Induced differentiation and culture method of callus of roughhaired holly

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to an induced differentiation and culture method of callus of roughhaired holly. The induced differentiation method of the roughhaired holly callus comprises the following steps: S1, washing and disinfecting roughhaired holly seedling leaves, and cutting to obtain roughhaired holly explants; s2, inoculating an induction culture medium with the holly root explant, and culturing to obtain a callus; s3, inoculating the callus into a differential culture medium for culturing to obtain adventitious buds of roughhaired holly; s4, inoculating the adventitious buds of the roughhaired holly into a proliferation culture medium for culturing to obtain clumpy buds of the roughhaired holly; and S5, inoculating the roughhaired holly clump buds into a rooting culture medium for culturing to obtain roughhaired holly seedlings. The method effectively improves the reproduction efficiency and economic benefits of the roughhaired holly, can be applied to large-scale and industrial culture of the roughhaired holly, and promotes development and utilization of roughhaired holly resources.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant tissue culture. Specifically, it relates to a method for inducing and differentiating callus of Ilex asprella and its culture method. Background Art

[0002] Ilex asprella is the dried root and stem of Ilex asprella (Hook. et Am.) Champ. ex Benth of the genus Ilex, also known as Ilex asprella, Ilex asprella, star tree, wild licorice, mountain plum root, etc., and is one of the main raw materials for Lingnan herbal tea. Currently, Ilex asprella is the main raw material for cold-relieving Chinese patent medicines. Its chemical components mainly include triterpenoids, phenolic acids, polysaccharides, volatile oils, etc., and it has a wide range of pharmacological effects such as anti-inflammatory, antipyretic, analgesic, anti-pathogenic microorganism, anti-tumor, anti-complement, anti-ulcer, and anti-Alzheimer's disease, and has extremely high medicinal and economic value.

[0003] Ilex asprella is distributed in many provinces in China. It likes warm and humid climate and has no strict requirements for soil. It can grow on fertile or barren land and is mostly found in sparse forests or shrubs on slopes. In recent years, the market demand for Ilex asprella has been increasing day by day. At present, there is a small amount of artificially cultivated Ilex asprella, but it still cannot meet the market demand.

[0004] Callus induction and differentiation have the characteristics of short cycle, high proliferation rate, and annual production, and are an effective way for rapid propagation of plant seedlings. Callus induction and differentiation are not restricted by location and season and can be continuously and rapidly propagated. Moreover, the operation is simple, the time used is short, the roots of mature seedlings are developed and of high quality, and it can effectively reduce production costs and improve the production efficiency of personnel. In order to achieve large-scale cultivation of Ilex asprella, it is very necessary to provide a method for inducing and differentiating callus of Ilex asprella and its culture method to meet the increasing market demand. Summary of the Invention

[0005] The primary object of the present invention is to provide a method for inducing and differentiating callus of Ilex asprella and its culture method, so as to achieve large-scale cultivation and planting of Ilex asprella.

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

[0007] One aspect of the present invention provides a method for inducing and differentiating callus of Ilex asprella, including the following steps: S1: Wash and disinfect the leaves of Ilex asprella seedlings, and then cut them to obtain Ilex asprella explants; S2: Inoculate the Ilex asprella explants into an induction medium and culture them at 25-30°C and a light intensity of 2000-3000 lux for 7-14 days to obtain callus; S3: Inoculate the callus into a differentiation medium and culture it for 10 - 20 days under the conditions of 25 - 30°C and a light intensity of 2000 - 3000 lux to obtain the indefinite buds of Ilex asprella; S4: Inoculate the indefinite buds of Ilex asprella into a proliferation medium and culture it for 15 - 25 days under the conditions of 25 - 30°C and a light intensity of 2000 - 3000 lux to obtain the cluster buds of Ilex asprella; S5: Inoculate the cluster buds of Ilex asprella into a rooting medium and culture it for 15 - 25 days under the conditions of 25 - 30°C and a light intensity of 2000 - 3000 lux to obtain the seedlings of Ilex asprella.

[0008] 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 of an inducer, 1 - 3 mg / L of 6 - BA, 0.1 - 0.2 mg / L of NAA, 25 - 32 g / L of sucrose, and 4 - 8 g / L of agar.

[0009] In a preferred embodiment of the present invention, the inducer is composed of a small - molecule inducer and polydopamine.

[0010] In a preferred embodiment of the present invention, the preparation method of the inducer includes the following steps: Dissolve 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. Place the obtained dopamine solution at 40 - 60°C and stir - react it for 12 - 24 h to obtain a polydopamine solution; Add a cross - linker and a small - molecule inducer to the polydopamine solution, stir at 30 - 40°C for 1 - 3 h, and then cool to room temperature and let it stand for 2 - 3 h to obtain the inducer.

[0011] In a preferred embodiment of the present invention, the polydopamine solution, the cross - linker, and the small - molecule inducer are added in an amount of 100 mL: 0.5 - 1 g: 1 - 3 mL by volume - mass ratio.

[0012] In a preferred embodiment of the present invention, the cross - linker is selected from di - isopropylbenzene peroxide or benzoyl peroxide.

[0013] 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.

[0014] 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.

[0015] In a preferred embodiment of the present invention, 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.

[0016] 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.

[0017] By means of the above technical solutions, the present invention has at least the following advantages: 1. By adding a small amount of inducer to the induction medium in the present invention, it is found through experiments that this inducer can promote the formation of callus, improve the induction rate of Ilex asprella callus, and has a promoting effect on the subsequent differentiation, proliferation, and rooting culture of callus. The inducer of the present invention is composed of a small molecule inducer and polydopamine. The small molecule inducer is a small molecule aniline organic compound. By combining with polydopamine, it can utilize the properties of polydopamine to organically combine various functional molecules or groups through PDA self-polymerization reaction, covalent modification, π-π stacking, and hydrogen bond and other forces, thereby realizing the synergistic effect between various active substances.

