A tissue culture method of lepidium virginicum

Abstract

The application provides a tissue culture method of smallflowered Dichanthium annulatum, which comprises the following steps: (1) collecting wild smallflowered Dichanthium annulatum seeds without diseases and insect pests, removing outer pulp, washing with running water, soaking in sterile water for 12-15 hours, and germinating on a culture dish with wet filter paper; (2) induction culture: inoculating hypocotyls and cotyledons of the germinated seeds into an induction culture medium containing an antibacterial agent for induction culture, so as to obtain callus containing multiple shoots; (3) proliferation subculture: inoculating the callus containing multiple shoots into a proliferation subculture medium containing an antibacterial agent for proliferation subculture, so as to obtain multiple shoots; (4) rooting culture: inoculating the multiple shoots into a rooting culture medium for rooting culture, so as to obtain tissue culture seedlings; and (5) seedling raising and transplanting: taking out the complete tissue culture seedlings after rooting culture, washing the culture medium in the roots, and transplanting. The method improves the survival rate of smallflowered Dichanthium annulatum, and the seedlings grow well.

Description

Technical Field

[0001] This invention belongs to the field of tissue culture technology, specifically relating to a method for tissue culture of *Symplocos buergeriana*. Background Technology

[0002] *Grewia microcarpa*, commonly known as the small-flowered shrub, is a species of plant belonging to the genus *Grewia* in the family Tiliaceae. This plant is primarily distributed in tropical and subtropical regions of Asia. It is a shrub or small tree, typically growing in low-altitude woodlands, thickets, or along riverbanks. In some areas, *Grewia microcarpa* may be used as an ornamental plant, and it may also possess certain medicinal value, though the specific medicinal efficacy needs to be determined based on local traditional medicine and modern scientific research. In natural ecosystems, it plays a role in maintaining biodiversity, providing habitat, and serving as a link in the food chain.

[0003] However, wild resources of *Symplocos simonii* are limited, and under natural conditions, seed germination rates are low, and offspring differentiation is severe, failing to maintain the superior characteristics of the parent plant and thus failing to meet the needs of landscaping, thereby restricting its widespread application. Therefore, establishing a tissue culture system for *Symplocos simonii* is of great significance for the rapid propagation and widespread application of its superior wild germplasm resources.

[0004] Therefore, there is an urgent need for a tissue culture method for *Symplocos buergeriana*. Summary of the Invention

[0005] The purpose of this invention is to provide a method for tissue culture of *Symplocos buergeriana*.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A method for tissue culture of *Symplocos buergeriana* includes the following steps:

[0008] (1) Collect wild seeds of *Symplocos buergeriana* free from pests and diseases, remove the outer pulp, rinse with running water, soak in sterile water for 12-15 hours, and germinate on a petri dish lined with moist filter paper.

[0009] (2) Induction culture: Hypocotyls and cotyledons of germinating seeds were inoculated into an induction medium containing antibacterial agent and induced to obtain callus tissue containing clustered buds.

[0010] (3) Proliferation and subculture: The callus tissue containing clustered buds was inoculated into a proliferation and subculture medium containing antibacterial agent and subjected to proliferation and subculture to obtain clustered seedlings;

[0011] (4) Rooting culture: The clump of seedlings is inoculated into a rooting medium containing an antibacterial agent and rooted to obtain tissue culture seedlings;

[0012] (5) Hardening and transplanting: Take out the complete tissue culture seedlings after rooting culture, wash the culture medium from the roots, and transplant them.

[0013] Furthermore, the induction medium is MS + sucrose 25-30 g / L + agar 15-25 g / L + 6-BA 0.5-1.0 mg / L + NAA 0.5-0.8 mg / L + antibacterial agent 100-150 ml / L.

[0014] Furthermore, the proliferation and subculture medium is MS + sucrose 15-20 g / L + agar 25-35 g / L + 6-BA 1-2 mg / L + NAA 1-2 mg / L + antibacterial agent 200-250 ml / L.

[0015] Furthermore, the rooting medium is 1 / 2 MS + sucrose 10-15 g / L + agar 15-25 g / L + NAA 0.4-0.8 mg / L + plant enzymatic hydrolysate 20-25 g / L.

[0016] Furthermore, the conditions for rooting culture are: temperature 25-27℃, light intensity 12-15h / d, and light intensity 1500-2000lx.

