the mixture was stirred at 40°C for 1 hour. The mixture was then cooled to room temperature and allowed to stand for 3 hours to obtain an inducer.

[0063] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration be 0.03 mg / L, 6-BA concentration be 1 mg / L, NAA concentration be 0.2 mg / L, sucrose concentration be 25 g / L, agar concentration be 8 g / L, sterilize at 121°C for 20 min to obtain induction medium.

[0064] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0065] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to achieve a hinokitiol concentration of 0.03 mg / L, N-ethyl-L-glutamine concentration of 0.5 mg / L, 2,4-D concentration of 0.1 mg / L, 5-hydroxy-IAA concentration of 2 mg / L, sucrose concentration of 10 g / L, and agar concentration of 8 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0066] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0067] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 2.5 mg / L, the NAA concentration 0.02 mg / L, and the fucoidan concentration 3 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0068] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0069] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.2 mg / L, IBA concentration 2 mg / L, vitamin C concentration 0.03 mg / L, and fucoidan concentration 3 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0070] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0071] Comparative Example 1

[0072] This comparative example provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0073] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0074] S2: Add 2-methyldiphenylamine, 6-BA, NAA, sucrose, and agar to MS medium to make the inducer concentrations 0.02 mg / L, 6-BA concentration 2 mg / L, NAA concentration 0.15 mg / L, sucrose concentration 28.5 g / L, and agar concentration 6 g / L. Sterilize at 121°C for 20 min to obtain an induction medium.

[0075] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0076] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a hinokitiol concentration of 0.065 mg / L, a N-ethyl-L-glutamine concentration of 0.35 mg / L, a 2,4-D concentration of 0.3 mg / L, a 5-hydroxy-IAA concentration of 1.5 mg / L, a sucrose concentration of 15 g / L, and agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0077] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0078] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0079] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0080] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0081] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0082] Comparative Example 2

[0083] This comparative example provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0084] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0085] S2: Add 6-BA, NAA, sucrose, and agar to MS medium to achieve a 6-BA concentration of 2 mg / L, an NAA concentration of 0.15 mg / L, a sucrose concentration of 28.5 g / L, and an agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain an induction medium.

[0086] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0087] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a hinokitiol concentration of 0.065 mg / L, a N-ethyl-L-glutamine concentration of 0.35 mg / L, a 2,4-D concentration of 0.3 mg / L, a 5-hydroxy-IAA concentration of 1.5 mg / L, a sucrose concentration of 15 g / L, and agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0088] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0089] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0090] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0091] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0092] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0093] Comparative Example 3

[0094] This comparative example provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0095] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0096] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 6 g / L. The obtained dopamine solution was stirred and reacted at 50°C for 18 hours to obtain a polydopamine solution; diisopropylbenzene peroxide and 2-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:0.75 g:2 mL, and the mixture was stirred at 35°C for 2 hours. The mixture was then cooled to room temperature and allowed to stand for 2.5 hours to obtain an inducer.

[0097] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration be 0.02 mg / L, 6-BA concentration be 2 mg / L, NAA concentration be 0.15 mg / L, sucrose concentration be 28.5 g / L, agar concentration be 6 g / L, sterilize at 121°C for 20 min to obtain induction medium.

[0098] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0099] S3: Add N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a differentiation medium with a concentration of 0.35 mg / L N-ethyl-L-glutamine, 0.3 mg / L 2,4-D, 1.5 mg / L 5-hydroxy-IAA, 15 g / L sucrose, and 6 g / L agar. Sterilize at 121°C for 20 min to obtain the differentiation medium.

[0100] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0101] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0102] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0103] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0104] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0105] Comparative Example 4

[0106] This comparative example provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0107] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0108] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 6 g / L. The obtained dopamine solution was stirred and reacted at 50°C for 18 hours to obtain a polydopamine solution; diisopropylbenzene peroxide and 2-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:0.75 g:2 mL, and the mixture was stirred at 35°C for 2 hours. The mixture was then cooled to room temperature and allowed to stand for 2.5 hours to obtain an inducer.