[0018] 2. By adding hinokitiol and N-ethyl-L-glutamine to the differentiation medium in the present invention, these two components cooperate with each other to reduce the browning rate and contamination rate. The differentiation medium of the present invention can promote the growth and reproduction of Ilex asprella callus, is beneficial to the generation of adventitious buds of Ilex asprella, can significantly improve the differentiation ability of cells, thereby increasing the differentiation rate of adventitious buds of Ilex asprella and realizing the high-frequency differentiation and regeneration of callus.

[0019] 3. The method of the present invention effectively improves the propagation efficiency and economic benefits of Ilex asprella, can be applied to the large-scale and industrialized cultivation of Ilex asprella, and promotes the development and utilization of Ilex asprella resources.

[0020] The above description is only an overview of the technical solution of the present invention. In order to understand the technical means of the present invention more clearly and implement it according to the content of the specification, the following describes the preferred embodiments of the present invention in detail. Detailed implementation manners

[0021] In order to make the technical means, creative features, achieved objectives and functions of the present invention easy 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 a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention.

[0022] Unless otherwise specified, the percentage content involved in the present invention refers to the mass percentage for solid-liquid mixing and solid-solid mixing, and the volume percentage for liquid-liquid mixing.

[0023] Unless otherwise specified, the percentage concentration involved in the present invention refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding the component.

[0024] The temperature parameters in the present invention, unless otherwise specifically limited, allow both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0025] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods.

[0026] Unless otherwise specified, the materials, reagents, etc. used in the following examples can all be obtained from commercial channels.

[0027] Example 1 This example provides a method for inducing and differentiating Ilex asprella callus, including the following steps: S1: Wash the leaves of Ilex asprella seedlings, first disinfect them with 75% ethanol solution by volume for 30 s, and then disinfect them with 0.1 wt% mercuric chloride aqueous solution (containing 1% Tween-80 by volume) for 3 min. Cut the disinfected Ilex asprella leaves to obtain Ilex asprella explants.

[0028] S2: Dissolve dopamine in Tris-HCl buffer with a pH value of 8 to obtain a dopamine solution with a concentration of 6 g / L. Place the obtained dopamine solution at 50 °C and stir for 18 h to obtain a polydopamine solution; add diisopropyl peroxide and 2-methyl diphenylamine to the polydopamine solution in an amount of 100 mL: 0.75 g: 2 mL by volume-mass ratio, stir at 35 °C for 2 h, and then cool to room temperature and let stand for 2.5 h to obtain an inducer.

[0029] An inducer, 6-BA, NAA, sucrose and agar were added to the MS medium, such that the concentration of the inducer was 0.02 mg / L, the concentration of 6-BA was 2 mg / L, the concentration of NAA was 0.15 mg / L, the concentration of sucrose was 28.5 g / L, and the concentration of agar was 6 g / L. It was sterilized at 121 °C for 20 min to obtain an induction medium.

[0030] The ex vitro plantlets of Ilex asprella were inoculated into the induction medium and cultured for 10 days at 28 °C under a light intensity of 2500 lux to obtain callus.

[0031] S3: Hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose and agar were added to the MS medium, such that the concentration of hinokitiol was 0.065 mg / L, the concentration of N-ethyl-L-glutamine was 0.35 mg / L, the concentration of 2,4-D was 0.3 mg / L, the concentration of 5-hydroxy-IAA was 1.5 mg / L, the concentration of sucrose was 15 g / L, and the concentration of agar was 6 g / L. It was sterilized at 121 °C for 20 min to obtain a differentiation medium.

[0032] The callus was inoculated into the differentiation medium and cultured for 15 days at 28 °C under a light intensity of 2500 lux to obtain adventitious buds of Ilex asprella.

[0033] S4: 6-BA, NAA and fucoidan were added to the MS medium, such that the concentration of 6-BA was 12 mg / L, the concentration of NAA was 0.06 mg / L, and the concentration of fucoidan was 2 g / L. It was sterilized at 121 °C for 20 min to obtain a proliferation medium.

[0034] The adventitious buds of Ilex asprella were inoculated into the proliferation medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain cluster buds of Ilex asprella.

[0035] S5: NAA, IBA, vitamin C and fucoidan were added to the 1 / 2MS medium, such that the concentration of NAA was 0.4 mg / L, the concentration of IBA was 1.5 mg / L, the concentration of vitamin C was 0.055 mg / L, and the concentration of fucoidan was 2 g / L. It was sterilized at 121 °C for 20 min to obtain a rooting medium.

[0036] The cluster buds of Ilex asprella were inoculated into the rooting medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain seedlings of Ilex asprella.

[0037] Example 2 This example provides a method for inducing and differentiating callus of Ilex asprella, including the following steps: S1: Wash the leaves of Ilex asprella seedlings, disinfect them with 75% ethanol solution by volume for 30 s first, then disinfect them with 0.1 wt% mercuric chloride aqueous solution (containing 1% Tween-80 by volume) for 3 min, and cut the disinfected Ilex asprella leaves to obtain Ilex asprella explants.

[0038] S2: Dissolve dopamine in Tris-HCl buffer solution with a pH value of 8 to obtain a dopamine solution with a concentration of 8 g / L. Place the obtained dopamine solution at 60 °C and stir for 12 h to obtain a polydopamine solution; add benzoyl peroxide and 3-methyldiphenylamine to the polydopamine solution in an amount of 100 mL:1 g:3 mL by volume-mass ratio, stir at 30 °C for 3 h, and then cool to room temperature and let stand for 2 h to obtain an inducer.

[0039] Add the inducer, 6-BA, NAA, sucrose, and agar to the MS medium so that the inducer concentration is 0.01 mg / L, the 6-BA concentration is 3 mg / L, the NAA concentration is 0.1 mg / L, the sucrose concentration is 32 g / L, and the agar concentration is 4 g / L. Sterilize at 121 °C for 20 min to obtain an induction medium.

