A culture medium for Longjing tea tree tissue and a culture method for Longjing tea tree tissue

Abstract

The present application discloses a culture medium of Longjing tea tree tissue and a culture method of Longjing tea tree tissue, the culture medium of Longjing tea tree tissue adopts MS culture medium, wherein, 0.3-1.1mg anti-browning agent, 1-3mg auxin, 0.4-0.9mg cytokinin, 15-40g carbon source, 3-9g agar and 0.3-1.5g inorganic material are added to each liter of the MS culture medium, and the inorganic material includes materials that can affect the degree of DNA methylation in the cell, regulate protein synthesis or adsorb harmful metabolites. The regenerated Longjing tea tree tissue cultivated by the culture medium and culture method of Longjing tea tree tissue of the present application has a high induction rate and a low browning rate, which provides a basis for the improvement and rapid breeding of Longjing tea tree tissue.

Description

Technical Field

[0001] The present application relates to the field of tissue culture medium, and in particular to a culture medium for Longjing tea tree tissue and a culture method for Longjing tea tree tissue. Background Art

[0002] Longjing tea is a famous green tea variety in the area around Hangzhou. Due to its excellent quality and unique aroma, it is known as the best green tea. However, the traditional Longjing tea production method has some defects, such as long seedling breeding cycle and susceptibility to diseases and insect pests. In order to obtain better quality Longjing tea, using tissue culture technology to quickly breed or improve the variety of Longjing tea is a better way.

[0003] However, in the process of tissue culture, browning is easily caused due to reasons such as pathogen erosion, changes in culture medium composition, and culture environment, which affects the growth and development of tissue cells and reduces the culture effect. Summary of the invention

[0004] In order to reduce the browning problem that occurs during tissue culture, the present application provides a culture medium for Longjing tea tree tissue and a preparation method thereof.

[0005] In a first aspect, a culture medium for Longjing tea tree tissue is provided, which adopts MS culture medium, wherein per liter of the MS culture medium, 0.3-1.1 mg anti-browning agent, 1-3 mg auxin, 0.4-0.9 mg cytokinin, 15-40 g carbon source, 3-9 g agar and 0.3-1.5 g inorganic material are added, and the inorganic material includes materials that can affect the degree of DNA methylation in cells, regulate protein synthesis or adsorb harmful metabolites.

[0006] Typically, but not limiting, the anti-browning agent includes one of polyvinyl pyrrolidone, cysteine, ascorbic acid or citric acid. The cytokinin includes one of benzylaminoadenine, kinetin, zeatin or mannitol. The auxin includes one of indoleacetic acid, indolebutyric acid or naphthylacetic acid. The carbon source includes one of sucrose, glucose or maltose.

[0007] By adopting the above technical solution, anti-browning agents, cytokinins, and auxins are added to the MS culture medium, which can promote cell division and proliferation while improving the anti-browning property of the tissue. The anti-browning agents include substances that can inhibit phenolic substances from being oxidized into quinone substances under the catalysis of enzymes, thereby reducing the degree of browning, such as polyvinyl pyrrolidone and cysteine. Anti-browning agents also include substances that can directly remove active oxygen in the tissue, participate in the internal circulation of the plant to remove the generated hydrogen peroxide, thereby protecting phenolic substances from oxidation, and reducing the damage caused by active oxygen generated by the body's metabolism, thereby reducing the problem of browning, such as ascorbic acid and citric acid.

[0008] Inorganic materials are added to MS culture medium. Inorganic materials have a wide range of effects as additives. Materials with antibacterial properties such as graphene, nanosilver, carbon nanotubes or activated carbon can be selected according to needs to improve the ability of tissues to resist external diseases, and increase the content of total phenols, flavonoids and hydroxycinnamic acid in cells, thereby improving the antioxidant properties and division ability of cells, thereby achieving an increase in tissue induction rate while reducing the degree of browning. Inorganic materials can be selected to affect the degree of DNA methylation in cells, regulate protein synthesis or adsorb harmful metabolites, including carbon nanotubes or activated carbon. In addition to increasing the content of phenolic compounds and flavonoid compounds in tissues, carbon nanotubes and activated carbon can affect the degree of DNA methylation and the synthesis pathway of proteins, thereby increasing the induction rate of tissues. Activated carbon can adsorb toxic metabolites produced during the process of tissue cell growth induction, reduce the accumulation of toxic substances, and thus reduce the occurrence of browning.

