A method for tissue culture regeneration of camellia sinensis cv. four ball tea

Abstract

The application discloses a four-ball tea tissue culture regeneration method, which comprises the following steps: firstly, treating the to-be-collected branch with Australian tea tree essential oil, and then sleeving the to-be-collected branch with a bag; secondly, collecting the to-be-collected branch, shearing, disinfecting and drying to obtain a stem section with an axillary bud; thirdly, preparing specific culture medium and specific culture conditions according to the culture requirements of each culture stage to carry out the tissue culture process of initial culture-axillary bud proliferation culture-small cluster bud culture-small cluster bud rooting to obtain four-ball tea tissue culture seedlings; and fourthly, carrying out sealing seedling raising and transplanting on the four-ball tea tissue culture seedlings to obtain high-quality four-ball tea seedlings. Compared with the prior art, the four-ball tea tissue culture seedlings are treated with Australian tea tree essential oil in combination with physical isolation to reduce the pollution rate and mortality rate of the initial culture of the four-ball tea explants, the sterile seedlings are obtained by combining specific disinfection methods and culture medium, the sterile seedling obtaining rate reaches 88.33%, the axillary bud proliferation is prepared by using specific culture medium and culture conditions, the axillary bud proliferation coefficient reaches 3.5, the induction rate reaches 90.00%, the small cluster bud proliferation coefficient is 4.5, the induction rate is 90.00%, and the rooting rate is 85.00%.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for the regeneration of *Tea spp.* through tissue culture. Background Technology

[0002] Camellia tetracocca Zhang is a species of tea belonging to the genus Camellia, subgenus Thea, section Thea, fifth cellar, Ser. Quiquelocularis. It is a rare ancient tea tree that grows in the dense forests of the mountains at an altitude of 1700-1950 meters in Puan County, Guizhou Province. Discovered and named "Four-Ball Tea" by Professor Zhang Hongda in 1981, it is a unique tea variety of Puan County, Guizhou Province. The "Golden Boat" black tea series, developed by the people of Puan using the tender shoots and leaves of Camellia tetracocca Zhang, not only boasts a golden-yellow liquor, mellow taste, fragrant aroma, and sweet aftertaste, but also is rich in various nutrients. If mass production is achieved, it will be highly popular with consumers.

[0003] Puan County boasts nearly 500 ancient tea trees and is one of the important origins of tea culture. Research and application of the unique Siqiu tea tree resources in Puan, Guizhou, can create distinctive tea products that represent the region's unique character, increase local income, and have significant implications for local economic and cultural development.

[0004] Tea semperflorens is a small tree. Currently, its germplasm resources are scarce, and it suffers from problems such as a long growth cycle and low natural and hybrid fruiting rates, hindering the industrialization of related tea products. Tissue culture propagation can quickly and efficiently preserve woody genetic characteristics to obtain a large number of reverting seedlings, which is one of the effective measures to solve the problems of long growth cycle and low natural and hybrid fruiting rates in tea semperflorens. Therefore, there is an urgent need to develop tissue culture technology for tea semperflorens to achieve the industrialization of tissue culture, seedling cultivation, research and development, and production. However, to date, no research or reports have been found on the in vitro propagation of plant organs related to tea semperflorens. Furthermore, the inventors, while studying the tissue culture of this species, discovered the following problems:

[0005] (1) Excellent ancient tea tree resources that are healthy, free from pests and diseases and grow vigorously are extremely scarce. The explants are very limited. The explant collection period affects the explant contamination and browning. The best collection period is in spring and summer. However, there is more rain in spring and summer, and wild plants carry a relatively large number and variety of pathogens. After collection and inoculation, they are easily contaminated and it is difficult to obtain sterile seedlings.

[0006] (2) During tissue culture, problems such as browning, vitrification, low budding rate, low propagation coefficient, slow growth rate, and difficulty in rooting are very likely to occur.

[0007] To solve the problem mentioned above (1), the inventors used tea tree branches with axillary buds in May and June as explants. One month in advance, the explant branches to be used were sprayed with tea tree essential oil and bagged. This reduced the probability of bacteria in the explants without harming the growth of axillary buds. Thiourea was added to the initial culture to resist browning and inhibit bacteria, thus reducing the pollution, browning and death of the explants. To solve the problem mentioned above (2), specific culture media and culture conditions were used to obtain tea tree tissue culture seedlings by initial culture - induction of axillary bud proliferation - induction and rejuvenation of small bud clusters - rooting of small bud clusters. Through tissue culture, hardening and post-transplantation management, a large number of tea tree seedlings were obtained in a short period of time. Summary of the Invention

[0008] The purpose of this invention is to provide a method for tissue culture regeneration of *Tetracentron sinense*, which aims to reduce explant contamination, browning, and mortality, increase induced budding rate and propagation coefficient, and rapidly root and harden seedlings to achieve a large-scale production of *Tetracentron sinense* seedlings in a short period of time.