[0017] Furthermore, the antibacterial agent, per 1L, comprises the following components in the following amounts: 200-300mL plant fermentation broth, 10-15g chitosan oligosaccharide, 10-20mL Tween 60, and the remainder is water;

[0018] Furthermore, the method for preparing the plant fermentation broth is as follows:

[0019] (1) Mix Coptis chinensis, Scutellaria baicalensis, Clematis armandii and Paeonia suffruticosa root bark in a mass ratio of 1:1.2-1.4:0.2-0.5:1.6-1.8, pulverize them through an 80-100 mesh sieve to obtain mixed micro powder;

[0020] (2) Mix 1 part by weight of the mixed micro powder with 8-10 parts by weight of water and perform ultrasonic treatment. The ultrasonic treatment temperature is 60-70℃, the frequency is 50-70KHz, and the time is 30-50min to obtain the ultrasonic product.

[0021] (3) Sterilize the ultrasonic product, add 0.02-0.04 parts by weight of compound bacteria, ferment, and pasteurize after fermentation to obtain the fermentation product;

[0022] (4) Filter the fermentation product to obtain filtrate, concentrate the filtrate to an extract with a relative density of 1.1-1.3, add 8-10 times the mass of water to the extract to obtain plant fermentation liquid.

[0023] Furthermore, the fermentation conditions are: anaerobic fermentation at 34-37℃ for 8-10 hours.

[0024] Furthermore, the compound bacteria is a mixture of Lactobacillus delbrueckii, Lactobacillus brevis, and Lactobacillus bulgaricus in a mass ratio of 1:1.4-1.7:0.3-0.6.

[0025] This invention utilizes conventional induction and subculture media, but the survival rate of *Pterocarya stenoptera* is not ideal. By adding a specific amount of antibacterial agent to the aforementioned media, the survival rate of *Pterocarya stenoptera* can be improved. The active components in the antibacterial agent, through synergistic action, reduce the risk of pathogenic microorganism infection in the *Pterocarya stenoptera* culture medium, alleviate oxidative stress in the tissues, and optimize the culture environment, thereby promoting healthy growth of *Pterocarya stenoptera* tissues and improving the survival rate. It also improves leaf growth. However, the growth height of *Pterocarya stenoptera* after transplanting is not ideal.

[0026] Furthermore, the method for preparing the plant enzymatic hydrolysate is as follows:

[0027] (1) Weigh 1-4 parts by weight of mint, 5-9 parts by weight of Sichuan pepper, 3-7 parts by weight of arborvitae leaves and 2-5 parts by weight of white willow bark, mix and grind them, and pass them through a 100-mesh sieve to obtain mixed powder;

[0028] (2) Mix the mixed powder, compound enzyme and water in a mass ratio of 10:0.1-0.2:60-70, soak and enzymatically hydrolyze at 40-45℃ for 6-8 hours, and heat at 120-122℃ for 15-20 minutes to inactivate the enzyme to obtain the enzymatic hydrolysate;

[0029] (3) Filter the enzymatic hydrolysate, concentrate the filtrate to an extract with a relative density of 1.1-1.3, add 8-10 times the mass of water to the extract to obtain plant enzymatic hydrolysate.

[0030] Furthermore, the complex enzyme is a mixture of cellulase, pectinase and neutral protease in a mass ratio of 0.3-0.6:1.2-1.5:1.

[0031] This invention adds plant enzymatic hydrolysate to the rooting medium, which can improve the growth height of *Symplocos buergeriana* after transplanting. Peppermint, *Litsea cubeba*, arborvitae leaves, and white willow bark all contain abundant natural active ingredients. Through enzymatic hydrolysis, these macromolecular compounds are broken down into smaller molecules that are more easily absorbed by plants, providing a rich source of nutrients. They also contain natural antibacterial components; adding the hydrolysate to the culture medium can reduce the invasion of pathogenic microorganisms, protect plant roots, and reduce root diseases. The hydrolysate contains plant growth regulators; adding it to the rooting medium effectively promotes root formation and growth. Strong roots allow plants to better absorb water and nutrients, promoting the growth of above-ground parts and increasing plant height.

[0032] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0033] 1. This invention provides a method for tissue culture of *Symplocos buergeriana*, which improves the survival rate of *Symplocos buergeriana*, resulting in better seedling growth and taller plants.

[0034] 2. This invention improves the survival rate of *Symplocos buergeriana* by adding a specific amount of antibacterial agent to the above-mentioned culture medium; at the same time, it improves the leaf growth of the plant.