[0009] The addition of inorganic materials can work synergistically with anti-browning agents, auxins, and cytokinins to reduce the degree of cell browning. After the addition of inorganic materials, the degree of cell browning will be significantly reduced, and the induction rate of cells will be further increased.

[0010] Preferably, the nanomaterial is activated carbon, and the particle size of the activated carbon is 1-30 nm.

[0011] By adopting the above technical scheme, activated carbon, due to its strong adsorption effect, will inhibit the transport of nutrients into cells and affect the internal and external circulation of cells, thereby causing certain inhibition on cell division, proliferation and differentiation. By optimizing the particle size of activated carbon, the activated carbon can fully exert its browning inhibition effect while reducing the inhibitory effect of activated carbon on cell growth.

[0012] Preferably, the inorganic material is a mixture of carbon nanotubes and activated carbon, and the weight ratio of the carbon nanotubes to the activated carbon is (0.5-0.8):(0.2-0.5).

[0013] By adopting the above technical solution, the MS medium is a solid medium. Due to the poor fluidity of the solid medium, the accumulation of harmful substances and the supply of nutrients are problems that affect the induction of cell browning.

[0014] When carbon nanotubes are used in conjunction with activated carbon, on the one hand, the strong adsorption effect of activated carbon can enrich nutrients and growth hormones, while carbon nanotubes have good biocompatibility and conductive properties, and can serve as a bridge between activated carbon and cells. Activated carbon transports nutrients and growth hormones into cells through carbon nanotubes, and cells excrete harmful metabolites into activated carbon through carbon nanotubes, thereby increasing the induction rate of cells and reducing the chance of browning.

[0015] On the other hand, the synergistic use of carbon nanotubes and activated carbon can also stabilize the pH value and ion concentration of MS culture medium, reduce the damage caused by the environment to cells, and thus reduce the occurrence of browning.

[0016] The synergistic effect of carbon nanotubes and activated carbon can have a more significant effect when the culture medium is a solid culture medium, such as the MS culture medium of the present application.

[0017] Preferably, the inorganic material comprises a mixture of nanosilver and carbon nanotubes, and the weight ratio of the nanosilver to the carbon nanotubes is (0.1-0.2):(0.8-0.9). Further preferably, the pore size of the carbon nanotubes can be selected from 10-20 nm, and the particle size of the nanosilver can be selected from 0.2-10 nm.

[0018] By adopting the above technical solution, when nanosilver and carbon nanotubes exist in the form of a mixture, the carbon nanotubes have a more porous structure and a larger specific surface area, and the nanosilver can be loaded on the carbon nanotubes, thereby improving the dispersibility of the nanosilver and further exerting the effect of the nanosilver.

[0019] Carbon nanotubes loaded with nanosilver can attach to the cell surface, forming an antibacterial layer on the cell surface, reducing the damage of pollutants or bacteria to the cells. Nanosilver can be dispersed in the culture medium to form silver ions, which will complex with harmful metabolites in the cells, reducing the toxic effects of harmful product accumulation on the cells, thereby synergizing with carbon nanotubes to reduce the occurrence of browning.

[0020] Nanosilver improves its compatibility with cells through carbon nanotubes, regulates hormone synthesis and signal transduction pathways within cells, increases the rate of cell division and growth, thereby improving cell metabolism to reduce the accumulation of harmful substances in cells and reduce the formation of browning.

[0021] Preferably, the auxin comprises indolebutyric acid, and the cytokinin comprises thidiazuron.

[0022] By adopting the above technical solution, indolebutyric acid has good stability and low cytotoxicity, can promote cell proliferation and differentiation for a long time, and reduce the accumulation of toxic substances, thereby improving the induction rate of cells and anti-browning performance.

[0023] Thidiazuron has a strong effect of promoting cell division and reproduction, and has high activity at low concentrations. In addition, Thidiazuron can also improve the stress resistance of cell tissues. In the case of inorganic materials including carbon nanotubes or activated carbon, it can synergistically improve the adaptability of cells to the environment, thereby increasing the induction rate while reducing browning.

