CN114568310B - Method for prolonging in-vitro survival time of tea tree germplasm resource cuttage branches - Google Patents

Method for prolonging in-vitro survival time of tea tree germplasm resource cuttage branches Download PDF

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CN114568310B
CN114568310B CN202210381541.8A CN202210381541A CN114568310B CN 114568310 B CN114568310 B CN 114568310B CN 202210381541 A CN202210381541 A CN 202210381541A CN 114568310 B CN114568310 B CN 114568310B
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adventitious bud
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culture
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CN114568310A (en
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段志芬
尚卫琼
李金龙
李慧
唐一春
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Tea Research Institute Yunnan Academy of Agricultural Sciences
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Abstract

The invention discloses a method for prolonging the in-vitro survival time of tea tree germplasm resource cuttage branches, and belongs to the technical field of in-vitro preservation of germplasm resources. The method comprises the following steps: (1) tea tree germplasm resource collection: collecting tea plant germplasm resources in the field, and performing fresh-keeping treatment; (2) preparing an explant: soaking and disinfecting the tea tree branches by adopting a disinfection solution A, a disinfection solution B and a disinfection solution C in sequence to obtain the explants; (3) adventitious bud induction culture: inoculating the explant into an adventitious bud induction culture medium, and culturing to obtain an adventitious bud; and (4) germplasm preservation and culture: inoculating the adventitious bud to a germ plasm preservation culture medium for preservation and culture, and subculturing once every 8 to 10 months. The method can reduce the subculture frequency of the adventitious bud, achieves the purpose of long-time preservation of germplasm resources (longer in vitro survival time), and lays a certain foundation for preservation and large-area popularization of excellent rare wild tea germplasm.

Description

Method for prolonging survival time of cuttage branches of tea plant germplasm resources in vitro
Technical Field
The invention relates to the technical field of in vitro preservation of germplasm resources, in particular to a method for prolonging in vitro survival time of tea tree germplasm resource cuttage branches.
Background
The tea tree is bush or small tree of Theaceae and Camellia, and has no hair on tender branch. Leathery, oblong or oval. The tea tree leaves can be used for making tea (different from tea-oil tree), the seeds can be used for extracting oil, the tea tree material is fine and dense, and the tea tree can be used for carving. In recent years, on one hand, the demand is gradually increased along with the cognition of the health care effect of tea by consumers, and the planting area of tea trees is continuously increased. On the other hand, tea plant germplasm resources are the material basis of production and utilization, variety innovation and biotechnology research, and are the potential of continuous development of the tea industry.
The germplasm resource is a national strategic resource, is a basic resource of productivity, is a material basis for species evolution, genetic research and plant breeding, is a core material of bioengineering technological innovation, and the preservation of the plant germplasm resource becomes a hot topic of global attention. After the tea tree germplasm resources are collected in different places, the utilization rate and the survival rate are lower due to the fact that branches lose water because of long transportation distance and long storage time, so that the waste of tea tree germplasm resources and economic loss are caused, and rare tea tree species cannot be popularized in a large area. How to effectively prolong the survival time of tea plant resources in vitro and effectively preserve excellent germplasm resources is a technical problem to be solved by technical personnel in the field for the continuous development of the tea industry.
Disclosure of Invention
The invention aims to provide a method for prolonging the in vitro survival time of a cuttage branch of a tea plant germplasm resource, so as to solve the problems in the prior art.
In order to achieve the purpose, the invention provides the following scheme: a method for prolonging the in-vitro survival time of tea plant germplasm resource cuttage branches comprises the following steps:
(1) Collecting tea plant germplasm resources: collecting tea plant germplasm resources, and performing fresh-keeping treatment;
(2) Preparing an explant: soaking and disinfecting the tea tree branches by adopting a disinfecting solution A, a disinfecting solution B and a disinfecting solution C in sequence to obtain explants;
the disinfection solution A is an ethanol solution; the disinfection solution B is a mixed aqueous solution of sodium hypochlorite and tween; the disinfection solution C is a mixed aqueous solution of polyvinylpyrrolidone, carbenicillin and streptomycin sulfate;
(3) Adventitious bud induction culture: inoculating the explant into an adventitious bud induction culture medium, and culturing to obtain an adventitious bud;
(4) And (3) germplasm preservation and culture: inoculating the adventitious bud to a germ plasm preservation culture medium for preservation and culture, and subculturing once every 8 to 10 months.