[0035] 3. The present invention adds plant enzyme hydrolysate to the rooting culture medium, which can improve the growth height of *Symplocos buergeriana* after transplanting. Attached Figure Description

[0036] Figure 1 Photographs of the callus tissue of *Symplocos buergeriana* obtained in Example 1;

[0037] Figure 2 Photographs of callus differentiation obtained from *Symplocos buergeriana* in Example 1;

[0038] Figure 3 This is a photograph of the tissue culture seedlings of *Symplocos buergeriana* obtained in Example 1. Detailed Implementation

[0039] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] The bacteria and enzymes used in this invention are all commercially available products, as detailed below:

[0041] β-glucanase, enzyme activity 40u / mg, Shanghai Yuanye Biotechnology Co., Ltd.; Cellulase, enzyme activity 50u / mg, Shanghai Yuanye Biotechnology Co., Ltd.

[0042] Neutral protease, enzyme activity 100u / mg, Shanghai Yuanye Biotechnology Co., Ltd.

[0043] Pectinase, enzyme activity 50u / mg, Shanghai Yuanye Biotechnology Co., Ltd.

[0044] Bromelain, enzyme activity 300u / mg, Shanghai Yuanye Biotechnology Co., Ltd.

[0045] Fig protease, enzyme activity 300u / mg, Shanghai Yuanye Biotechnology Co., Ltd.

[0046] Lactobacillus delbrueckii, accession number: CCTCC AB 200048. Deposited at the China Center for Type Culture Collection.

[0047] Lactobacillus brevis, accession number: SHBCC D14346. Deposited at the Shanghai Center for Microbiological Collections.

[0048] Lactobacillus bulgaricus, accession number: CCTCC CB 20082295. Deposited at the China Center for Type Culture Collection.

[0049] Lactobacillus plantarum, accession number: CCTCC AB 2010210. Deposited at the China Center for Type Culture Collection.

[0050] Bifidobacterium longum, accession number: CCTCC HB 20082718. Deposited at the China Center for Type Culture Collection.

[0051] Lactobacillus casei, accession number: SHBCC D 24737. Deposited at the Shanghai Center for Microbiological Collections.

[0052] Example 1

[0053] This embodiment provides a method for tissue culture of *Symplocos buergeriana*, including the following steps:

[0054] (1) Collect wild seeds of *Symplocos buergeriana* free from pests and diseases, remove the outer pulp, rinse with running water, soak in sterile water for 12 hours, and germinate on a petri dish lined with moist filter paper.

[0055] (2) Induction culture: Hypocotyls and cotyledons of germinating seeds were inoculated into an induction medium containing an antibacterial agent for induction culture to obtain callus tissue containing clustered buds (e.g. Figure 1 (as shown);

[0056] (3) Proliferation and subculture: The callus tissue containing clustered shoots was inoculated into a proliferation and subculture medium containing an antibacterial agent and subjected to proliferation and subculture to obtain clustered shoots (e.g., Figure 2 (as shown);

[0057] (4) Rooting culture: The clump of seedlings is inoculated into a rooting medium containing an antibacterial agent for rooting culture to obtain tissue culture seedlings (e.g., Figure 3 (as shown);

[0058] (5) Hardening and transplanting: Take out the complete tissue culture seedlings after rooting culture, wash the culture medium from the roots, and transplant them.

[0059] The induction medium consisted of MS medium containing 27 g / L sucrose, 20 g / L agar, 0.8 mg / L 6-BA, 0.6 mg / L NAA, and 120 ml / L antibacterial agent.

[0060] The proliferation and subculture medium consisted of MS medium containing 17 g / L sucrose, 30 g / L agar, 1.4 mg / L 6-BA, 1.5 mg / L NAA, and 220 ml / L antibacterial agent.

[0061] The rooting medium consisted of 1 / 2 MS medium containing 12 g / L sucrose, 22 g / L agar, 0.6 mg / L NAA, and 22 g / L plant enzymatic hydrolysate.

[0062] The conditions for rooting culture were: temperature 26℃, light intensity 14h / d, and light intensity 1600lx.