[0009] To achieve the above objectives, the present invention provides a method for tissue culture regeneration of *Tea spp.*, the method comprising:

[0010] S1, Explant treatment: First, treat the branches to be collected with Australian tea tree essential oil and cover the branches with a bag. Then, collect the branches, cut, disinfect and dry them to obtain stem segments with axillary buds.

[0011] S2, Initial culture: Take the stem segment with axillary buds and inoculate it into the initial culture medium for initial culture to obtain axillary buds. The initial culture medium includes MS and thiourea.

[0012] S3, Axillary bud proliferation culture: Take the stem segment with axillary buds from step S2 and inoculate it into the induction medium for culture;

[0013] S4, Small bud culture: Cut off the axillary buds from step S3, inoculate the axillary buds into the induction medium for induction culture to obtain bud clusters, divide the bud clusters into small bud clusters with 3-6 adventitious buds, inoculate the small bud clusters into the rejuvenation medium for culture to obtain robust small bud clusters;

[0014] S5, Rooting of small bud clusters: The robust small bud clusters are inoculated into a rooting medium for culture, the rooting medium comprising 1 / 2MS, amino acid ester, and sodium dodecylbenzene sulfonate.

[0015] S6, sealing and hardening off seedlings and transplanting.

[0016] According to the above technical solution, the inoculation material for axillary bud proliferation culture in this invention is the stem segment with axillary buds in the initial culture stage, which realizes axillary bud proliferation while avoiding the formation of callus tissue at the inoculation site, thereby improving the axillary bud proliferation efficiency and shortening the proliferation time.

[0017] Preferably, in the above technical solution, the induction culture medium is MS + 80-100 mg / L Ca(NO3)2 + 6-8 mg / L BA + 3-4 mg / L IAA mg / L + 0.01-0.05 mg / L triiodobenzoic acid + 0.1-1 mg / L triacontanol.

[0018] Preferably, in the above technical solution, in step S4, the induction culture specifically involves culturing for 7-10 days at a temperature of 26-30℃ and a light intensity of 800-1000 Lx, and then culturing under a light intensity of 3000-6000 Lx and a light duration of 8-12 h.

[0019] Preferably, in the above technical solution, in the step of inoculating the small bud clusters into a rejuvenation medium for cultivation, the rejuvenation medium is MS + 0.6-0.8 mg / L 6-BA + 0.04-0.08 mg / L 1BA.

[0020] Preferably, in the above technical solution, the robust small shoots are inoculated into a rooting medium for cultivation, wherein the rooting medium is 1 / 2MS + 40-50 mg / L Ca(NO3)2 + 1-1.5 mg / L IAA + 1-1.5 mg / L IBA + 0.3-0.5 mg / L aminoethyl ester + 0.02-0.05 mg / L sodium dodecylbenzenesulfonate.

[0021] According to the above technical solution, by adding amino acid ester and sodium dodecylbenzene sulfonate, and adjusting the concentration ratio of Ca(NO3)2, IAA, IBA, amino acid ester and sodium dodecylbenzene sulfonate, the rapid rooting of the small buds of *Tetracentron sinense* can be induced, and the root elongation of the small buds of *Tetracentron sinense* can be promoted.

[0022] Preferably, in the above technical solution, in steps S3, S4 and S5, the cultivation is specifically carried out under the conditions of light intensity of 3000-6000 Lx and light duration of 8-12 h.

[0023] Preferably, in the above technical solution, in the step of inoculating into the initial culture medium for initial culture, the initial culture medium is MS + 6-8 mg / L BA + 3-4 mg / L IAA + 0.01-0.05 mg / L thiourea.

[0024] Preferably, in the above technical solution, the step of inoculating into the initial culture medium for initial culture specifically involves culturing for 5-10 days at a temperature of 26-30℃ and a light intensity of 300-500 Lx, followed by culturing under a light intensity of 2000-3000 Lx and a light duration of 8-12 h.

[0025] Preferably, in the above technical solution, the steps of explant treatment are as follows:

[0026] One to one and a half months before explant collection, Australian tea tree essential oil was sprayed onto the stem segments, leaves, and axillary buds of the branches to be collected. The branches to be collected were then covered with bags. The branches to be collected were collected in May and June. The branches to be collected were cut into stem segments with 1 to 2 axillary buds, disinfected, and dried to obtain stem segments with axillary buds.

[0027] Preferably, in the above technical solution, the disinfection and drying steps are as follows:

[0028] Soak the stem segments with axillary buds in 75% alcohol for 10-30 seconds with stirring, then rinse with sterile water; then soak in 0.1% mercuric chloride solution for 6-12 minutes, stirring every 2-3 minutes during soaking, rinse with sterile water, blot dry the surface moisture of the stem segments with axillary buds with sterile filter paper, and then cut off 0.3-0.5 cm from both ends of the stem segments with axillary buds to obtain stem segments with 1-2 axillary buds.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] (1) One method involves treating the plants with Australian tea tree oil combined with physical isolation using plastic bags. One month before harvesting, the plants are sprayed with Australian tea tree oil and then covered with plastic bags to reduce the contamination and mortality rates during initial cultivation. The second method uses mercuric chloride for disinfection. 2+ It binds to negatively charged proteins, causing bacterial protein denaturation and enzyme inactivation. Adding thiourea to the initial culture medium has certain bactericidal and anti-browning effects, thereby reducing explant contamination rate and browning mortality.