[0040] Inoculate the Ilex asprella explants into the induction medium and culture them under the conditions of 28 °C and a light intensity of 2500 lux for 10 days to obtain callus.

[0041] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium so that the hinokitiol concentration is 0.1 mg / L, the N-ethyl-L-glutamine concentration is 0.2 mg / L, the 2,4-D concentration is 0.5 mg / L, the 5-hydroxy-IAA concentration is 1 mg / L, the sucrose concentration is 20 g / L, and the agar concentration is 4 g / L. Sterilize at 121 °C for 20 min to obtain a differentiation medium.

[0042] Inoculate the callus into the differentiation medium and culture it under the conditions of 28 °C and a light intensity of 2500 lux for 15 days to obtain adventitious buds of Ilex asprella.

[0043] S4: Add 6-BA, NAA, and fucoidan to the MS medium so that the 6-BA concentration is 1.5 mg / L, the NAA concentration is 0.08 mg / L, and the fucoidan concentration is 1 g / L. Sterilize at 121 °C for 20 min to obtain a proliferation medium.

[0044] Inoculate the adventitious buds of Ilex asprella into the proliferation medium and culture them under the conditions of 28 °C and a light intensity of 2500 lux for 20 days to obtain cluster buds of Ilex asprella.

[0045] S5: Add NAA, IBA, vitamin C, and fucoidan to 1 / 2 MS medium to make the NAA concentration 0.6 mg / L, the IBA concentration 1 mg / L, the vitamin C concentration 0.08 mg / L, and the fucoidan concentration 1 g / L. Sterilize at 121 °C for 20 min to obtain a rooting medium.

[0046] Inoculate the axillary buds of Ilex asprella in the rooting medium and culture them for 20 days under the conditions of 28 °C and a light intensity of 2500 lux to obtain Ilex asprella seedlings.

[0047] Example 3 This example provides a method for inducing and differentiating callus of Ilex asprella, including the following steps: S1: Wash the leaves of Ilex asprella seedlings, disinfect them with 75% ethanol solution by volume for 30 s first, and then disinfect them with 0.1 wt% mercuric chloride aqueous solution (containing 1% Tween - 80 by volume) for 3 min. Cut the disinfected Ilex asprella leaves to obtain Ilex asprella explants.

[0048] Dissolve dopamine in Tris - HCl buffer with a pH value of 8 to obtain a dopamine solution with a concentration of 2 g / L. Place the obtained dopamine solution at 40 °C and stir - react for 24 h to obtain a polydopamine solution; add diisopropylbenzene peroxide and 4 - methoxy - 4'-methyl diphenylamine to the polydopamine solution in an amount of 100 mL:0.5 g:1 mL by volume - mass ratio, stir at 40 °C for 1 h, and then cool to room temperature and let it stand for 3 h to obtain an inducer.

[0049] Add the inducer, 6 - BA, NAA, as well as sucrose and agar to MS medium to make the inducer concentration 0.03 mg / L, the 6 - BA concentration 1 mg / L, the NAA concentration 0.2 mg / L, the sucrose concentration 25 g / L, and the agar concentration 8 g / L. Sterilize at 121 °C for 20 min to obtain an induction medium.

[0050] Inoculate the Ilex asprella explants into the induction medium and culture them for 10 days under the conditions of 28 °C and a light intensity of 2500 lux to obtain callus.

[0051] Add hinokitiol, N - ethyl - L - glutamine, 2,4 - D, 5 - hydroxy - IAA, as well as sucrose and agar to MS medium to make the hinokitiol concentration 0.03 mg / L, the N - ethyl - L - glutamine concentration 0.5 mg / L, the 2,4 - D concentration 0.1 mg / L, the 5 - hydroxy - IAA concentration 2 mg / L, the sucrose concentration 10 g / L, and the agar concentration 8 g / L. Sterilize at 121 °C for 20 min to obtain a differentiation medium.

[0052] The callus was inoculated into the differentiation medium and cultured for 15 days at 28 °C under a light intensity of 2500 lux to obtain the adventitious buds of Ilex asprella.

[0053] S4: 6-BA, NAA and fucoidan were added to the MS medium such that the concentration of 6-BA was 2.5 mg / L, the concentration of NAA was 0.02 mg / L, and the concentration of fucoidan was 3 g / L. It was sterilized at 121 °C for 20 min to obtain the proliferation medium.

[0054] The adventitious buds of Ilex asprella were inoculated into the proliferation medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain the cluster buds of Ilex asprella.

[0055] S5: NAA, IBA, vitamin C and fucoidan were added to the 1 / 2MS medium such that the concentration of NAA was 0.2 mg / L, the concentration of IBA was 2 mg / L, the concentration of vitamin C was 0.03 mg / L, and the concentration of fucoidan was 3 g / L. It was sterilized at 121 °C for 20 min to obtain the rooting medium.

[0056] The cluster buds of Ilex asprella were inoculated into the rooting medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain the seedlings of Ilex asprella.

[0057] Comparative Example 1 This comparative example provides a method for inducing and differentiating the callus of Ilex asprella, including the following steps: S1: After washing the leaves of the Ilex asprella seedlings, they were first disinfected with a 75% (v / v) ethanol solution for 30 s, and then disinfected with a 0.1 wt% aqueous mercuric chloride solution (containing 1% (v / v) Tween-80) for 3 min. The disinfected Ilex asprella leaves were cut to obtain the explants of Ilex asprella.

[0058] S2: 2-methyl diphenylamine, 6-BA, NAA, sucrose and agar were added to the MS medium such that the concentration of the inducer was 0.02 mg / L, the concentration of 6-BA was 2 mg / L, the concentration of NAA was 0.15 mg / L, the concentration of sucrose was 28.5 g / L, and the concentration of agar was 6 g / L. It was sterilized at 121 °C for 20 min to obtain the induction medium.