[0109] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration be 0.02 mg / L, 6-BA concentration be 2 mg / L, NAA concentration be 0.15 mg / L, sucrose concentration be 28.5 g / L, agar concentration be 6 g / L, sterilize at 121°C for 20 min to obtain induction medium.

[0110] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0111] S3: Add hinokitiol, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to achieve a hinokitiol concentration of 0.065 mg / L, a 2,4-D concentration of 0.3 mg / L, a 5-hydroxy-IAA concentration of 1.5 mg / L, a sucrose concentration of 15 g / L, and an agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0112] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0113] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0114] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0115] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0116] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0117] Comparative Example 5

[0118] This comparative example provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0119] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0120] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 6 g / L. The obtained dopamine solution was stirred and reacted at 50°C for 18 hours to obtain a polydopamine solution; diisopropylbenzene peroxide and 2-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:0.75 g:2 mL, and the mixture was stirred at 35°C for 2 hours. The mixture was then cooled to room temperature and allowed to stand for 2.5 hours to obtain an inducer.

[0121] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration be 0.02 mg / L, 6-BA concentration be 2 mg / L, NAA concentration be 0.15 mg / L, sucrose concentration be 28.5 g / L, agar concentration be 6 g / L, sterilize at 121°C for 20 min to obtain induction medium.

[0122] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0123] S3: Add 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a 2,4-D concentration of 0.3 mg / L, a 5-hydroxy-IAA concentration of 1.5 mg / L, a sucrose concentration of 15 g / L, and an agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0124] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0125] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0126] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0127] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0128] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0129] Comparative Example 6

[0130] This comparative example provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0131] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0132] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 6 g / L. The obtained dopamine solution was stirred and reacted at 50°C for 18 hours to obtain a polydopamine solution; diisopropylbenzene peroxide and 2-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:0.75 g:2 mL, and the mixture was stirred at 35°C for 2 hours. The mixture was then cooled to room temperature and allowed to stand for 2.5 hours to obtain an inducer.

[0133] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration be 0.05 mg / L, 6-BA concentration be 2 mg / L, NAA concentration be 0.15 mg / L, sucrose concentration be 28.5 g / L, agar concentration be 6 g / L, sterilize at 121°C for 20 min to obtain induction medium.

[0134] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0135] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a hinokitiol concentration of 0.065 mg / L, a N-ethyl-L-glutamine concentration of 0.35 mg / L, a 2,4-D concentration of 0.3 mg / L, a 5-hydroxy-IAA concentration of 1.5 mg / L, a sucrose concentration of 15 g / L, and agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0136] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0137] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0138] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0139] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0140] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0141] Comparative Example 7

[0142] This comparative example provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0143] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0144] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 6 g / L. The obtained dopamine solution was stirred and reacted at 50°C for 18 hours to obtain a polydopamine solution; diisopropylbenzene peroxide and 2-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:0.75 g:2 mL, and the mixture was stirred at 35°C for 2 hours. The mixture was then cooled to room temperature and allowed to stand for 2.5 hours to obtain an inducer.

[0145] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration be 0.02 mg / L, 6-BA concentration be 2 mg / L, NAA concentration be 0.15 mg / L, sucrose concentration be 28.5 g / L, agar concentration be 6 g / L, sterilize at 121°C for 20 min to obtain induction medium.

[0146] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0147] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a hinokitiol concentration of 0.01 mg / L, N-ethyl-L-glutamine concentration of 0.6 mg / L, 2,4-D concentration of 0.3 mg / L, 5-hydroxy-IAA concentration of 1.5 mg / L, sucrose concentration of 15 g / L, and agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0148] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0149] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0150] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0151] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0152] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0153] Comparative Example 8

[0154] This comparative example provides a method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, comprising the following steps:

[0155] S1: Wash the leaves of the jujube seedlings and disinfect them with a 75% ethanol solution by volume for 30 seconds. Then disinfect them with a 0.1 wt% aqueous solution of mercuric chloride (containing 1% Tween-80 by volume) for 3 minutes. Cut the disinfected jujube leaves to obtain jujube explants.