[0031] (2) Using specific culture media and culture conditions, clustered four-ball tea seedlings were quickly obtained by inducing axillary bud proliferation, inducing and rejuvenating small bud clusters, rooting small bud clusters, sealing and hardening seedlings, and then transplanting. Attached Figure Description

[0032] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings.

[0033] Figure 1 This is a diagram showing the treatment of *Tea japonica* branches before explant collection in a specific embodiment of the present invention;

[0034] Figure 2 This is a diagram showing the treatment of *Tea japonica* branches after explant collection in a specific embodiment of the present invention;

[0035] Figure 3 This is a diagram of the initial culture stage of *Tea spores* tissue culture in a specific embodiment;

[0036] Figure 4 This is a diagram of the axillary bud proliferation culture stage of *Tea spp.* in a specific embodiment;

[0037] Figure 5 This is a diagram illustrating the induction of clustered buds during the small bud culture stage of *Tea spp.* in a specific embodiment.

[0038] Figure 6 This is a diagram illustrating the rejuvenation of *Tea florida* tissue culture during the small bud culture stage in a specific embodiment.

[0039] Figure 7 This is a diagram of the rejuvenation of small buds in the small bud culture stage of *Tea spp.* in a specific embodiment;

[0040] Figure 8 This is a diagram of tissue culture of *Tea florida* in a specific embodiment;

[0041] Figure 9 This is a diagram of the *Tea florida* tissue culture at the rooting stage of small buds in a specific embodiment;

[0042] Figure 10 This is a diagram of the *Tea spp.* tissue culture at the rooting stage of small buds in a specific embodiment. Detailed Implementation

[0043] The technical solutions in the embodiments of the present invention have been clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] Example 1

[0045] A method for tissue culture regeneration of *Tea spp.*, the method comprising:

[0046] S1, Explant treatment:

[0047] Select healthy, pest-free, and robust wild tea tree species. One month before harvesting, spray the stems, leaves, and axillary buds of the branches to be harvested with 15% Australian tea tree essential oil. Immediately cover the branches to be harvested with plastic bags to combine the physical isolation of Australian tea tree essential oil and plastic bags, thereby reducing the contamination rate and mortality rate of the initial cultivation.

[0048] Every year in May and June, explants are collected between 10:00 and 12:00 on days with more than 3 consecutive sunny days, while the explants are still in plastic bags. The explants are healthy branches that have been bagged and are ready for collection. The base of the collected branches is wrapped with a clean, damp towel and placed in a wide-mouthed cup with an ice pack before being transported to the laboratory.

[0049] Remove the leaves and old stem segments from the branches, rinse off surface dust with tap water, and cut stem segments 2.5-3.0 cm long with 1-2 axillary buds. Wash the segments 2-3 times with distilled or purified water and place them on a clean bench. Soak the stem segments in 75% alcohol for 15 seconds and stir. Rinse with sterile water 3-4 times. Then soak the explants in 0.1% mercuric chloride solution for 8 minutes, stirring with a glass rod every 2-3 minutes during the soaking period. Rinse with sterile water 3-4 times.

[0050] Use sterile filter paper to absorb the surface moisture of the sterilized stem segments, and cut off any remaining leaves and 0.5cm from the top and bottom of the stem segments. Cut the stem segments into 1.5-2.5cm long sections with 1-2 axillary buds for later use.

[0051] S2, Initial Culture:

[0052] The initial culture medium was prepared by MS + 8 mg / L BA + 4 mg / L IIA + 0.05 mg / L thiourea + 3% sucrose + 5.5 g / L agar powder, and sterilized by autoclaving and cooling.

[0053] Take stem segments from 1-2 axillary buds and inoculate them into the above-mentioned initial culture medium. Culture them for 6 days at a temperature of 26-30℃ and a light intensity of 300 Lx. Then, place them under a light intensity of 2500 Lx and a light duration of 12 h for initial culture until the axillary buds grow to 2 cm.

[0054] S3, axillary bud proliferation culture:

[0055] Preparation of induction medium: MS + 100 mg / L Ca(NO3)2 + 8 mg / L BA + 4 mg / L IAA

[0056] mg / L + 0.05 mg / L triiodobenzoic acid + 0.2 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder;

[0057] The axillary buds obtained in step S2, together with the initial stem segments, were inoculated onto the above-mentioned induction medium and cultured under a light intensity of 5000 Lx and a light duration of 10 h until a large number of axillary buds proliferated at the axillary bud site.

[0058] S4, Small Bud Cultivation:

[0059] Preparation of induction medium: MS + 100 mg / L Ca(NO3)2 + 6 mg / L BA + 4 mg / L IAA + 0.05 mg / L triiodobenzoic acid + 0.5 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder;

[0060] Cut off the axillary buds from step S3, inoculate the axillary buds shown in the above induction medium, and culture them for 7 days at a temperature of 26-30℃ and a light intensity of 800 Lx. Then, culture them under a light intensity of 5000 Lx and a light duration of 10 h until a large number of adventitious buds are induced at the base of the inoculated axillary buds, thus obtaining cluster buds.