[0059] The explants of Ilex asprella were inoculated into the induction medium and cultured for 10 days at 28 °C under a light intensity of 2500 lux to obtain the callus.

[0060] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, as well as sucrose and agar to the MS medium, such that the concentration of hinokitiol is 0.065 mg / L, the concentration of N-ethyl-L-glutamine is 0.35 mg / L, the concentration of 2,4-D is 0.3 mg / L, the concentration of 5-hydroxy-IAA is 1.5 mg / L, the concentration of sucrose is 15 g / L, and the concentration of agar is 6 g / L. Sterilize at 121 °C for 20 min to obtain a differentiation medium.

[0061] Inoculate the callus into the differentiation medium and culture it for 15 days under the conditions of 28 °C and a light intensity of 2500 lux to obtain the adventitious buds of Radix Ilicis Asprellae.

[0062] S4: Add 6-BA, NAA, and fucoidan to the MS medium, such that the concentration of 6-BA is 12 mg / L, the concentration of NAA is 0.06 mg / L, and the concentration of fucoidan is 2 g / L. Sterilize at 121 °C for 20 min to obtain a proliferation medium.

[0063] Inoculate the adventitious buds of Radix Ilicis Asprellae into the proliferation medium and culture it for 20 days under the conditions of 28 °C and a light intensity of 2500 lux to obtain the cluster buds of Radix Ilicis Asprellae.

[0064] S5: Add NAA, IBA, vitamin C, and fucoidan to the 1 / 2 MS medium, such that the concentration of NAA is 0.4 mg / L, the concentration of IBA is 1.5 mg / L, the concentration of vitamin C is 0.055 mg / L, and the concentration of fucoidan is 2 g / L. Sterilize at 121 °C for 20 min to obtain a rooting medium.

[0065] Inoculate the cluster buds of Radix Ilicis Asprellae into the rooting medium and culture it for 20 days under the conditions of 28 °C and a light intensity of 2500 lux to obtain the seedlings of Radix Ilicis Asprellae.

[0066] Comparative Example 2 This comparative example provides a method for inducing and differentiating the callus of Radix Ilicis Asprellae, including the following steps: S1: Wash the leaves of the seedlings of Radix Ilicis Asprellae, first disinfect them with a 75% (v / v) ethanol solution for 30 s, and then disinfect them with a 0.1 wt% aqueous mercuric chloride solution (containing 1% (v / v) Tween-80) for 3 min. Cut the disinfected leaves of Radix Ilicis Asprellae to obtain the explants of Radix Ilicis Asprellae.

[0067] S2: Add 6-BA, NAA, as well as sucrose and agar to the MS medium, such that the concentration of 6-BA is 2 mg / L, the concentration of NAA is 0.15 mg / L, the concentration of sucrose is 28.5 g / L, and the concentration of agar is 6 g / L. Sterilize at 121 °C for 20 min to obtain an induction medium.

[0068] The Ilex asprella explants were inoculated into the induction medium and cultured for 10 days at 28°C under a light intensity of 2500 lux to obtain callus.

[0069] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, as well as sucrose and agar to the MS medium, such that the concentration of hinokitiol is 0.065 mg / L, the concentration of N-ethyl-L-glutamine is 0.35 mg / L, the concentration of 2,4-D is 0.3 mg / L, the concentration of 5-hydroxy-IAA is 1.5 mg / L, the concentration of sucrose is 15 g / L, and the concentration of agar is 6 g / L. Sterilize at 121°C for 20 min to obtain the differentiation medium.

[0070] The callus was inoculated into the differentiation medium and cultured for 15 days at 28°C under a light intensity of 2500 lux to obtain adventitious buds of Ilex asprella.

[0071] S4: Add 6-BA, NAA, and fucoidan to the MS medium, such that the concentration of 6-BA is 12 mg / L, the concentration of NAA is 0.06 mg / L, and the concentration of fucoidan is 2 g / L. Sterilize at 121°C for 20 min to obtain the proliferation medium.

[0072] The adventitious buds of Ilex asprella were inoculated into the proliferation medium and cultured for 20 days at 28°C under a light intensity of 2500 lux to obtain cluster buds of Ilex asprella.

[0073] S5: Add NAA, IBA, vitamin C, and fucoidan to the 1 / 2MS medium, such that the concentration of NAA is 0.4 mg / L, the concentration of IBA is 1.5 mg / L, the concentration of vitamin C is 0.055 mg / L, and the concentration of fucoidan is 2 g / L. Sterilize at 121°C for 20 min to obtain the rooting medium.

[0074] The cluster buds of Ilex asprella were inoculated into the rooting medium and cultured for 20 days at 28°C under a light intensity of 2500 lux to obtain Ilex asprella seedlings.

[0075] Comparative Example 3 This comparative example provides a method for inducing and differentiating callus of Ilex asprella, including the following steps: S1: Wash the leaves of Ilex asprella seedlings, first disinfect them with a 75% (v / v) ethanol solution for 30 s, and then disinfect them with a 0.1 wt% aqueous mercuric chloride solution (containing 1% (v / v) Tween-80) for 3 min. Cut the disinfected Ilex asprella leaves to obtain Ilex asprella explants.

[0076] S2: Dissolve dopamine in Tris-HCl buffer with a pH value of 8 to obtain a dopamine solution with a concentration of 6 g / L. Place the obtained dopamine solution in a stirred reaction at 50 °C for 18 h to obtain a polydopamine solution. Add cumene hydroperoxide and 2-methyl diphenylamine to the polydopamine solution in an amount according to the volume-mass ratio of 100 mL: 0.75 g: 2 mL, stir at 35 °C for 2 h, and then cool to room temperature and let stand for 2.5 h to obtain an inducer.