[0063] The antibacterial agent, per 1L, comprises the following components in the following amounts: 250mL plant fermentation broth, 12g chitosan oligosaccharide, 15mL Tween 60, and the remainder is water;

[0064] The method for preparing the plant fermentation broth is as follows:

[0065] (1) Mix Coptis chinensis, Scutellaria baicalensis, Clematis armandii and Paeonia suffruticosa root bark in a mass ratio of 1:1.3:0.4:1.7, pulverize and pass through a 100-mesh sieve to obtain mixed micro powder;

[0066] (2) Mix 1 part by mass of the mixed micro powder with 9 parts by mass of water and perform ultrasonic treatment. The ultrasonic treatment temperature is 65℃, the frequency is 60KHz, and the time is 40min to obtain the ultrasonic product.

[0067] (3) Sterilize the ultrasonic product, add 0.03 parts by weight of compound bacteria, ferment, and pasteurize after fermentation to obtain the fermentation product;

[0068] (4) Filter the fermentation product to obtain filtrate, concentrate the filtrate to an extract with a relative density of 1.2, add water with a mass of 9 times that of the extract to obtain plant fermentation liquid.

[0069] The fermentation conditions were: anaerobic fermentation at 35℃ for 9 hours.

[0070] The compound bacteria are a mixture of Lactobacillus delbrueckii, Lactobacillus brevis, and Lactobacillus bulgaricus in a mass ratio of 1:1.5:0.5.

[0071] The method for preparing the plant enzymatic hydrolysate is as follows:

[0072] (1) Weigh 2 parts by weight of mint, 7 parts by weight of Sichuan pepper, 5 parts by weight of arborvitae leaves and 3 parts by weight of white willow bark, mix and grind them, and pass them through a 100-mesh sieve to obtain mixed powder;

[0073] (2) Mix the powder, compound enzyme and water in a mass ratio of 10:0.15:67, soak and enzymatically hydrolyze at 42℃ for 7h, and heat at 121℃ for 15min to inactivate the enzyme to obtain the enzymatic hydrolysate.

[0074] (3) Filter the enzymatic hydrolysate, concentrate the filtrate to an extract with a relative density of 1.2, add water with a mass of 9 times that of the extract to obtain plant enzymatic hydrolysate.

[0075] The complex enzyme is a mixture of cellulase, pectinase and neutral protease in a mass ratio of 0.5:1.4:1.

[0076] Example 2

[0077] This embodiment provides a method for tissue culture of *Symplocos buergeriana*, including the following steps:

[0078] (1) Collect wild seeds of *Symplocos buergeriana* free from pests and diseases, remove the outer pulp, rinse with running water, soak in sterile water for 12 hours, and germinate on a petri dish lined with moist filter paper.

[0079] (2) Induction culture: Hypocotyls and cotyledons of germinating seeds were inoculated into an induction medium containing antibacterial agent and induced to obtain callus tissue containing clustered buds.

[0080] (3) Proliferation and subculture: The callus tissue containing clustered buds was inoculated into a proliferation and subculture medium containing antibacterial agent and subjected to proliferation and subculture to obtain clustered seedlings;

[0081] (4) Rooting culture: The clump of seedlings is inoculated into a rooting medium containing an antibacterial agent and rooted to obtain tissue culture seedlings;

[0082] (5) Hardening and transplanting: Take out the complete tissue culture seedlings after rooting culture, wash the culture medium from the roots, and transplant them.

[0083] The induction medium consisted of MS medium containing 30 g / L sucrose, 15 g / L agar, 1.0 mg / L 6-BA, 0.5 mg / L NAA, and 150 ml / L antibacterial agent.

[0084] The proliferation and subculture medium consisted of MS medium containing 15 g / L sucrose, 35 g / L agar, 2 mg / L 6-BA, 2 mg / L NAA, and 200 ml / L antibacterial agent.

[0085] The rooting medium consisted of 1 / 2 MS medium containing 15 g / L sucrose, 25 g / L agar, 0.8 mg / L NAA, and 20 g / L plant enzymatic hydrolysate.

[0086] The conditions for rooting culture were: temperature 27℃, light intensity 12h / d, and light intensity 1500lx.

[0087] The antibacterial agent, per 1L, comprises the following components in the following amounts: 200mL plant fermentation broth, 15g chitosan oligosaccharide, 10mL Tween 60, and the remainder is water;

[0088] The method for preparing the plant fermentation broth is as follows:

[0089] (1) Mix Coptis chinensis, Scutellaria baicalensis, Clematis armandii and Paeonia suffruticosa root bark in a mass ratio of 1:1.2:0.5:1.6, pulverize them through an 80-mesh sieve to obtain mixed micro powder;

[0090] (2) Mix 1 part by mass of the mixed micro powder with 10 parts by mass of water and perform ultrasonic treatment. The ultrasonic treatment temperature is 60℃, the frequency is 70KHz, and the time is 50min to obtain the ultrasonic product.