[0024] When indolebutyric acid and thidiazuron are used in synergy, indoleacetic acid promotes the formation and proliferation of tissues, while thidiazuron promotes the growth rate of tissues, thereby synergistically improving the regeneration rate of plant tissues. Indoleacetic acid can promote the differentiation and growth of root cells, while thidiazuron can promote the differentiation and growth of stem cells. The two can synergistically improve the subsequent differentiation of tissue cells.

[0025] Preferably, the cytokinin further comprises mannitol, and the weight ratio of thidiazuron to mannitol is (0.7-0.9): (0.1-0.3).

[0026] By adopting the above technical solution, mannitol can play a role in regulating osmotic pressure. When using MS culture medium with high nutrient concentration, mannitol can reduce cell water loss and reduce cell damage. When synergistically acting with benzathiapiprolon, it further improves the adaptability of cells to the environment and reduces browning. Mannitol can also synergistically promote the formation of tissue cells with indolebutyric acid and benzathiapiprolon and improve the induction rate of cells.

[0027] In a second aspect, a method for culturing Longjing tea tree tissue comprises the following steps:

[0028] S1: explant pretreatment: the explant is treated with 75% ethanol for 25-35 seconds, and then treated with a sodium hypochlorite solution with a mass fraction of 0.1-0.15% for 4-6 minutes to obtain the pretreated explant;

[0029] S2: Tissue culture: The pretreated explants are inoculated into the culture medium of Longjing tea tree tissue under the conditions of light intensity of 3000-4000lx and culture temperature of (25±2)°C to obtain regenerated Longjing tea tree tissue.

[0030] Typically, but not limiting, the explants may be selected from stem segments, old leaves, and young leaves.

[0031] By adopting the above technical solution, the explants are first disinfected with 75% ethanol and sodium hypochlorite with low toxicity, which can reduce the damage of the disinfectant to the explants and reduce the formation of browning of the explants during subsequent cultivation. In addition, the pretreatment process is optimized to improve the disinfection effect and reduce the possibility of explant contamination, thereby increasing the induction rate of subsequent tissues and reducing the generation of browning. By adopting the above tissue culture process parameters, regenerated Longjing tea tree tissue with a high induction rate and a low browning rate can be obtained.

[0032] Preferably, the explants are young and tender stem segments.

[0033] By adopting the above technical scheme, the young stems of Longjing tea contain lower phenolic substances and are less sensitive to oxidation. Therefore, during the cultivation process, the occurrence of browning can be reduced, and the stem tissue has better inducibility, which can improve the induction rate during the cultivation process.

[0034] Preferably, in step S2, the time for regenerating Longjing tea tree tissue culture is 68-82 hours.

[0035] Preferably, in step S2, the photoperiod is 16-20 h of light and 4-8 h of darkness.

[0036] By adopting the above scheme, adjusting the appropriate light cycle and culture time can increase the induction rate of the tissue and reduce the contamination of the cells by environmental factors, thereby increasing the induction rate while reducing the formation of browning.

[0037] In summary, this application has the following beneficial effects:

[0038] 1. Adding inorganic materials including activated carbon or carbon nanotubes to the MS culture medium, and combining it with hormones at appropriate concentrations, can improve the growth and metabolism of tissue cells while reducing the accumulation of toxic substances in the cells, thereby increasing the induction rate while reducing the formation of browning.

[0039] 2. The inorganic material is a mixture of activated carbon and carbon nanotubes. The two can not only play a complementary role (the inhibitory effect of activated carbon is neutralized by carbon nanotubes), but also play a significant role in promoting cell growth when the culture medium is a solid culture medium.

[0040] 3. The inorganic material is a mixture of nanosilver and carbon nanotubes, and the pore size of the carbon nanotubes is optimized so that the carbon nanotubes can load nanosilver, which gives the cells strong antibacterial properties and regulates the synthesis of beneficial hormones inside the cells, increases the induction rate of the cells and reduces browning. DETAILED DESCRIPTION

[0041] The present application is further described in detail below with reference to the embodiments.