Further, in the step (1), the fresh-keeping treatment specifically includes: wrapping the lower ends of the branches with absorbent cotton, and spraying a preservative.
Further, the preservative comprises the following components in parts by mass: 2 parts of cane sugar, 0.5 part of 1-methylcyclopropene, 1 part of lignin, 5 parts of honeysuckle extract, 4 parts of carrot juice, 4 parts of aloe extract, 3 parts of ethanol and 1 part of diatomite.
The adopted preservative can prolong the preservation time of the tea tree branches, has strong pertinence and lasting effect, reduces the toxic action on the tea tree branches while keeping the activity of the tea tree branches, and the honeysuckle extract in the preservative not only has excellent antioxidant effect, but also can inhibit harmful microorganisms on the tea tree branches; the aloe extract has direct inhibition effect on various pathogenic bacteria, and can also reduce the loss of nutrient substances of tea tree branches.
Further, in the step (2), the volume fraction of the ethanol solution is 70 to 75 percent; the volume fraction of sodium hypochlorite in the disinfection solution B is 0.8-1.2%, and the volume fraction of tween is 0.1-0.2%; the concentration of polyvinylpyrrolidone in the disinfection solution C is 30 to 50mg/L, the concentration of carbenicillin is 20 to 60mg/L, and the concentration of streptomycin sulfate is 10 to 20mg/L.
Further, in the step (2), the tea tree branches are soaked in the disinfection solution A for 15-20 s, soaked in the disinfection solution B for 1-2min and soaked in the disinfection solution C for 10-15min.
Further, in the step (3), the formula of the adventitious bud induction medium is as follows: taking a 1/2MS culture medium as a basic culture medium, and adding 0.5-1.0 mg/L6-BA + 0.2-0.4 mg/L LNAA + 0.1-0.2mg/L ascorbic acid + 0.2-0.4 g/L active carbon + 10-15g/L mannitol + 1-2g/L corn husk extract.
The tea tree is a perennial herb, the content of phenolic substances in the tissue of the tea tree is high, when the tissue is cultured, because the explant is cut to cause a wound, the isolation between the polyphenolic substances and a substrate PPO is broken, under a proper condition, the polyphenolic substances and the substrate PPO are oxidized to generate brown quinones, and the quinone substances gradually diffuse into the whole culture medium to finally cause the browning of the explant. The corn bran extract in the adventitious bud induction culture medium is rich in substances such as ferulic acid and the like, can effectively inhibit the activity of polyphenol oxidase, further reduce the occurrence rate of oxidation browning of the explant, and can obviously reduce the occurrence rate of oxidation browning of the explant by combining with the adsorption effect of activated carbon.
Further, in the step (3), the culture conditions specifically include: culturing for 30 to 45d under the conditions that the temperature is 23 to 25 ℃, the illumination intensity is 1500 to 2000Lx and the illumination time is 12 to 14h/d.
Further, in the step (4), the formula of germplasm preservation and culture is as follows: taking a 1/2MS culture medium as a basic culture medium, and adding 0.2 to 0.5mg/L of 5-nitroguaiacol sodium, 0.2 to 0.5mg/L of cyanocoumarin, 0.1 to 0.3mg/L of paclobutrazol, 0.3 to 0.6mg/L of vitamin C, 3 to 5g of reed extract and 1 to 2g of lemon essential oil.
The reed extract and the lemon essential oil in the germ plasm preservation culture can effectively inhibit the invasion of endogenous bacteria to adventitious buds during the adventitious bud preservation period, thereby improving the preservation survival rate of the adventitious buds.
The 5-nitroguaiacol sodium has the effects of promoting root development, promoting dry matter formation and transporting to storage organs; the vitamin C can delay the growth speed of the adventitious bud;
further, in the step (4), the conditions for the preservation culture specifically include: the culture is carried out under the conditions that the temperature is 13 to 15 ℃, the illumination intensity is 600 to 800Lx, and the illumination time is 8 to 10h/d.