[0077] Add the inducer, 6-BA, NAA, as well as sucrose and agar to the MS medium so that the inducer concentration is 0.02 mg / L, the 6-BA concentration is 2 mg / L, the NAA concentration is 0.15 mg / L, the sucrose concentration is 28.5 g / L, and the agar concentration is 6 g / L. Sterilize at 121 °C for 20 min to obtain an induction medium.

[0078] Inoculate the Ilex asprella explants into the induction medium and culture them under the conditions of 28 °C and a light intensity of 2500 lux for 10 days to obtain callus.

[0079] Add N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, as well as sucrose and agar to the MS medium so that the N-ethyl-L-glutamine concentration is 0.35 mg / L, the 2,4-D concentration is 0.3 mg / L, the 5-hydroxy-IAA concentration is 1.5 mg / L, the sucrose concentration is 15 g / L, and the agar concentration is 6 g / L. Sterilize at 121 °C for 20 min to obtain a differentiation medium.

[0080] Inoculate the callus into the differentiation medium and culture them under the conditions of 28 °C and a light intensity of 2500 lux for 15 days to obtain Ilex asprella adventitious buds.

[0081] Add 6-BA, NAA, and fucoidan to the MS medium so that the 6-BA concentration is 12 mg / L, the NAA concentration is 0.06 mg / L, and the fucoidan concentration is 2 g / L. Sterilize at 121 °C for 20 min to obtain a proliferation medium.

[0082] Inoculate the Ilex asprella adventitious buds into the proliferation medium and culture them under the conditions of 28 °C and a light intensity of 2500 lux for 20 days to obtain Ilex asprella cluster buds.

[0083] Add NAA, IBA, vitamin C, and fucoidan to the 1 / 2MS medium so that the NAA concentration is 0.4 mg / L, the IBA concentration is 1.5 mg / L, the vitamin C concentration is 0.055 mg / L, and the fucoidan concentration is 2 g / L. Sterilize at 121 °C for 20 min to obtain a rooting medium.

[0084] Inoculate the adventitious buds of Ilex asprella in the rooting medium and culture them for 20 days at 28°C under a light intensity of 2500 lux to obtain Ilex asprella seedlings.

[0085] Comparative Example 4 This comparative example provides a method for inducing and differentiating callus of Ilex asprella, including the following steps: S1: Wash the leaves of Ilex asprella seedlings, first disinfect them with 75% ethanol solution by volume for 30 s, then disinfect them with a 0.1 wt% mercuric chloride aqueous solution (containing 1% Tween - 80 by volume) for 3 min, and cut the disinfected Ilex asprella leaves to obtain Ilex asprella explants.

[0086] S2: Dissolve dopamine in Tris - HCl buffer with a pH value of 8 to obtain a dopamine solution with a concentration of 6 g / L. Place the obtained dopamine solution at 50°C and stir - react for 18 h to obtain a polydopamine solution; add cumene hydroperoxide and 2 - methyl - diphenylamine to the polydopamine solution in an amount of 100 mL:0.75 g:2 mL by volume - mass ratio, stir at 35°C for 2 h, and then cool to room temperature and let it stand for 2.5 h to obtain an inducer.

[0087] Add the inducer, 6 - BA, NAA, sucrose, and agar to the MS medium so that the inducer concentration is 0.02 mg / L, the 6 - BA concentration is 2 mg / L, the NAA concentration is 0.15 mg / L, the sucrose concentration is 28.5 g / L, and the agar concentration is 6 g / L. Sterilize at 121°C for 20 min to obtain an induction medium.

[0088] Inoculate the Ilex asprella explants into the induction medium and culture them for 10 days at 28°C under a light intensity of 2500 lux to obtain callus.

[0089] S3: Add hinokitiol, 2,4 - D, 5 - hydroxy - IAA, sucrose, and agar to the MS medium so that the hinokitiol concentration is 0.065 mg / L, the 2,4 - D concentration is 0.3 mg / L, the 5 - hydroxy - IAA concentration is 1.5 mg / L, the sucrose concentration is 15 g / L, and the agar concentration is 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0090] Inoculate the callus into the differentiation medium and culture them for 15 days at 28°C under a light intensity of 2500 lux to obtain adventitious buds of Ilex asprella.

[0091] S4: Add 6 - BA, NAA, and fucoidan to the MS medium so that the 6 - BA concentration is 12 mg / L, the NAA concentration is 0.06 mg / L, and the fucoidan concentration is 2 g / L. Sterilize at 121°C for 20 min to obtain a proliferation medium.

[0092] The adventitious buds of Ilex asprella were inoculated into the proliferation medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain Ilex asprella cluster buds.

[0093] S5: NAA, IBA, vitamin C and fucoidan were added to the 1 / 2MS medium so that the concentration of NAA was 0.4 mg / L, the concentration of IBA was 1.5 mg / L, the concentration of vitamin C was 0.055 mg / L, and the concentration of fucoidan was 2 g / L. It was sterilized at 121 °C for 20 min to obtain the rooting medium.

[0094] The Ilex asprella cluster buds were inoculated into the rooting medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain Ilex asprella seedlings.

[0095] Comparative Example 5 This comparative example provided a method for inducing and differentiating Ilex asprella callus, including the following steps: S1: After washing the leaves of Ilex asprella seedlings, they were first disinfected with a 75% (v / v) ethanol solution for 30 s, and then disinfected with a 0.1 wt% mercuric chloride aqueous solution (containing 1% (v / v) Tween-80) for 3 min. The disinfected Ilex asprella leaves were cut to obtain Ilex asprella explants.

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

[0097] The inducer, 6-BA, NAA, sucrose and agar were added to the MS medium so that the concentration of the inducer was 0.02 mg / L, the concentration of 6-BA was 2 mg / L, the concentration of NAA was 0.15 mg / L, the concentration of sucrose was 28.5 g / L, and the concentration of agar was 6 g / L. It was sterilized at 121 °C for 20 min to obtain the induction medium.