[0042] Example

[0043] Example 1, a culture medium for Longjing tea tree tissue and a culture method thereof,

[0044] A culture medium for Longjing tea tree tissue is prepared by the following steps: 0.7 mg of ascorbic acid, 2 mg of indolebutyric acid, 0.65 mg of benzathine, 27.5 g of sucrose, 6 g of agar and 0.9 g of activated carbon are added to 1 L of MS basic culture medium in sequence, stirred evenly, adjusted the pH to 6.2, sterilized by high pressure at 125°C for 10 min, and taken out after standing to room temperature to obtain the culture medium for Longjing tea tree tissue.

[0045] Example 2, a culture medium for Longjing tea tree tissue and a culture method thereof,

[0046] A culture medium for Longjing tea tree tissue is prepared by the following steps: 1.1 mg of ascorbic acid, 1 mg of indolebutyric acid, 0.4 mg of benzathine, 40 g of sucrose, 9 g of agar and 1.5 g of activated carbon are added to 1 L of MS basic culture medium in sequence, stirred evenly, adjusted the pH to 6, sterilized by high pressure at 125°C for 10 min, and taken out after standing to room temperature to obtain the culture medium for Longjing tea tree tissue.

[0047] Example 3, a culture medium for Longjing tea tree tissue and a culture method thereof,

[0048] A culture medium for Longjing tea tree tissue is prepared by the following steps: 0.3 mg of ascorbic acid, 3 mg of indolebutyric acid, 0.9 mg of benzathine, 15 g of sucrose, 3 g of agar and 0.3 g of activated carbon are added to 1 L of MS basic culture medium in sequence, stirred evenly, adjusted the pH to 6.4, sterilized by high pressure at 125°C for 10 min, and taken out after standing to room temperature to obtain the culture medium for Longjing tea tree tissue.

[0049] Example 4, a culture medium for Longjing tea tree tissue, is different from Example 1 in that the activated carbon is replaced by a mixture of equal amounts of carbon nanotubes and activated carbon, and the weight ratio of the carbon nanotubes to the activated carbon is 0.5:0.5.

[0050] Example 5, a culture medium for Longjing tea tree tissue, which is different from Example 1 in that the activated carbon is replaced by a mixture of equal amounts of carbon nanotubes and activated carbon, and the weight ratio of the carbon nanotubes to the activated carbon is 0.8:0.2.

[0051] Example 6, a culture medium for Longjing tea tree tissue, which is different from Example 1 in that the activated carbon is replaced by a mixture of equal amounts of carbon nanotubes and nanosilver, and the weight ratio of the carbon nanotubes to the nanosilver is 0.8:0.2.

[0052] The pore size of the carbon nanotube is selected from 10-20 nm, and the particle size of the nanosilver is selected from 0.2-10 nm.

[0053] Example 7, a culture medium for Longjing tea tree tissue, which is different from Example 1 in that the activated carbon is replaced by a mixture of equal amounts of carbon nanotubes and nanosilver, and the weight ratio of the carbon nanotubes to the nanosilver is 0.9:0.1.

[0054] The pore size of the carbon nanotube is selected from 10-20 nm, and the particle size of the nanosilver is selected from 0.2-10 nm.

[0055] Example 8, a culture medium for Longjing tea tree tissue, which is different from Example 1 in that the cytokinin is replaced by a mixture of equal amounts of thidiazuron and mannitol, and the weight ratio of thidiazuron to mannitol is 0.7:0.3.

[0056] Example 9, a culture medium for Longjing tea tree tissue, which is different from Example 1 in that the cytokinin is replaced by a mixture of equal amounts of thidiazuron and mannitol, and the weight ratio of thidiazuron to mannitol is 0.9:0.1.

[0057] Embodiment 10, a method for culturing Longjing tea tree tissue, comprising the following steps:

[0058] S1: Explant pretreatment: 1.2 cm young stem segments of Longjing No. 43 were cut and treated with 75% ethanol for 30 seconds, and then treated with 0.15% sodium hypochlorite solution for 5 minutes to obtain pretreated explants;

[0059] S2: Tissue culture: The pretreated explants were inoculated into the culture medium of Longjing tea tree tissue. The culture conditions were a light intensity of 3500 lx, a photoperiod of 18 h light and 6 h dark, a culture temperature of 25°C, and cultured for 75 h to obtain regenerated Longjing tea tree tissue.