The invention discloses the following technical effects:
by adopting the method, the occurrence rate of explant oxidation browning in the adventitious bud induction process can be remarkably reduced, the induction rate of the adventitious bud is improved, the survival rate of the adventitious bud can reach 88.2% after being stored for 10 months, the rooting rate of the adventitious bud stored by the method can reach more than 99.7% after germplasm rejuvenation and rooting culture, the survival rate after transplanting reaches 95.1%, the number of subculture of the adventitious bud can be reduced, the purpose of long-time preservation of germplasm resources (longer in vitro survival time) is achieved, and a certain foundation is laid for the preservation and large-area popularization of excellent rare wild tea germplasm.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The specification and examples are exemplary only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
Example 1
A method for prolonging the survival time of in vitro cutting branches of tea plant germplasm resources comprises the following steps:
(1) Collecting tea plant germplasm resources: collecting young and tender branches of Pu' er tea trees with normal development and no plant diseases and insect pests in the wild, shearing off 1-2 internodes of the lower ends of the branches, wrapping the lower ends of the branches with absorbent cotton, wrapping the absorbent cotton with a preservative film, and spraying a preservative on the branches, wherein the branches are wetted; and (4) filling the treated branches into a paper box, punching 5 small ventilation holes with the diameter of 2cm on the periphery of the paper box, and sealing by using an adhesive tape.
The preservative comprises the following components: 2 parts of cane sugar, 0.5 part of 1-methyl cyclopropene, 1 part of lignin, 5 parts of honeysuckle extract, 4 parts of carrot juice, 4 parts of aloe extract, 3 parts of ethanol (absolute ethyl alcohol), 1 part of diatomite and 200 parts of water.
The preparation method of the honeysuckle extract comprises the following steps: the honeysuckle flower extract is prepared by crushing honeysuckle flower to about 50 meshes to obtain honeysuckle flower powder, then uniformly mixing the honeysuckle flower powder and water according to a mass ratio of 1.
The preparation method of the carrot juice comprises the following steps: uniformly mixing carrot and water in a mass ratio of 1.
The preparation method of the aloe extract comprises the following steps: cleaning and peeling aloe leaves, and keeping pulp parts; cutting into 2.0cm cuboid pieces, treating in 90 deg.C water bath for 45min to obtain mixed solution, filtering with 400 mesh sieve, and filtering with 0.22 μm filter membrane to obtain filtrate, i.e. Aloe extract.
(2) Preparing an explant: cutting collected tea tree branches placed for 5 days into stem segments containing 3 growing points, sequentially soaking the stem segments in a disinfection solution A for 15s, in a disinfection solution B for 1min, in a disinfection solution C for 12min, and then washing with sterile water for 3-4 times to obtain explants.
The disinfection solution A is ethanol water solution with the volume fraction of 70%.
The volume fraction of sodium hypochlorite in the disinfection solution B is 1%, the volume fraction of Tween 80 is 0.1%, and water is used as a solvent.
The concentration of polyvinylpyrrolidone in the disinfection solution C is 50mg/L, the concentration of carbenicillin is 20mg/L, the concentration of streptomycin sulfate is 20mg/L, and water is used as a solvent.
(3) Adventitious bud induction culture: inoculating the explant prepared in the step (2) into an adventitious bud induction culture medium, and then culturing for 40d under the conditions of 24 ℃ of temperature, 1500Lx of illumination intensity and 12h/d of illumination time to obtain the adventitious bud.
The preparation method of the adventitious bud induction culture medium specifically comprises the following steps: taking 1/2MS culture medium as basic culture medium, adding 0.5mg/L6-BA +0.4mg/LNAA +0.1mg/L ascorbic acid +0.3g/L active carbon +12g/L mannitol +1g/L corn bran extract, adjusting pH value to 6.0, and sterilizing at 121 deg.C for 20min.
The preparation method of the corn bran extract comprises the following steps: adding 4000mL of water into 500g of corn bran, cooking for 2.5h at 100 ℃, drying at 50 ℃ until the moisture content is 0.5wt% to obtain a dried corn bran, and mixing the dried corn bran and 70% by volume of ethanol in a mass ratio of 1:10 evenly mixing, heating and refluxing for 2 hours to obtain an extracting solution, and performing rotary evaporation on the extracting solution to remove ethanol to obtain the corn bran extract.
(4) Germplasm preservation and culture: inoculating the adventitious bud to a germ plasm preservation culture medium, and culturing at 14 deg.C under the condition of illumination intensity of 600Lx and illumination time of 8h/d, and subculturing once every 10 months.
The preparation method of the germplasm preservation culture medium comprises the following steps: taking a 1/2MS culture medium as a basic culture medium, adding 0.2mg/L of 5-nitroguaiacol sodium, 0.4mg/L of cynanchum glaucescens, 0.2mg/L of paclobutrazol, 0.3mg/L of vitamin C, 4g of reed extract and 1g of lemon essential oil, adjusting the pH value to 6.0, and then sterilizing at 121 ℃ for 20min.