[0156] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH of 8 to obtain a dopamine solution with a concentration of 6 g / L. The obtained dopamine solution was stirred and reacted at 50°C for 18 hours to obtain a polydopamine solution; diisopropylbenzene peroxide and 2-methyldiphenylamine were added to the polydopamine solution in a volume mass ratio of 100 mL:0.75 g:2 mL, and the mixture was stirred at 35°C for 2 hours. The mixture was then cooled to room temperature and allowed to stand for 2.5 hours to obtain an inducer.

[0157] Inducer, 6-BA, NAA, sucrose and agar were added to MS medium to make the inducer concentration be 0.02 mg / L, 6-BA concentration be 2 mg / L, NAA concentration be 0.15 mg / L, sucrose concentration be 28.5 g / L, agar concentration be 6 g / L, sterilize at 121°C for 20 min to obtain induction medium.

[0158] The explants of Prunus armeniaca were inoculated into an induction medium and cultured at 28°C and a light intensity of 2500 lux for 10 days to obtain callus tissue.

[0159] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium to obtain a hinokitiol concentration of 0.2 mg / L, a N-ethyl-L-glutamine concentration of 0.1 mg / L, a 2,4-D concentration of 0.3 mg / L, a 5-hydroxy-IAA concentration of 1.5 mg / L, a sucrose concentration of 15 g / L, and agar concentration of 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0160] The callus tissue was inoculated into a differentiation medium and cultured at 28° C. and a light intensity of 2500 lux for 15 days to obtain adventitious buds of the herb.

[0161] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the 6-BA concentration 12 mg / L, the NAA concentration 0.06 mg / L, and the fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0162] The adventitious buds of the Herba Lycoris Radiata were inoculated into a proliferation medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain Herba Lycoris Radiata cluster buds.

[0163] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.4 mg / L, IBA concentration 1.5 mg / L, vitamin C concentration 0.055 mg / L, and fucoidan concentration 2 g / L. Sterilize at 121°C for 20 min to obtain rooting medium.

[0164] The buds of the shrub were inoculated into a rooting medium and cultured at 28° C. and a light intensity of 2500 lux for 20 days to obtain shrub seedlings.

[0165] Experiment 1: Effects of different methods of induction and differentiation of callus tissue of Prunus mume on the tissue induction rate and bud differentiation rate of Prunus mume.

[0166] First, steps S1 to S2 in Examples 1-3 and Comparative Examples 1-8 were used to induce culture of the scutellaria explants, wherein the number of scutellaria explants inoculated into the induction medium was 50, and their size and morphology remained substantially the same. After induction culture, the callus induction rate (%) was calculated, where the callus induction rate (%) = (number of explants forming callus / total number of inoculated explants) × 100%.

[0167] Next, the calli of the shrub were differentiated and cultured using step S3 of Examples 1-3 and Comparative Examples 1-8 to obtain adventitious buds of the shrub. Thirty shrub calli were inoculated into the differentiation medium, and their size and morphology remained substantially uniform. After differentiation culture, the emergence of the adventitious buds of the shrub was counted, where bud differentiation rate (%) = number of callus pieces that differentiated and sprouted / total number of callus pieces inoculated × 100%.

[0168] The results are shown in Table 1.

[0169] Table 1 Effects of different induction and differentiation methods on callus induction rate and bud differentiation rate of Prunus armeniaca

[0170]

[0171] As can be seen from the results in Table 1, compared with Comparative Examples 1-2 and 6, the induction medium of the present invention can significantly increase the induction rate of the callus of the schizonepeta tenuifolia. In particular, compared with Comparative Examples 1-2 and Example 1, it can be seen that the inducer has a significant effect on the induction rate of the callus of the schizonepeta tenuifolia. In another aspect, compared with Comparative Examples 3-4 and 7-8, the differentiation medium of the present invention can increase the differentiation rate of the adventitious buds of the schizonepeta tenuifolia.

[0172] Experiment 2: Effects of different methods of induction and differentiation of cyperus callus on the survival rate of cyperus seedlings

[0173] Steps S1 to S5 in Example 1 and Comparative Examples 1-8 were respectively used to induce, differentiate, proliferate, and root culture the explants of the hyacinthus annuus to obtain hyacinthus annuus seedlings, wherein the number of hyacinthus annuus explants inoculated into the induction medium in step S2 was 20, and their size and morphology remained substantially the same. The growth of the hyacinthus annuus seedlings obtained in each group after cultivation in steps S1 to S5 was statistically analyzed, and the results are shown in Table 2.