[0060] The particle size of the activated carbon is 1-30 nm. The culture medium of the Longjing tea tree tissue is the culture medium of the Longjing tea tree tissue of Example 3.

[0061] Embodiment 11, a method for culturing Longjing tea tree tissue, comprising the following steps:

[0062] A method for culturing Longjing tea tree tissue comprises the following steps:

[0063] S1: Explant pretreatment: 1.2 cm young stem segments of Longjing No. 43 were cut and treated with 75% ethanol for 35 seconds, and then treated with 0.1% sodium hypochlorite solution for 6 minutes to obtain pretreated explants;

[0064] S2: Tissue culture: The pretreated explants were inoculated into the culture medium of Longjing tea tree tissue. The culture conditions were a light intensity of 4000 lx, a photoperiod of 16 h light and 8 h darkness, a culture temperature of 27°C, and cultured for 68 h to obtain regenerated Longjing tea tree tissue.

[0065] The particle size of the activated carbon is 1-30 nm, and the culture medium of the Longjing tea tree tissue adopts the culture medium of the Longjing tea tree tissue of Example 2.

[0066] Example 12, a method for culturing Longjing tea tree tissue, comprising the following steps:

[0067] A method for culturing Longjing tea tree tissue comprises the following steps:

[0068] S1: Explant pretreatment: 1.2 cm young stem segments of Longjing No. 43 were cut and treated with 75% ethanol for 25 seconds, and then treated with 0.15% sodium hypochlorite solution for 4 minutes to obtain pretreated explants;

[0069] S2: Tissue culture: The pretreated explants were inoculated into the culture medium of Longjing tea tree tissue. The culture conditions were a light intensity of 3000 lx, a photoperiod of 20 h light and 4 h dark, a culture temperature of 23°C, and cultured for 82 h to obtain regenerated Longjing tea tree tissue.

[0070] The particle size of the activated carbon is 1-30 nm, and the culture medium of the Longjing tea tree tissue adopts the culture medium of the Longjing tea tree tissue of Example 3.

[0071] Example 13, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Example 4.

[0072] Example 14, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Example 5.

[0073] Example 15, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Example 6.

[0074] Example 16, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Example 7.

[0075] Example 17, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Example 8.

[0076] Example 18, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Example 9.

[0077] Example 19, a method for culturing Longjing tea tree tissue, is different from Example 10 in that in step S2, the culturing time of the regenerated Longjing tea tree tissue is 60 hours.

[0078] Example 20, a method for culturing Longjing tea tree tissue, is different from Example 10 in that in step S2, the culturing time of the regenerated Longjing tea tree tissue is 90 hours.

[0079] Example 21, a method for culturing Longjing tea tree tissue, is different from Example 10 in that in step S2, the photoperiod is 24 hours of illumination.

[0080] Example 22, a culture medium for Longjing tea tree tissue and a culture method thereof, which is different from Example 10 in that in step S2, the photoperiod is 12 hours of light and 12 hours of darkness.

[0081] Example 23, a method for culturing Longjing tea tree tissue, differs from Example 10 in that the explant is the top shoot of Longjing No. 43 grown in the current year.

[0082] Comparative Example

[0083] Comparative Example 1 is a culture medium for Longjing tea tree tissue, which differs from Example 1 in that the activated carbon is replaced by an equal amount of vermiculite powder.

[0084] Comparative Example 2 is a culture medium for Longjing tea tree tissue, which is different from Example 1 in that the auxin is replaced by an equal amount of cytokinin.

[0085] Comparative Example 3 is a culture medium for Longjing tea tree tissue, which is different from Example 1 in that cytokinin is replaced by an equal amount of auxin.

[0086] Comparative Example 4, a culture medium for Longjing tea tree tissue, is different from Example 1 in that 0.1 mg anti-browning agent, 0.5 mg auxin, 1.3 mg cytokinin, 22.5 g sucrose, 6 g agar and 2 g activated carbon are added to each liter of MS culture medium.