[0098] The Ilex asprella explants were inoculated into the induction medium and cultured for 10 days at 28 °C under a light intensity of 2500 lux to obtain callus.

[0099] 2,4-D, 5-hydroxy-IAA, sucrose and agar were added to the MS medium so that the concentration of 2,4-D was 0.3 mg / L, the concentration of 5-hydroxy-IAA was 1.5 mg / L, the concentration of sucrose was 15 g / L, and the concentration of agar was 6 g / L. It was sterilized at 121 °C for 20 min to obtain the differentiation medium.

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

[0101] S4: 6-BA, NAA and fucoidan were added to the MS medium so that the concentration of 6-BA was 12 mg / L, the concentration of NAA was 0.06 mg / L, and the concentration of fucoidan was 2 g / L. It was sterilized at 121 °C for 20 min to obtain the proliferation medium.

[0102] The indefinite buds of Ilex asprella were inoculated into the proliferation medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain the cluster buds of Ilex asprella.

[0103] S5: NAA, IBA, vitamin C and fucoidan were added to the 1 / 2MS medium so that the concentration of NAA was 0.4 mg / L, the concentration of IBA was 1.5 mg / L, the concentration of vitamin C was 0.055 mg / L, and the concentration of fucoidan was 2 g / L. It was sterilized at 121 °C for 20 min to obtain the rooting medium.

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

[0105] Comparative Example 6 This comparative example provided a method for inducing and differentiating the callus of Ilex asprella, including the following steps: S1: After washing the leaves of the Ilex asprella seedlings, they were first disinfected with a 75% (v / v) ethanol solution for 30 s, and then disinfected with a 0.1 wt% mercuric chloride aqueous solution (containing 1% (v / v) Tween-80) for 3 min. The disinfected Ilex asprella leaves were cut to obtain the explants of Ilex asprella.

[0106] S2: Dopamine was dissolved in a Tris-HCl buffer solution with a pH value of 8 to obtain a dopamine solution with a concentration of 6 g / L. The obtained dopamine solution was placed at 50 °C and stirred for 18 h to obtain a polydopamine solution; dicumyl peroxide and 2-methyl diphenylamine were added to the polydopamine solution in an amount of 100 mL:0.75 g:2 mL by volume-mass ratio, stirred at 35 °C for 2 h, and then cooled to room temperature and left to stand for 2.5 h to obtain the inducer.

[0107] The inducer, 6-BA, NAA, as well as sucrose and agar were added to the MS medium so that the concentration of the inducer was 0.05 mg / L, the concentration of 6-BA was 2 mg / L, the concentration of NAA was 0.15 mg / L, the concentration of sucrose was 28.5 g / L, and the concentration of agar was 6 g / L. It was sterilized at 121 °C for 20 min to obtain the induction medium.

[0108] The Ilex asprella explants were inoculated into the induction medium and cultured for 10 days at 28 °C under a light intensity of 2500 lux to obtain callus.

[0109] S3: To the MS medium, hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose and agar were added such that the concentration of hinokitiol was 0.065 mg / L, the concentration of N-ethyl-L-glutamine was 0.35 mg / L, the concentration of 2,4-D was 0.3 mg / L, the concentration of 5-hydroxy-IAA was 1.5 mg / L, the concentration of sucrose was 15 g / L, and the concentration of agar was 6 g / L. It was sterilized at 121 °C for 20 min to obtain the differentiation medium.

[0110] The callus was inoculated into the differentiation medium and cultured for 15 days at 28 °C under a light intensity of 2500 lux to obtain adventitious buds of Ilex asprella.

[0111] S4: To the MS medium, 6-BA, NAA and fucoidan were added such that the concentration of 6-BA was 12 mg / L, the concentration of NAA was 0.06 mg / L, and the concentration of fucoidan was 2 g / L. It was sterilized at 121 °C for 20 min to obtain the proliferation medium.

[0112] The adventitious buds of Ilex asprella were inoculated into the proliferation medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain cluster buds of Ilex asprella.

[0113] S5: To the 1 / 2MS medium, NAA, IBA, vitamin C and fucoidan were added such that the concentration of NAA was 0.4 mg / L, the concentration of IBA was 1.5 mg / L, the concentration of vitamin C was 0.055 mg / L, and the concentration of fucoidan was 2 g / L. It was sterilized at 121 °C for 20 min to obtain the rooting medium.

[0114] The cluster buds of Ilex asprella were inoculated into the rooting medium and cultured for 20 days at 28 °C under a light intensity of 2500 lux to obtain Ilex asprella seedlings.

[0115] Comparative Example 7 This comparative example provides a method for inducing and differentiating callus of Ilex asprella, including the following steps: S1: After washing the leaves of Ilex asprella seedlings, they were first disinfected with a 75% (v / v) ethanol solution for 30 s, and then disinfected with a 0.1 wt% aqueous mercuric chloride solution (containing 1% (v / v) Tween-80) for 3 min. The disinfected Ilex asprella leaves were cut to obtain Ilex asprella explants.

[0116] S2: Dissolve dopamine in Tris-HCl buffer solution with a pH value of 8 to obtain a dopamine solution with a concentration of 6 g / L. Place the obtained dopamine solution in a stirred reaction at 50 °C for 18 h to obtain a polydopamine solution; add cumene hydroperoxide and 2-methyl diphenylamine to the polydopamine solution in an amount of 100 mL: 0.75 g: 2 mL by volume-mass ratio, stir at 35 °C for 2 h, and then cool to room temperature and let stand for 2.5 h to obtain an inducer.

[0117] Add the inducer, 6-BA, NAA, as well as sucrose and agar to the MS medium, such that the inducer concentration is 0.02 mg / L, the 6-BA concentration is 2 mg / L, the NAA concentration is 0.15 mg / L, the sucrose concentration is 28.5 g / L, and the agar concentration is 6 g / L. Sterilize at 121 °C for 20 min to obtain an induction medium.