The preparation method of the reed extract comprises the following steps: collecting reed leaves, shearing, uniformly mixing the reed leaves with distilled water according to a mass ratio of 1.
Example 2
(1) Collecting tea plant germplasm resources: collecting young and tender branches of Pu' er tea trees which grow normally and do not have diseases and insect pests in the field, shearing off 1-2 internode old and rough leaves at the lower ends of the branches, wrapping the lower ends of the branches with absorbent cotton, wrapping the absorbent cotton with a preservative film, and spraying a preservative on the branches until the branches are wet; and (4) filling the treated branches into a paper box, punching 5 small ventilation holes with the diameter of 2cm on the periphery of the paper box, and sealing by using an adhesive tape.
The preservative comprises the following components: 2 parts of cane sugar, 0.5 part of 1-methyl cyclopropene, 1 part of lignin, 5 parts of honeysuckle extract, 4 parts of carrot juice, 4 parts of aloe extract, 3 parts of ethanol, 1 part of diatomite and 200 parts of water.
The preparation method of the honeysuckle extract comprises the following steps: the honeysuckle flower extract is prepared by crushing honeysuckle flower to about 50 meshes to obtain honeysuckle flower powder, then uniformly mixing the honeysuckle flower powder and water according to a mass ratio of 1.
The preparation method of the carrot juice comprises the following steps: uniformly mixing carrot and water in a mass ratio of 1.
The preparation method of the aloe extract comprises the following steps: cleaning and peeling aloe leaves, and keeping pulp parts; cutting into 2.0cm cuboid pieces, treating in 90 deg.C water bath for 45min to obtain mixed solution, filtering with 400 mesh sieve, and filtering with 0.22 μm filter membrane to obtain filtrate, i.e. Aloe extract.
(2) Preparing an explant: and (3) cutting the collected tea tree branches placed for 5 days into stems containing 3 growing points, sequentially soaking the stems in the disinfection solution A for 15s, in the disinfection solution B for 1min, in the disinfection solution C for 12min, and then washing with sterile water for 3 to 4 times to obtain explants.
The disinfection solution A is ethanol water solution with 75 percent of volume fraction.
The volume fraction of sodium hypochlorite in the disinfection solution B is 0.8%, the volume fraction of Tween 80 is 0.2%, and water is used as a solvent.
The concentration of polyvinylpyrrolidone in the disinfection solution C is 30mg/L, the concentration of carbenicillin is 60mg/L, the concentration of streptomycin sulfate is 10mg/L, and water is used as a solvent.
(3) Adventitious bud induction culture: inoculating the explant prepared in the step (2) into an adventitious bud induction culture medium, and then culturing for 35d under the conditions of 24 ℃ of temperature, 2000Lx of illumination intensity and 14h/d of illumination time to obtain the adventitious bud.
The preparation method of the adventitious bud induction culture medium comprises the following specific steps: taking a 1/2MS culture medium as a basic culture medium, adding 1.0mg/L6-BA, 0.2mg/LNAA, 0.2mg/L ascorbic acid, 0.2g/L active carbon, 10g/L mannitol and 2g/L corn bran extract, adjusting the pH value to 6.0, and then sterilizing at 121 ℃ for 20min.
The preparation method of the corn bran extract comprises the following steps: adding 4000mL of water into 500g of corn husks, cooking for 2.5h at 100 ℃, drying at 50 ℃ until the moisture content is 0.5wt% to obtain dried corn husks, and mixing the dried corn husks with 70% by volume of ethanol in a mass ratio of 1:10 evenly mixing, heating and refluxing for 2 hours to obtain an extracting solution, and performing rotary evaporation on the extracting solution to remove ethanol to obtain the corn bran extract.
(4) And (3) germplasm preservation and culture: inoculating the adventitious bud to a germ plasm preservation culture medium, and culturing at 14 deg.C under illumination intensity of 800Lx for 10h/d, and subculturing every 10 months.
The preparation method of the germplasm preservation culture medium comprises the following steps: taking a 1/2MS culture medium as a basic culture medium, adding 0.5mg/L of 5-nitroguaiacol sodium, 0.2mg/L of cynanchum glaucescens, 0.3mg/L of paclobutrazol, 0.6mg/L of vitamin C, 3g of reed extract and 2g of lemon essential oil, adjusting the pH value to 6.0, and then sterilizing at 121 ℃ for 20min.