[0091] (3) Sterilize the ultrasonic product, add 0.02 parts by weight of compound bacteria, ferment, and pasteurize after fermentation to obtain the fermentation product;

[0092] (4) Filter the fermentation product to obtain filtrate, concentrate the filtrate to an extract with a relative density of 1.3, add water at 8 times the mass of the extract to obtain plant fermentation liquid.

[0093] The fermentation conditions were: anaerobic fermentation at 37°C for 8 hours.

[0094] The compound bacteria are a mixture of Lactobacillus delbrueckii, Lactobacillus brevis, and Lactobacillus bulgaricus in a mass ratio of 1:1.7:0.3.

[0095] The method for preparing the plant enzymatic hydrolysate is as follows:

[0096] (1) Weigh 1 part by weight of mint, 9 parts by weight of Sichuan pepper, 3 parts by weight of arborvitae leaves and 5 parts by weight of white willow bark, mix and grind them, and pass them through a 100-mesh sieve to obtain mixed powder;

[0097] (2) Mix the mixed powder, compound enzyme and water in a mass ratio of 10:0.1:70, soak and enzymatically hydrolyze at 45°C for 6 hours, and heat at 122°C for 15 minutes to inactivate the enzyme to obtain the enzymatic hydrolysate.

[0098] (3) Filter the enzymatic hydrolysate, concentrate the filtrate to an extract with a relative density of 1.3, add water at 8 times the mass of the extract to obtain plant enzymatic hydrolysate.

[0099] The complex enzyme is a mixture of cellulase, pectinase and neutral protease in a mass ratio of 0.3:1.2:1.

[0100] Comparative Example 1

[0101] The difference between this comparative example and Example 1 is that the mass ratio of Coptis chinensis, Scutellaria baicalensis, Clematis armandii, and Paeonia suffruticosa root bark is 1:1:1:1.

[0102] Comparative Example 2

[0103] The difference between this comparative example and Example 1 is that the compound bacteria is a mixture of Lactobacillus delbrueckii, Lactobacillus brevis, and Lactobacillus bulgaricus in a mass ratio of 1:1:1.

[0104] Comparative Example 3

[0105] The difference between this comparative example and Example 1 is that the compound bacteria is a mixture of Lactobacillus plantarum, Bifidobacterium longum and Lactobacillus casei in a mass ratio of 1:1.5:0.5.

[0106] Comparative Example 4

[0107] The difference between this comparative example and Example 1 is as follows: 7 parts by weight of peppermint, 2 parts by weight of Sichuan pepper, 1 part by weight of arborvitae leaves, and 7 parts by weight of white willow bark were weighed, mixed and pulverized, and passed through a 100-mesh sieve to obtain a mixed powder.

[0108] Comparative Example 5

[0109] The difference between this comparative example and Example 1 is that the complex enzyme is a mixture of cellulase, pectinase and neutral protease in a mass ratio of 1:1:1.

[0110] Comparative Example 6

[0111] The difference between this comparative example and Example 1 is that the complex enzyme is a mixture of β-glucanase, bromelain, and fig protease in a mass ratio of 0.5:1.4:1.

[0112] Performance testing

[0113] The tissue culture seedlings from Examples 1-2 and Comparative Examples 1-6 were transplanted, with 60 seedlings per group. After transplanting, the seedlings were thoroughly watered to help them establish themselves. For two weeks after transplanting, the temperature was maintained at 20-27℃, humidity at 80-85%, and light intensity at 5000-5500 LX. Two weeks after transplanting, the seedlings entered normal care and management. The following is a description of the seedlings after three months of care.

[0114] Table 1 Performance Test Results

[0115]

[0116] The performance test results above show that the methods in Examples 1-2 resulted in a high survival rate and good seedling growth for *Symplocos buergeriana*, with Example 1 showing the most outstanding overall performance.

[0117] The comparative examples, however, did not employ the necessary technical solutions, resulting in significantly inferior performance compared to the examples. In comparative examples 1-3, the antibacterial agent was changed, leading to a decrease in survival rate, demonstrating that the formulation of the antibacterial agent has a significant impact on the survival rate. In comparative examples 4-6, the lack of plant enzyme hydrolysate resulted in an impact on plant height. These experimental results further demonstrate the importance of the technical solutions defined in this invention for its technical effectiveness.