[0087] Comparative Example 5, a method for culturing Longjing tea tree tissue, prepared by the following steps:

[0088] S1: Explant pretreatment: 1.2 cm young stem segments of Longjing No. 43 were cut and treated with 75% ethanol for 30 seconds, and then treated with 0.15% sodium hypochlorite solution for 5 minutes to obtain pretreated explants;

[0089] S2: Tissue culture: The pretreated explants were inoculated into the culture medium of Longjing tea tree tissue. The culture conditions were a light intensity of 3500 lx, a photoperiod of 18 h light and 6 h dark, a culture temperature of 25°C, and cultured for 75 h to obtain regenerated Longjing tea tree tissue.

[0090] The culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Comparative Example 1.

[0091] Comparative Example 6, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Comparative Example 2.

[0092] Comparative Example 7, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Comparative Example 3.

[0093] Comparative Example 8, a method for culturing Longjing tea tree tissue, is different from Example 10 in that the culture medium of Longjing tea tree tissue adopts the culture medium of Longjing tea tree tissue of Comparative Example 4.

[0094] Comparative Example 9 is a method for culturing Longjing tea tree tissue, which is different from Example 10 in that the explant is directly subjected to tissue culture without the pretreatment of step S1.

[0095] Performance testing

[0096] Test 1: contamination rate test, contamination rate (%) = number of contaminated explants / total number of explants × 100%;

[0097] Test 2: Test of induction rate, induction rate (%) = number of explants producing callus / number of inoculated explants × 100%.

[0098] Test 3: browning rate test, browning rate (%) = number of browned explants / total number of explants × 100%.

[0099] The performance test results of Examples 10-23 and Comparative Examples 5-9 are shown in Table 1:

[0100] Group Pollution rate / % Induction rate / % Browning rate / % Example 10 3.92 82.64 15.39 Embodiment 11 3.56 80.44 15.97 Example 12 5.17 79.31 16.54 Example 13 4.79 92.67 5.54 Embodiment 14 5.74 93.16 4.92 Embodiment 15 3.16 92.39 6.33 Example 16 4.08 91.97 6.04 Embodiment 17 3.77 84.16 11.85 Embodiment 18 3.94 85.23 12.07 Embodiment 19 3.48 53.29 6.59 Embodiment 20 10.87 82.79 21.34 Embodiment 21 3.87 75.42 19.48 Embodiment 22 4.03 76.57 18.82 Embodiment 23 3.97 66.39 31.47 Comparative Example 5 21.3 63.25 52.39 Comparative Example 6 6.67 62.98 23.55 Comparative Example 7 7.49 59.37 30.62 Comparative Example 8 3.42 43.18 20.49 Comparative Example 9 34.66 32.58 62.37

[0101] Analysis of test results:

[0102] (1) It can be seen from the combination of Examples 1-14 and Comparative Examples 1-4 and Table 1 that the Longjing tea tree tissue culture configured in the present application, in combination with the corresponding culture method, can produce regenerated Longjing tea tree tissue with a low browning rate and a high induction rate. The reason is that the inorganic material, the anti-browning agent, the auxin and the cytokinin can synergistically improve the stress resistance of tissue cells, improve the metabolic activity of tissue cells, thereby improving the induction rate of tissue cells and reducing the generation of browning.

[0103] (2) Combining Examples 1-7, Comparative Example 1 and Table 1, it can be seen that using different inorganic materials as additives has a significant effect on the browning and induction of tissue cells. Among them, when a mixture of carbon nanotubes and activated carbon and a mixture of carbon nanotubes and nanosilver are used as additives, the regenerated Longjing tea tree tissue has a lower contamination rate, browning rate and a higher induction rate. The reason is that whether it is carbon nanotubes, activated carbon or nanosilver, adding them alone can only produce better effects in one aspect, while the combination of two can produce a synergistic effect while eliminating the adverse effects of a single component (for example, carbon nanotubes can reduce the inhibition of activated carbon on induction and make full use of the strong adsorption and large specific surface area advantages of activated carbon).

[0104] (3) In combination with Examples 1-3, Examples 8-9 and Table 1, it can be seen that when the cytokinin adopts a mixture of thidiazuron and mannitol, the browning rate of the regenerated Longjing tea tree tissue is further reduced, and the induction rate is slightly increased. The reason is that mannitol can play a role in regulating osmotic pressure. When using MS medium with a higher nutrient concentration, mannitol can reduce cell water loss and reduce cell damage. When synergistically acting with benzilcarb, the adaptability of cells to the environment is further improved, and browning is reduced. Mannitol can also promote the formation of tissue cells in coordination with indolebutyric acid and benzilcarb, and improve the induction rate of cells.