[0061] Prepare the rejuvenation medium: MS medium + 0.8 mg / L 6-BA + 0.05 mg / L IBA + 3% sucrose + 4.5 g / L agar powder;

[0062] The bud clusters were divided into smaller bud clusters with 3-6 adventitious buds each. The smaller bud clusters were inoculated into the above-mentioned rejuvenation medium and cultured under the conditions of light intensity of 5000 Lx and light duration of 10 h until some of the smaller bud clusters reached a height of 2.0-3.5 cm and grew vigorously.

[0063] S5, rooting of small buds:

[0064] Prepare rooting medium: 1 / 2 MS + 50 mg / L Ca(NO3)2 + 1.5 mg / L IAA + 1.5 mg / L IBA + 0.5 mg / L aminoethyl ester + 0.05 mg / L sodium dodecylbenzenesulfonate + 1.5% sucrose + 5.5 g / L agar powder;

[0065] The small buds with a height of 2.0-3.5cm and vigorous growth were inoculated into the rooting medium and cultured under the conditions of light intensity of 5000Lx and light duration of 10h.

[0066] S6, Sealing and Hardening: When the roots of the tissue culture seedlings are 4-5cm long, transfer the rooted bottle seedlings to a seedling shed with a shading rate of 75-80% for 30 days, and then transplant them into non-woven fabric seedling bags (9cm×3cm or 10cm×12cm). The seedling substrate is a lightweight substrate prepared by mixing coconut coir, perlite and peat moss in a 2:1:2 ratio. Fill the non-woven seedling bags with the substrate and carefully remove the *Tea japonica* tissue culture seedlings from the bottles. After washing off the culture medium, transfer them into the non-woven seedling bags. For the first 7 days after transplanting, maintain a shading rate of 75-80% and a substrate humidity of 80-90%. After 7 days, maintain a shading rate of 50-75%. After 21 days, maintain a shading rate of 20-40%. Spray water once a day for 30 minutes each time. After the tea seedlings have grown new leaves, spray the leaves with a 0.1-0.3% compound fertilizer (N:P2O5:K2O=15:15:15) aqueous solution, and fertilize once every 15 days.

[0067] Table 1 shows the mortality and contamination status of the initial culture in Example 1, axillary bud proliferation, proliferation coefficient of cluster buds, average bud height of small cluster buds, rooting time, average root length and rooting rate 14 days after rooting.

[0068] Table 1 Project Indicators for Each Stage of Example 1

[0069]

[0070]

[0071] Example 2

[0072] A method for tissue culture regeneration of *Tea spp.*, the method comprising:

[0073] S1, Explant treatment: Basically the same as in Example 1, the difference being that the disinfection method is slightly different:

[0074] Remove the leaves and old stem segments from the branches, rinse off surface dust with tap water, and cut stem segments 2.5-3.0 cm long with 1-2 axillary buds. Wash the stem segments 2-3 times with distilled or purified water and place them on a clean bench. Soak the stem segments in 75% alcohol for 30 seconds and stir. Rinse with sterile water 3-4 times. Then soak the explants in 0.3% mercuric chloride solution for 12 minutes, stirring with a glass rod every 2-3 minutes during the soaking period. Rinse with sterile water 3-4 times.

[0075] S2, Initial Culture:

[0076] The initial culture medium was prepared by MS + 6 mg / L BA + 3 mg / L IIA + 0.03 mg / L thiourea + 3% sucrose + 5.5 g / L agar powder, and sterilized by autoclaving and cooling.

[0077] Take stem segments from 1-2 axillary buds and inoculate them into the above-mentioned initial culture medium. Culture them for 5 days at a temperature of 26-30℃ and a light intensity of 500 Lx. Then, place them under a light intensity of 3000 Lx and a light duration of 8 hours for initial culture until the axillary buds grow to 2 cm.

[0078] S3, axillary bud proliferation culture:

[0079] Preparation of induction medium: MS + 100 mg / L Ca(NO3)2 + 8 mg / L BA + 4 mg / L IAA

[0080] mg / L + 0.03 mg / L triiodobenzoic acid + 0.3 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder;

[0081] The axillary buds obtained in step S2, together with the initial stem segments, were inoculated onto the above-mentioned induction medium and cultured under the conditions of light intensity of 4000 Lx and light duration of 12 h until a large number of axillary buds proliferated at the axillary bud site.

[0082] S4, Small Bud Cultivation:

[0083] Preparation of induction medium: MS + 100 mg / L Ca(NO3)2 + 6 mg / L BA + 4 mg / L IAA

[0084] mg / L + 0.03 mg / L triiodobenzoic acid + 0.8 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder;

[0085] Cut off the axillary buds from step S3, inoculate the axillary buds shown in the above induction medium, and culture them for 7 days at a temperature of 26-30℃ and a light intensity of 1000 Lx. Then, culture them under a light intensity of 4000 Lx and a light duration of 12 h until a large number of adventitious buds are induced at the base of the inoculated axillary buds, thus obtaining a cluster of buds.