[0118] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for tissue culture of *Pteris vittata*, characterized in that, Includes the following steps: (1) Collect wild seeds of *Symplocos buergeriana* free from pests and diseases, remove the outer pulp, rinse with running water, soak in sterile water for 12-15 hours, and germinate on a petri dish lined with moist filter paper. (2) Induction culture: Hypocotyls and cotyledons of germinating seeds were inoculated into an induction medium containing an antibacterial agent for induction culture to obtain callus tissue containing clustered buds; the induction medium was MS + sucrose 25-30 g / L + agar 15-25 g / L + 6-BA 0.5-1.0 mg / L + NAA 0.5-0.8 mg / L + antibacterial agent 100-150 ml / L; (3) Proliferation and subculture: Callus tissue containing clustered shoots was inoculated into a proliferation and subculture medium containing antibacterial agent and subjected to proliferation and subculture to obtain clustered shoots; the proliferation and subculture medium was MS + sucrose 15-20g / L + agar 25-35g / L + 6-BA 1-2mg / L + NAA 1-2mg / L + antibacterial agent 200-250ml / L; (4) Rooting culture: The clump of seedlings is inoculated into the rooting medium and rooted to obtain tissue culture seedlings; the rooting medium is 1 / 2 MS + sucrose 10-15 g / L + agar 15-25 g / L + NAA 0.4-0.8 mg / L + plant enzyme hydrolysate 20-25 g / L; (5) Hardening and transplanting: Take out the complete tissue culture seedlings after rooting culture, wash the culture medium off the roots, and transplant them; The antibacterial agent, per 1L, comprises the following components in the following amounts: 200-300mL plant fermentation broth, 10-15g chitosan oligosaccharide, 10-20mL Tween 60, and the remainder is water; The preparation method of plant fermentation broth is as follows: (1) Mix Coptis chinensis, Scutellaria baicalensis, Clematis armandii and Paeonia suffruticosa root bark in a mass ratio of 1:1.2-1.4:0.2-0.5:1.6-1.8, pulverize them through an 80-100 mesh sieve to obtain mixed micro powder; (2) Mix 1 part by weight of the mixed micro powder with 8-10 parts by weight of water and perform ultrasonic treatment. The ultrasonic treatment temperature is 60-70℃, the frequency is 50-70KHz, and the time is 30-50min to obtain the ultrasonic product. (3) Sterilize the ultrasonic product, add 0.02-0.04 parts by weight of compound bacteria, ferment, and pasteurize after fermentation to obtain the fermentation product; the compound bacteria is a mixture of Lactobacillus delbrueckii, Lactobacillus brevis and Lactobacillus bulgaricus in a mass ratio of 1:1.4-1.7:0.3-0.

6. (4) Filter the fermentation product to obtain filtrate, concentrate the filtrate to an extract with a relative density of 1.1-1.3, add 8-10 times the mass of water to the extract to obtain plant fermentation liquid; The preparation method of plant enzymatic hydrolysate is as follows: (1) Weigh 1-4 parts by weight of mint, 5-9 parts by weight of Sichuan pepper, 3-7 parts by weight of arborvitae leaves and 2-5 parts by weight of white willow bark, mix and grind them, and pass them through a 100-mesh sieve to obtain mixed powder; (2) Mix the powder, compound enzyme and water in a mass ratio of 10:0.1-0.2:60-70, enzymatically hydrolyze at 40-45℃ for 6-8 hours, and inactivate the enzyme by heating at 120-122℃ for 15-20 minutes to obtain the enzymatic hydrolysate; the compound enzyme is a mixture of cellulase, pectinase and neutral protease in a mass ratio of 0.3-0.6:1.2-1.5:

1. (3) Filter the enzymatic hydrolysate, concentrate the filtrate to an extract with a relative density of 1.1-1.3, add 8-10 times the mass of water to the extract to obtain plant enzymatic hydrolysate.

2. The method for tissue culture of *Symplocos buergeriana* according to claim 1, characterized in that, The conditions for rooting culture are: temperature 25-27℃, light intensity 12-15h / d, and light intensity 1500-2000lx.

3. The method for tissue culture of *Symplocos buergeriana* according to claim 1, characterized in that, The fermentation conditions are: anaerobic fermentation at 34-37℃ for 8-10 hours.

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