[0174] Table 2 Average root length and seedling length of Xiagangmei seedlings under different induction differentiation culture methods

[0175]

[0176] As can be seen from the results in Table 2, compared with Comparative Examples 1 to 8, the growth of the jujube seedlings obtained by the method in Example 1 of the present invention is the best. This shows that the method of the present invention can obtain jujube seedlings with good growth.

[0177] In addition, by training the jujube seedlings obtained in each group (20 in total) by the same method and then transplanting them for 10 days, it can be found that the transplant survival rate of the jujube seedlings in Example 1 of the present invention can reach 95%, which is significantly higher than the survival rate of the jujube seedlings in Comparative Examples 1-8. The results are shown in Table 3.

[0178] Table 3 The number and survival rate of surviving Atractylodes lanceolata seedlings induced by different methods

[0179]

[0180] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any technician familiar with this profession can make slight changes or modifications to equivalent embodiments of the methods and technical contents disclosed above without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for inducing differentiation of callus tissue of Rhizoma Citri Reticulatae, characterized in that: The following steps are involved: S1: Wash and disinfect the leaves of the safflower seedlings, and then cut them to obtain safflower explants; S2: Inoculate the explants of P. chebula into the induction medium and culture them at 25-30°C and 2000-3000 lux for 7-14 days to obtain callus tissue; S3: The callus tissue was inoculated into a differentiation medium and cultured at 25-30°C and a light intensity of 2000-3000 lux for 10-20 days to obtain adventitious buds of the plant. S4: inoculating the adventitious buds of the Chinese yew into a proliferation medium, and culturing them at 25-30°C and a light intensity of 2000-3000 lux for 15-25 days to obtain clusters of Chinese yew buds; S5: Inoculate the buds of the Chinese yew clump into a rooting medium and culture them at 25-30°C and a light intensity of 2000-3000 lux for 15-25 days to obtain Chinese yew seedlings; In step S2, the induction medium is composed of: MS medium containing 0.01-0.03 mg / L inducer, 1-3 mg / L 6-BA, 0.1-0.2 mg / L NAA, 25-32 g / L sucrose, and 4-8 g / L agar; The inducer is a compound of a small molecule inducer and polydopamine; In step S3, the differentiation medium is composed of: MS medium containing 0.03-0.1 mg / L hinokitiol, 0.2-0.5 mg / L N-ethyl-L-glutamine, 0.1-0.5 mg / L 2,4-D, 1-2 mg / L 5-hydroxy-IAA, 10-20 g / L sucrose, and 4-8 g / L agar; The small molecule inducer is selected from any one of diphenylamine, 2-methyldiphenylamine, 3-methyldiphenylamine, 4-methoxy-4'-methyldiphenylamine, 3-methoxydiphenylamine, and 2,4,4'-trimethyldiphenylamine; The preparation method of the inducer comprises the following steps: dissolving dopamine in an alkaline solution with a pH value of 8-9 to obtain a dopamine solution with a concentration of 2-8 g / L, placing the obtained dopamine solution at 40-60° C. and stirring for reaction for 12-24 hours to obtain a polydopamine solution; adding a crosslinking agent and a small molecule inducer to the polydopamine solution, stirring at 30-40° C. for 1-3 hours, then cooling to room temperature and standing for 2-3 hours to obtain the inducer; the polydopamine solution, the crosslinking agent and the small molecule inducer are added in a volume-to-mass ratio of 100 mL: 0.5-1 g: 1-3 mL; the crosslinking agent is selected from dicumyl peroxide or benzoyl peroxide.

2. The differentiation induction method according to claim 1, characterized in that: In step S4, the proliferation medium is an MS medium containing 1.5-2.5 mg / L 6-BA, 0.02-0.08 mg / L NAA, and 1-3 g / L fucoidan.

3. The differentiation induction method according to claim 1, characterized in that In step S5, the rooting medium is a 1 / 2MS medium containing 0.2-0.6 mg / L NAA, 1-2 mg / L IBA, 0.03-0.08 mg / L vitamin C, and 1-3 g / L fucoidan.

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