[0118] Inoculate the Ilex asprella explants into the induction medium and culture them under the conditions of 28 °C and a light intensity of 2500 lux for 10 days to obtain callus.

[0119] Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, as well as sucrose and agar to the MS medium, such that the hinokitiol concentration is 0.01 mg / L, the N-ethyl-L-glutamine concentration is 0.6 mg / L, the 2,4-D concentration is 0.3 mg / L, the 5-hydroxy-IAA concentration is 1.5 mg / L, the sucrose concentration is 15 g / L, and the agar concentration is 6 g / L. Sterilize at 121 °C for 20 min to obtain a differentiation medium.

[0120] Inoculate the callus into the differentiation medium and culture them under the conditions of 28 °C and a light intensity of 2500 lux for 15 days to obtain adventitious buds of Ilex asprella.

[0121] Add 6-BA, NAA, and fucoidan to the MS medium, such that the 6-BA concentration is 12 mg / L, the NAA concentration is 0.06 mg / L, and the fucoidan concentration is 2 g / L. Sterilize at 121 °C for 20 min to obtain a proliferation medium.

[0122] Inoculate the adventitious buds of Ilex asprella into the proliferation medium and culture them under the conditions of 28 °C and a light intensity of 2500 lux for 20 days to obtain cluster buds of Ilex asprella.

[0123] Add NAA, IBA, vitamin C, and fucoidan to the 1 / 2MS medium, such that the NAA concentration is 0.4 mg / L, the IBA concentration is 1.5 mg / L, the vitamin C concentration is 0.055 mg / L, and the fucoidan concentration is 2 g / L. Sterilize at 121 °C for 20 min to obtain a rooting medium.

[0124] Inoculate the adventitious buds of Ilex asprella in the rooting medium and culture them for 20 days at 28°C under a light intensity of 2500 lux to obtain Ilex asprella seedlings.

[0125] Comparative Example 8 This comparative example provides a method for inducing and differentiating callus of Ilex asprella, including the following steps: S1: Wash the leaves of Ilex asprella seedlings, first disinfect them with 75% ethanol solution by volume for 30 s, then disinfect them with 0.1 wt% mercuric chloride aqueous solution (containing 1% by volume of Tween-80) for 3 min, and cut the disinfected Ilex asprella leaves to obtain Ilex asprella explants.

[0126] S2: Dissolve dopamine in Tris-HCl buffer with a pH value of 8 to obtain a dopamine solution with a concentration of 6 g / L. Place the obtained dopamine solution at 50°C and stir for 18 h to obtain a polydopamine solution; add diisopropylbenzene peroxide and 2-methyl diphenylamine to the polydopamine solution in an amount of 100 mL:0.75 g:2 mL by volume-mass ratio, stir at 35°C for 2 h, and then cool to room temperature and let stand for 2.5 h to obtain an inducer.

[0127] Add the inducer, 6-BA, NAA, sucrose, and agar to the MS medium so that the inducer concentration is 0.02 mg / L, the 6-BA concentration is 2 mg / L, the NAA concentration is 0.15 mg / L, the sucrose concentration is 28.5 g / L, and the agar concentration is 6 g / L. Sterilize at 121°C for 20 min to obtain an induction medium.

[0128] Inoculate the Ilex asprella explants into the induction medium and culture them for 10 days at 28°C under a light intensity of 2500 lux to obtain callus.

[0129] S3: Add hinokitiol, N-ethyl-L-glutamine, 2,4-D, 5-hydroxy-IAA, sucrose, and agar to the MS medium so that the hinokitiol concentration is 0.2 mg / L, the N-ethyl-L-glutamine concentration is 0.1 mg / L, the 2,4-D concentration is 0.3 mg / L, the 5-hydroxy-IAA concentration is 1.5 mg / L, the sucrose concentration is 15 g / L, and the agar concentration is 6 g / L. Sterilize at 121°C for 20 min to obtain a differentiation medium.

[0130] Inoculate the callus into the differentiation medium and culture them for 15 days at 28°C under a light intensity of 2500 lux to obtain adventitious buds of Ilex asprella.

[0131] S4: Add 6-BA, NAA, and fucoidan to the MS medium to make the concentration of 6-BA 12 mg / L, the concentration of NAA 0.06 mg / L, and the concentration of fucoidan 2 g / L. Sterilize at 121 °C for 20 min to obtain the proliferation medium.

[0132] Inoculate the Ilex asprella adventitious buds into the proliferation medium and culture them for 20 days under the conditions of 28 °C and a light intensity of 2500 lux to obtain the Ilex asprella cluster buds.

[0133] S5: Add NAA, IBA, vitamin C, and fucoidan to the 1 / 2MS medium to make the concentration of NAA 0.4 mg / L, the concentration of IBA 1.5 mg / L, the concentration of vitamin C 0.055 mg / L, and the concentration of fucoidan 2 g / L. Sterilize at 121 °C for 20 min to obtain the rooting medium.

[0134] Inoculate the Ilex asprella cluster buds into the rooting medium and culture them for 20 days under the conditions of 28 °C and a light intensity of 2500 lux to obtain the Ilex asprella seedlings.

[0135] Experiment 1: Effects of different induction and differentiation methods of Ilex asprella callus on the induction rate and bud differentiation rate of Ilex asprella tissue.

[0136] First, use the steps S1-S2 in Examples 1-3 and Comparative Examples 1-8 to induce and culture the Ilex asprella explants respectively. The number of Ilex asprella explants inoculated into the induction medium is 50, and their sizes and morphologies are basically the same. After the induction culture, count the induction situation of the callus. Among them, the callus induction rate (%) = (the number of explants forming callus / the total number of inoculated explants) × 100%.