[0086] Prepare the rejuvenation medium: MS medium + 0.8 mg / L 6-BA + 0.04 mg / L IBA + 3% sucrose + 4.5 g / L agar powder;

[0087] The bud clusters were divided into smaller bud clusters with 3-6 adventitious buds each. The smaller bud clusters were inoculated into the above-mentioned rejuvenation medium and cultured under the conditions of light intensity of 4000 Lx and light duration of 12 h until some of the smaller bud clusters reached a height of 2.0-3.5 cm and grew vigorously.

[0088] S5, rooting of small buds:

[0089] Prepare rooting medium: 1 / 2 MS + 50 mg / L Ca(NO3)2 + 1.5 mg / L IAA + 1.5 mg / L IBA + 0.3 mg / L aminoethyl ester + 0.02 mg / L sodium dodecylbenzenesulfonate + 1.5% sucrose + 5.5 g / L agar powder;

[0090] The healthy buds with a height of 2.0-3.5 cm were inoculated into rooting medium and cultured under the conditions of light intensity of 4000 Lx and light duration of 12 h.

[0091] S6, Sealing and hardening off the seedlings: Same as in Example 1.

[0092] Example 3

[0093] A method for tissue culture regeneration of *Tea spp.*, the method comprising:

[0094] S1, Explant treatment: Basically the same as in Example 1, the difference being that the disinfection method is slightly different:

[0095] Remove the leaves and old stem segments from the branches, rinse off surface dust with tap water, and cut stem segments 2.5-3.0 cm long with 1-2 axillary buds. Wash the segments 2-3 times with distilled or purified water and place them on a clean bench. Soak the stem segments in 75% alcohol for 30 seconds and stir. Rinse with sterile water 3-4 times. Then soak the explants in 0.1% mercuric chloride solution for 12 minutes, stirring with a glass rod every 2-3 minutes during the soaking period. Rinse with sterile water 3-4 times.

[0096] S2, Initial Culture:

[0097] The initial culture medium was prepared by MS + 8 mg / L BA + 4 mg / L IIA + 0.01 mg / L thiourea + 3% sucrose + 5.5 g / L agar powder, and sterilized by autoclaving and cooling.

[0098] Take stem segments from 1-2 axillary buds and inoculate them into the above-mentioned initial culture medium. Culture them for 10 days at a temperature of 26-30℃ and a light intensity of 300 Lx. Then, place them under a light intensity of 2000 Lx and a light duration of 12 h for initial culture until the axillary buds grow to 2 cm.

[0099] S3, axillary bud proliferation culture:

[0100] Preparation of induction medium: MS + 90 mg / L Ca(NO3)2 + 6 mg / L BA + 3 mg / L IAA

[0101] mg / L + 0.05 mg / L triiodobenzoic acid + 0.2 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder;

[0102] The axillary buds obtained in step S2, together with the initial stem segments, were inoculated onto the above-mentioned induction medium and cultured under the conditions of light intensity of 6000 Lx and light duration of 8 h until a large number of axillary buds proliferated at the axillary bud site.

[0103] S4, Small Bud Cultivation:

[0104] Preparation of induction medium: MS + 90 mg / L Ca(NO3)2 + 6 mg / L BA + 4 mg / L IAA

[0105] mg / L + 0.05 mg / L triiodobenzoic acid + 0.5 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder;

[0106] Cut off the axillary buds from step S3, inoculate the axillary buds shown in the above induction medium, and culture them for 7 days at a temperature of 26-30℃ and a light intensity of 800 Lx. Then, culture them under a light intensity of 6000 Lx and a light duration of 8 hours until a large number of adventitious buds are induced at the base of the inoculated axillary buds, thus obtaining cluster buds.

[0107] Prepare the rejuvenation medium: MS medium + 0.6 mg / L 6-BA + 0.04 mg / L IBA + 3% sucrose + 4.5 g / L agar powder;

[0108] The bud clusters were divided into smaller bud clusters with 3-6 adventitious buds each. The smaller bud clusters were inoculated into the above-mentioned rejuvenation medium and cultured under the conditions of light intensity of 6000 Lx and light duration of 8 h until some of the smaller bud clusters reached a height of 2.0-3.5 cm and grew vigorously.

[0109] S5, rooting of small buds:

[0110] Preparation of rooting medium: 1 / 2 MS + 40 mg / L Ca(NO3)2 + 1 mg / L IAA + 1 mg / L IBA

[0111] +0.5 mg / L aminoethyl ester + 0.05 mg / L sodium dodecylbenzene sulfonate + 1.5% sucrose + 5.5 g / L agar powder;

[0112] The healthy buds with a height of 2.0-3.5 cm were inoculated into rooting medium and cultured under the conditions of light intensity of 6000 Lx and light duration of 8 h.

[0113] S6, Sealing and hardening off the seedlings: Same as in Example 1.