[0105] (4) In combination with Examples 1-3, Examples 12-14, Comparative Example 5 and Table 1, it can be seen that the choice of photoperiod, the choice of explant, the pretreatment of explant, and the choice of culture time will affect the browning rate, contamination rate, and induction rate of the regenerated Longjing tea tree tissue. The reason is that the explants are not sterilized and can carry a variety of viruses. Although they do not affect the plant, when tissue culture is carried out, due to the fragility of tissue cells and the poor antibacterial properties of the culture medium, it is easy to cause pollution, thereby causing browning, so it is very necessary for the explants to be disinfected in a suitable manner. In addition, since the application adds inorganic materials, it will affect the metabolic growth of tissue cells. Therefore, it is necessary to select suitable culture conditions for cultivation according to the culture medium of the application and the selected explants, so as to obtain a regenerated Longjing tea tree tissue with a low contamination rate and a high induction rate.

[0106] (5) In combination with Examples 1-3, Comparative Example 4 and Table 1, it can be seen that the culture medium of Longjing tea tree tissue improved by the present application has a significant effect on the increase of Longjing regeneration tissue induction rate, the decrease of pollution rate and browning rate. The reason is that: the activity of auxin, anti-browning agent and cytokinin is very strong, and a small amount can play a very significant role, and excessive dosage may cause irreversible damage to cell growth, thereby having a counter-effect. In addition, activated carbon has a strong inhibitory effect on the induction of tissue cells. If the amount of activated carbon, auxin, anti-browning agent and cytokinin added is not appropriate, it will affect the compatibility between them, and play a harmful effect of damaging cell induction and further causing browning.

[0107] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. An application of a culture medium of Longjing tea tree tissue in Longjing tea tree tissue culture, characterized in that: The MS culture medium is used, wherein the MS culture medium consists of an MS basal culture medium, 0.3-1.1 mg / L anti-browning agent, 1-3 mg / L auxin, 0.4-0.9 mg / L cytokinin, 15-40 g / L carbon source, 3-9 g / L agar and 0.9-1.5 g / L inorganic material; the inorganic material is a mixture of nanosilver and carbon nanotubes or a mixture of carbon nanotubes and activated carbon, the weight ratio of the nanosilver and carbon nanotubes is (0.1-0.2):(0.8-0.9), the weight ratio of the carbon nanotubes and activated carbon is (0.5-0.8):(0.2-0.5), wherein the pore diameter of the carbon nanotubes is 10-20 nm, and the particle diameter of the activated carbon is 1-30 nm; the auxin is indolebutyric acid, and the cytokinin is thidiazuron or consists of thidiazuron and mannitol.

2. The use of a culture medium for Longjing tea tree tissue according to claim 1 in Longjing tea tree tissue culture, characterized in that: The weight ratio of thidiazuron to mannitol is (0.7-0.9): (0.1-0.3).

3. A method for culturing Longjing tea tree tissue, characterized in that: The steps include: S1: Explant pretreatment: The explants were treated with 75% ethanol for 25-35 seconds, and then treated with a 0.1-0.15% sodium hypochlorite solution for 4-6 minutes to obtain the pretreated explants; S2: Tissue culture: The pretreated explant is inoculated into the culture medium of the Longjing tea tree tissue in the application described in claim 1 or 2, and the culture conditions are a light intensity of 3000-4000lx and a culture temperature of (25±2)°C to obtain regenerated Longjing tea tree tissue.

4. The method for culturing Longjing tea tree tissue according to claim 3, characterized in that: The explants are young and tender stem segments.

5. The method for culturing Longjing tea tree tissue according to claim 3, characterized in that: In step S2, the time for regenerating Longjing tea tree tissue culture is 68-82 hours.

6. The method for culturing Longjing tea tree tissue according to claim 3, characterized in that: In step S2, the photoperiod is 16-20 hours of light and 4-8 hours of darkness.