The preparation method of the reed extract comprises the following steps: collecting reed leaves, shearing, uniformly mixing the reed leaves with distilled water according to a mass ratio of 1.
Comparative example 1
The difference from the example 1 is that the step (1) is specifically as follows: collecting young and tender branches of Pu' er tea trees which grow normally and do not have diseases and insect pests in the field, shearing off 1-2 internode old and rough leaves at the lower ends of the branches, wrapping the lower ends of the branches with absorbent cotton, wrapping the absorbent cotton with a preservative film, filling the treated branches into a carton, punching 5 small ventilation holes with the diameter of 2cm on the periphery of the carton, and sealing the carton with an adhesive tape.
Comparative example 2
The difference from example 1 is that the antistaling agent in step (1) does not contain honeysuckle extract.
Comparative example 3
The difference from the example 1 is that the step (2) is specifically: and cutting the collected tea tree branches placed for 5 days into stem segments containing 3 growing points, sequentially soaking the stem segments in the disinfection solution A for 15s, soaking the stem segments in the disinfection solution C for 12min, and then washing the stem segments with sterile water for 3 to 4 times to obtain the explant.
Comparative example 4
The method is the same as the embodiment 1, except that the step (2) specifically comprises the following steps: and cutting the collected tea tree branches placed for 5 days into stem segments containing 3 growing points, sequentially soaking the stem segments in a disinfection solution A for 15s, soaking the stem segments in a disinfection solution B for 1min, and then washing the stem segments with sterile water for 3 to 4 times to obtain the explant.
Comparative example 5
The difference from example 1 is that the disinfecting solution C does not contain polyvinylpyrrolidone.
Comparative example 6
The same as example 1 except that the adventitious bud induction medium in step (3) does not contain a corn husk extract.
Comparative example 7
The same as example 1 except that the adventitious bud induction medium in step (3) does not contain activated carbon.
Comparative example 8
The method is the same as example 1 except that the germplasm preservation medium in the step (4) does not contain reed extract and lemon essential oil.
Comparative example 9
The method is the same as example 1 except that the germplasm storage medium in step (4) does not contain sodium 5-nitroguaiacol.
Effect example 1
The survival rates of the adventitious buds after the storage in the step (4) in the examples 1 to 2 and the comparative examples 1 to 9 (after the subculture for 1 time and 10 months) were counted, and the results are shown in Table 1.
TABLE 1 survival rate of adventitious bud
Figure DEST_PATH_IMAGE001
Figure DEST_PATH_IMAGE002
Effect example 2
The incidence rate of the explant oxidation browning after the culture in the step (3) in the examples 1 to 2 and the comparative examples 1 to 9 is counted, and the result is shown in the table 2.
Figure DEST_PATH_IMAGE003
Effect example 3
The induction rates of the adventitious buds cultured in the step (3) in examples 1 to 2 and comparative examples 1 to 9 were counted, and the results are shown in Table 3.
TABLE 3
Figure DEST_PATH_IMAGE004
Effect example 4
(1) Germplasm rejuvenation: after the tea tree seedlings are stored by the methods of examples 1 to 2 and comparative examples 1 to 9, the adventitious buds are transferred to a rejuvenation culture medium and cultured for 30 days under the conditions of the temperature of 25 ℃, the illumination intensity of 2000Lx and the illumination time of 12h/d to obtain the tea tree seedlings.
The preparation method of the rejuvenation culture medium comprises the following steps: MS culture medium is used as basic culture medium, 5mg of 6-BA, 0.35mg of kinetin and 0.03mg of indoleacetic acid are added, and the pH value is adjusted to be 6.0.
(2) Rooting culture: and (3) transferring the tea tree seedlings into a rooting culture medium, culturing for 25d under the conditions that the temperature is 25 ℃, the illumination intensity is 2500Lx and the illumination time is 15h/d to induce rooting, and calculating the rooting rate, wherein the result is shown in a table 4.
The preparation method of the rooting medium comprises the following steps: taking 1/2MS culture medium as basic culture medium, adding 0.8mg/L indoleacetic acid and 1.0g/L active carbon, and adjusting pH value to 5.8.
(3) Transplanting the tea tree seedlings obtained by culturing in the step (2), and counting the survival rate of the tea tree seedlings after 2 months of transplanting, wherein the results are shown in a table 4.