[0114] Comparative Example 1

[0115] A method for treating *Tea spp.* explants, the method being as follows:

[0116] Select healthy, pest-free, and robust wild tea tree varieties of the Siqiu variety. One month before harvesting, spray the stems, leaves, and axillary buds of the branches to be harvested with 75% alcohol, and immediately cover the branches to be harvested with plastic bags.

[0117] Every year in May and June, explants are collected between 10:00 and 12:00 on days with more than 3 consecutive sunny days, while the explants are still in plastic bags. The explants are healthy branches that have been bagged and are ready for collection. The base of the collected branches is wrapped with a clean, damp towel and placed in a wide-mouthed cup with an ice pack before being transported to the laboratory.

[0118] Remove the leaves and old stem segments from the branches, rinse off the surface dust with tap water, and cut the stem segments to 2.5-3.0 cm in length with 1-2 axillary buds. Wash the stem segments 2-3 times with distilled or purified water and place them on a clean bench. Soak the stem segments in 75% alcohol for 15 seconds and rinse them with sterile water 3-4 times. Then soak the explants in 0.1% mercuric chloride solution for 8 minutes, stirring with a glass rod every 2-3 minutes during the soaking period, and rinse them with sterile water 3-4 times.

[0119] After sterilizing, absorb the surface moisture of the stem segments with sterile filter paper, and cut off any remaining leaves and 0.5cm from the top and bottom of the stem segments. Cut the stem segments into 1.5-2.5cm long segments with 1-2 axillary buds. Inoculate the stem segments into MS medium containing 8 mg / L BA, 4 mg / L IAA, and 0.05 mg / L thiourea. Culture the segments at 26-30℃ and 300 Lx light intensity for 6 days, and then culture them at 2500 Lx light intensity for 12 hours.

[0120] Comparative Example 2

[0121] A method for treating *Tea spp.* explants, the method being as follows:

[0122] Select healthy, disease-free, and robust wild tea tree species. Every year from May to June, collect explants by keeping them in plastic bags between 10:00 and 12:00 after three consecutive sunny days. The explants are the robust branches to be collected after being bagged. Wrap the base of the collected branches with a clean, damp towel and place them in a wide-mouthed cup with an ice pack for transport to the laboratory.

[0123] Remove the leaves and old stem segments from the branches, rinse off surface dust with tap water, and cut stem segments 2.5-3.0 cm long with 1-2 axillary buds. Wash the segments 2-3 times with distilled or purified water and place them on a clean bench. Soak the stem segments in 75% alcohol for 15 seconds and stir. Rinse with sterile water 3-4 times. Then soak the explants in 0.1% sodium hypochlorite solution for 8 minutes, stirring with a glass rod every 2-3 minutes during the soaking period. Rinse with sterile water 3-4 times.

[0124] After sterilizing, absorb the surface moisture of the stem segments with sterile filter paper, and cut off any remaining leaves and 0.5cm from the top and bottom of the stem segments, cutting them into 1.5-2.5cm long stem segments with 1-2 axillary buds. Inoculate the stem segments into MS medium + 8mg / L BA + 4mg / L IAA, and culture them for 6 days at a temperature of 26-30℃ and a light intensity of 300Lx, and then culture them under a light intensity of 2500Lx and a light duration of 12h.

[0125] Comparative Example 3

[0126] A method for treating *Tea spp.* explants, the method being as follows:

[0127] Select healthy, pest-free, and robust wild tea tree species. One month before harvesting, spray the stems and leaves of the branches to be harvested with 15% Australian tea tree essential oil. Immediately cover the branches to be harvested with plastic bags to combine Australian tea tree essential oil with physical isolation, thereby reducing the contamination rate and mortality rate of the initial cultivation.

[0128] Every year in May and June, explants are collected between 10:00 and 12:00 after three consecutive sunny days. The explants are healthy branches that have been bagged and are ready for collection. The base of the collected branches is wrapped with a clean, damp towel and placed in a wide-mouthed cup with an ice pack before being transported to the laboratory.

[0129] Remove the leaves and old stem segments from the branches, rinse off surface dust with tap water, and cut stem segments 2.5-3.0 cm long with 1-2 axillary buds. Wash the segments 2-3 times with distilled or purified water and place them on a clean bench. Soak the stem segments in 75% alcohol for 15 seconds and rinse with sterile water 3-4 times. Then soak the explants in 0.1% mercuric chloride solution for 8 minutes, stirring with a glass rod every 2-3 minutes during the soaking period, and rinse with sterile water 3-4 times.

[0130] After sterilizing, absorb the surface moisture of the stem segments with sterile filter paper, and cut off any remaining leaves and 0.5cm from the top and bottom of the stem segments. Cut the stem segments into 1.5-2.5cm long segments with 1-2 axillary buds. Inoculate the stem segments into MS medium with 8mg / L BA and 4mg / L IAA, and culture them for 6 days at a temperature of 26-30℃ and a light intensity of 300Lx. Then, culture them under a light intensity of 2500Lx and a light duration of 12h.