[0137] Second, use the steps S3 in Examples 1-3 and Comparative Examples 1-8 to differentiate and culture the Ilex asprella callus to obtain the Ilex asprella adventitious buds. The number of Ilex asprella callus inoculated into the differentiation medium is 30, and their sizes and morphologies are basically the same. After the differentiation culture, count the budding situation of the Ilex asprella adventitious buds. Among them, the bud differentiation rate (%) = the number of callus blocks with differentiated buds / the total number of callus blocks inoculated × 100%.

[0138] The results are shown in Table 1.

[0139] Table 1 Effects of different induction and differentiation methods on the callus induction rate and bud differentiation rate of Ilex asprella

[0140] 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 improve the induction rate of Ilex asprella callus. Among them, it can be seen from a comparison between Comparative Examples 1-2 and Example 1 that the inducer has a significant effect on improving the induction rate of Ilex asprella callus. On the other hand, compared with Comparative Examples 3-4 and 7-8, the differentiation medium of the present invention can increase the differentiation rate of adventitious buds of Ilex asprella.

[0141] Experiment 2: Effects of different induction and differentiation methods of Ilex asprella callus on the survival rate of Ilex asprella seedlings The explants of Ilex asprella were successively induced, differentiated, proliferated, and rooted by using the steps S1-S5 in Example 1 and Comparative Examples 1-8 to obtain Ilex asprella seedlings. Among them, the number of Ilex asprella explants inoculated into the induction medium in step S2 was 20, and their sizes and morphologies were basically the same. After culturing by steps S1-S5, the growth conditions of the obtained Ilex asprella seedlings in each group were counted, and the results are shown in Table 2.

[0142] Table 2 Average root length and seedlings of Ilex asprella under different induction and differentiation culture methods

[0143] As can be seen from the results in Table 2, compared with Comparative Examples 1-8, the growth conditions of the Ilex asprella seedlings obtained by the method of Example 1 of the present invention are the best. This shows that the method of the present invention can obtain Ilex asprella seedlings with better growth.

[0144] In addition, it can be found that after the Ilex asprella seedlings obtained in each group (20 in number) were acclimatized and transplanted by the same method and planted for 10 days, the transplant survival rate of the Ilex asprella seedlings in the group of Example 1 of the present invention can reach 95%, which is significantly higher than the survival rate of the Ilex asprella seedlings in Comparative Examples 1-8. The results are shown in Table 3.

[0145] Table 3 Survival number and survival rate of Ilex asprella seedlings induced by different methods

[0146] The above is only a preferred embodiment of the present invention, and it does not impose any form of limitation on the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the above-disclosed methods and technical contents within the scope of the technical solution of the present invention to obtain equivalent embodiments with equivalent changes. However, as long as the content does not depart from the technical solution of the present invention, any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for inducing and differentiating the callus of Ilex asprella, characterized in that, It includes the following steps: S1: Wash and disinfect the leaves of Ilex asprella seedlings, then cut them to obtain Ilex asprella explants. S2: Inoculate the Ilex asprella explants into an induction medium and culture them for 7 - 14 days under the conditions of 25 - 30°C and a light intensity of 2000 - 3000 lux to obtain callus. S3: Inoculate the callus into a differentiation medium and culture it for 10 - 20 days under the conditions of 25 - 30°C and a light intensity of 2000 - 3000 lux to obtain adventitious buds of Ilex asprella. S4: Inoculate the adventitious buds of Ilex asprella into a proliferation medium and culture them for 15 - 25 days under the conditions of 25 - 30°C and a light intensity of 2000 - 3000 lux to obtain clustered shoots of Ilex asprella. S5: Inoculate the clustered shoots of Ilex asprella into a rooting medium and culture them for 15 - 25 days under the conditions of 25 - 30°C and a light intensity of 2000 - 3000 lux to obtain Ilex asprella seedlings. In step S2, the induction medium is an MS medium containing 0.01 - 0.03 mg / L of an inducer, 1 - 3 mg / L of 6 - BA, 0.1 - 0.2 mg / L of NAA, 25 - 32 g / L of sucrose, and 4 - 8 g / L of agar. The inducer is composed of a small - molecule inducer and polydopamine. In step S3, the differentiation medium is an MS medium containing 0.03 - 0.1 mg / L of hinokitiol, 0.2 - 0.5 mg / L of N - ethyl - L - glutamine, 0.1 - 0.5 mg / L of 2,4 - D, 1 - 2 mg / L of 5 - hydroxy - IAA, 10 - 20 g / L of sucrose, and 4 - 8 g / L of agar.

2. The induced differentiation method according to claim 1, characterized in that The preparation method of the inducer includes the following steps: Dissolve 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. Place the obtained dopamine solution at 40 - 60°C and stir - react for 12 - 24 h to obtain a polydopamine solution. Add a cross - linker and a small - molecule inducer to the polydopamine solution, stir at 30 - 40°C for 1 - 3 h, and then cool to room temperature and let it stand for 2 - 3 h to obtain the inducer.

3. The induction and differentiation method according to claim 2, characterized in that The polydopamine solution, cross - linker, and small - molecule inducer are added in an amount of 100 mL: 0.5 - 1 g: 1 - 3 mL by volume - mass ratio.

4. The induced differentiation method according to claim 2 or 3, characterized in that, The cross - linker is selected from diisopropylbenzene peroxide or benzoyl peroxide.

5. The induction and differentiation method according to claim 2 or 3, characterized in that 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.

6. The induced differentiation method according to claim 1, wherein In step S4, the proliferation medium is an MS medium containing 1.5 - 2.5 mg / L of 6 - BA, 0.02 - 0.08 mg / L of NAA, and 1 - 3 g / L of fucoidan.

7. The induced differentiation 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 of NAA, 1 - 2 mg / L of IBA, 0.03 - 0.08 mg / L of vitamin C, and 1 - 3 g / L of fucoidan.

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