Figure DEST_PATH_IMAGE005
From tables 1 to 4, it can be confirmed that by adopting the methods of embodiments 1 to 2 of the present invention, the occurrence rate of the oxidation browning of the explant in the adventitious bud induction process can be significantly reduced, the induction rate of the adventitious bud can be improved, and after the adventitious bud is stored for 10 months, the survival rate of the adventitious bud can be maintained within 86.5 to 88.2%, and the rooting rate of the adventitious bud stored by the method of the present invention after the germplasm rejuvenation and rooting culture can be more than 99.7%, the survival rate after transplantation can be up to 95.1%, the number of subcultures of the adventitious bud can be reduced, the purpose of long-term storage of rare resources (longer in vitro survival time) can be achieved, and a certain foundation can be laid for the storage and large-area popularization of excellent wild tea plant germplasm.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.

Claims (1)

1. A method for prolonging the survival time of in vitro cutting branches of tea plant germplasm resources is characterized by comprising the following steps:
(1) Collecting tea plant germplasm resources: collecting tea plant germplasm resources, and performing fresh-keeping treatment;
(2) Preparing an explant: soaking and disinfecting the tea tree branches by adopting a disinfecting solution A, a disinfecting solution B and a disinfecting solution C in sequence to obtain explants;
the disinfection solution A is an ethanol solution; the disinfection solution B is a mixed aqueous solution of sodium hypochlorite and tween; the disinfection solution C is a mixed aqueous solution of polyvinylpyrrolidone, carbenicillin and streptomycin sulfate;
(3) Adventitious bud induction culture: inoculating the explant into an adventitious bud induction culture medium, and culturing to obtain an adventitious bud;
(4) And (3) germplasm preservation and culture: inoculating the adventitious bud to a germ plasm preservation culture medium for preservation and culture, and subculturing once every 10 months;
in the step (1), the fresh-keeping treatment specifically comprises: wrapping the lower ends of the branches with absorbent cotton, and spraying a preservative;
the preservative is prepared from the following components in parts by mass: 2 parts of cane sugar, 0.5 part of 1-methyl cyclopropene, 1 part of lignin, 5 parts of honeysuckle extract, 4 parts of carrot juice, 4 parts of aloe extract, 3 parts of absolute ethyl alcohol, 1 part of diatomite and 200 parts of water;
in the step (2), the volume fraction of the ethanol solution is 70%; the volume fraction of sodium hypochlorite in the disinfection solution B is 1%, and the volume fraction of tween is 0.1%; the concentration of polyvinylpyrrolidone in the disinfection solution C is 50mg/L, the concentration of carbenicillin is 20mg/L, and the concentration of streptomycin sulfate is 20mg/L;
in the step (2), the tea tree branches are soaked in the disinfection solution A for 15s, soaked in the disinfection solution B for 1min and soaked in the disinfection solution C for 12min;
in the step (3), the formula of the adventitious bud induction culture medium is as follows: taking a 1/2MS culture medium as a basic culture medium, and adding 0.5mg/L6-BA +0.4mg/LNAA +0.1mg/L ascorbic acid +0.3g/L active carbon +12g/L mannitol +1g/L corn bran extract;
the preparation method of the corn bran extract comprises the following steps: adding 4000mL of water into 500g of corn husks, cooking for 2.5h at 100 ℃, drying at 50 ℃ until the moisture content is 0.5wt% to obtain dried corn husks, and mixing the dried corn husks with 70% by volume of ethanol in a mass ratio of 1:10 evenly mixing, heating, refluxing and extracting for 2 hours to obtain an extracting solution, and performing rotary evaporation on the extracting solution to remove ethanol to obtain a corn bran extract;
in the step (3), the culture conditions specifically include: culturing at 24 deg.C under illumination intensity of 1500Lx for 40d under illumination time of 12 h/d;
in the step (4), the formula of germplasm preservation and culture is as follows: taking a 1/2MS culture medium as a basic culture medium, and adding 0.2mg/L of 5-nitroguaiacol sodium, 0.4mg/L of cynanchum otophyllum, 0.2mg/L of paclobutrazol, 0.3mg/L of vitamin C, 4g of reed extract and 1g of lemon essential oil;
in the step (4), the conditions for preservation and culture specifically include: storing and culturing at 14 deg.C under illumination intensity of 600Lx for 8 h/d;
the tea plant germplasm resource is Pu' er wild big tea trees.
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