[0131] The explants of *Tetracentron sinense* were treated according to the methods of Comparative Examples 1, 2, and 3. During the culture process, the mortality and contamination of the explants were monitored. The mortality rate, contamination rate, and sterile seedling acquisition rate were calculated and compared with those of Example 1 (initial culture stage). The results are shown in Table 2 below. The formulas for contamination rate, browning rate, and sterile seedling acquisition rate are as follows:

[0132] Contamination rate (%) = (Number of contaminated strains / Total number of inoculated strains) × 100%;

[0133] Mortality rate (%) = (Number of browned and dead plants / Total number of inoculated plants) × 100%;

[0134] Sterile vaccine acquisition rate (%) = (uncontaminated and non-browned / total number of inoculated vaccines) × 100%.

[0135] Table 2. Effects of different explant treatments on browning rate, contamination rate, and sterile seedling acquisition rate.

[0136] Group mortality rate(%) Pollution rate (%) Sterile vaccine acquisition rate (%) Example 1 5.00(3 / 60) 6.67(4 / 60) 88.33(53 / 60) Comparative Example 1 10.00(6 / 60) 16.67(10 / 60) 73.33(44 / 60) Comparative Example 2 18.33(11 / 60) 35.00(21 / 60) 46.67(28 / 60) Comparative Example 3 15.00(9 / 60) 26.67(16 / 60) 58.33(35 / 60)

[0137] The results showed that aseptic seedlings obtained by bagging branches treated with Australian tea tree essential oil before collection were significantly better than those obtained by treating branches with alcohol. Further investigation revealed that mercuric chloride was effective in disinfecting the explants of *Tea japonica*, possibly due to the presence of Hg. 2+It can bind to negatively charged proteins, causing bacterial protein denaturation and enzyme inactivation; spraying the stems and leaves of the branches to be collected with 15% Australian tea tree essential oil and bagging them before collection can effectively reduce the contamination rate in the initial culture stage and improve the rate of obtaining sterile seedlings; adding 0.05 mg / L thiourea to the culture medium has certain anti-browning and antibacterial effects. Its anti-browning effect may be that thiourea inhibits catalase activity, reduces its oxidation of phenolic substances, and thus reduces browning.

[0138] Comparative Example 4

[0139] Investigating the effects of explant collection time on fungal abundance and bud regeneration capacity.

[0140] The explants were collected in three phases: March-April, May-June, and July-August. The explant treatment method was the same as in Example 1. The results are shown in Table 3.

[0141] Table 3. Effects of explant collection time on mortality, contamination rate, and sterile seedling acquisition rate.

[0142] mortality rate Pollution rate sterile vaccine acquisition rate March-April 13.33(8 / 60) 6.67(4 / 60) 80.00(48 / 60) May-June 5.00(3 / 60) 6.67(4 / 60) 88.33(53 / 60) July-August 21.67(13 / 60) 28.33(17 / 60) 50.00(30 / 60)

[0143] The contamination rate was lower in March-April and May-June, resulting in a higher rate of sterile seedlings. However, the sterile seedling rate was even better in May-June. This may be because the axillary buds have a shorter growth period in March-April, making them more fragile and leading to a higher mortality rate after disinfection with alcohol and mercuric chloride, thus resulting in a lower sterile seedling rate. The inventors found that the explants of *Tetracentron sinense* collected in May-June had the best maturity, with minimal impact from disinfection, and induced axillary bud germination quickly, efficiently, and with less browning. Using suitable explant materials combined with the use of *Melaleuca alternifolia* essential oil and plastic bag packaging allowed the axillary buds to grow while reducing the problem of microbial contamination during the rainy season, ultimately resulting in the best sterile seedling rate.

[0144] Comparative Example 5

[0145] The inoculation subjects were tissue culture seedlings obtained by the initial culture stage of Example 1. They were transferred to an induction medium consisting of MS + 8 mg / L BA + 4 mg / L IAA + 2 mg / L TDZ + 3% sucrose + 4.5 g / L agar powder and cultured under a light intensity of 5000 Lx and a light duration of 10 h.

[0146] Comparative Example 6

[0147] The inoculation subjects were tissue culture seedlings obtained by the initial culture stage of Example 1. They were transferred to an induction medium consisting of MS + 8 mg / L BA + 4 mg / L IAA + 3% sucrose + 4.5 g / L agar powder and cultured under a light intensity of 5000 Lx and a light duration of 10 h.

[0148] Comparative Example 7

[0149] The inoculation subjects were tissue culture seedlings obtained by the initial culture stage of Example 1. They were transferred to an induction medium consisting of MS + 70 mg / L Ca(NO3)2 + 8 mg / L BA + 4 mg / L IAA + 0.05 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder and cultured under a light intensity of 5000 Lx and a light duration of 10 h.

[0150] Comparative Example 8

[0151] The inoculation subjects were tissue culture seedlings obtained by the initial culture stage of Example 1. They were transferred to an induction medium consisting of MS + 100 mg / L Ca(NO3)2 + 8 mg / L BA + 4 mg / L IAA + 0.2 mg / L triacontanol + 3% sucrose + 4.5 g / L agar powder and cultured under a light intensity of 5000 Lx and a light duration of 10 h.

[0152] The axillary bud proliferation coefficients and induction rates of comparative examples 5, 6, 7, and 8 were statistically analyzed and compared with the axillary bud proliferation culture of example 1. The results are shown in Table 4.

[0153] Table 4. Proliferation coefficients and induction rates of Examples 1 and Comparative Examples 5-8

[0154] Proliferation coefficient Induction rate (%) Example 1 3.5 90.00％(36 / 40) Comparative Example 5 2.5 57.50％(23 / 40) Comparative Example 6 1.5 47.50％(19 / 40) Comparative Example 7 2.0 67.50％(27 / 40) Comparative Example 8 3.0 77.50％(31 / 40)

[0155] Comparative Example 9

[0156] Small, vigorous shoots with a height of 2.0-3.5 cm were transferred to rooting medium 1 / 2 MS + 50 mg / L Ca(NO3)2 + 1.5 mg / L IAA + 1.5 mg / L IBA + 0.5 mg / L riboflavin + 1.5% sucrose + 5.5 g / L agar powder and cultured under light intensity of 5000 Lx and light duration of 10 h.

[0157] Comparative Example 10

[0158] Healthy bud clusters with a height of 2.0-3.5 cm were transferred to rooting medium 1 / 2 MS + 50 mg / L Ca(NO3)2 + 1.5 mg / L IAA + 1.5 mg / L IBA + 0.5 mg / L amino acid ester + 1.5% sucrose + 5.5 g / L agar powder and cultured under light intensity of 5000 Lx and light duration of 10 h.

[0159] The rooting status of Comparative Examples 9 and 10 was statistically analyzed and compared with that of the small bud rooting culture in Example 1. The results are shown in Table 5.

[0160] Table 5. Rooting of small shoot clusters in Example 1 and Comparative Examples 9-10

[0161] Rooting time Average root length after 14 days (cm) Rooting rate (%) Example 1 Rooting in 25 days 1cm 85.00％(34 / 40) Comparative Example 9 Rooting in 55 days 0.40cm 57.50％(23 / 40) Comparative Example 10 Rooting in 30 days 0.75cm 72.50％(29 / 40)

[0162] This invention can be implemented in various ways and is not limited to the embodiments described. Those skilled in the art will understand that the invention can be implemented in other specific ways without changing the technical concept or essential features. Therefore, it should be understood that the embodiments described above are exemplary and not intended to limit the invention.

Claims

1. A method for tissue culture regeneration of *Tea spp.*, characterized in that, The method includes: S1, Explant treatment: 1-1.5 months before explant collection, spray Australian tea tree essential oil on the stem segments, leaves and axillary buds of the branches to be collected, then cover the branches to be collected with bags, collect the branches to be collected in May-June, cut the branches to be collected into stem segments with 1-2 axillary buds, disinfect and dry them to obtain stem segments with axillary buds; S2, Initial culture: Take the stem segment with axillary buds and inoculate it into the initial culture medium for initial culture to obtain axillary buds. The initial culture medium is MS + 6-8 mg / L BA + 3-4 mg / L IAA + 0.01-0.05 mg / L thiourea. S3, Axillary bud proliferation culture: The stem segments with axillary buds described in step S2 are inoculated into an induction medium for culture. The induction medium is MS + 80-100 mg / L Ca(NO3)2 + 6-8 mg / L BA + 3-4 mg / L IAA + 0.01-0.05 mg / L triiodobenzoic acid + 0.1-1 mg / L triacontanol; S4, Small Bud Cluster Culture: Axillary buds from step S3 are cut off and inoculated into an induction medium for induction culture to obtain bud clusters. The bud clusters are divided into small bud clusters with 3-6 adventitious buds each. The small bud clusters are inoculated into a rejuvenation medium for culture to obtain robust small bud clusters. The rejuvenation medium is MS + 0.6-0.8 mg / L 6-BA + 0.04-0.08 mg / L 1-BA. S5, Rooting of small bud clusters: The robust small bud clusters are inoculated into a rooting medium for culture. The rooting medium is 1 / 2MS + 40-50 mg / L Ca(NO3)2 + 1-1.5 mg / L IAA + 1-1.5 mg / L IBA + 0.3-0.5 mg / L aminoethyl ester + 0.02-0.05 mg / L sodium dodecylbenzenesulfonate. S6, sealing and hardening off seedlings and transplanting.

2. The method for tissue culture regeneration of *Tea spp.* as described in claim 1, characterized in that, In step S4, the induction culture specifically involves culturing at a temperature of 26-30℃ and a light intensity of 800-1000 Lx for 7-10 days, followed by culturing under a light intensity of 3000-6000 Lx and a light duration of 8-12 h.

3. The method for tissue culture regeneration of *Tea spp.* as described in claim 1, characterized in that, In steps S3, S4 and S5, the cultivation is specifically carried out under the conditions of light intensity of 3000-6000 Lx and light duration of 8-12 h.

4. The method for tissue culture regeneration of *Tea spp.* as described in claim 1, characterized in that, In the step of inoculating into the initial culture medium for initial culture, the initial culture specifically involves culturing at a temperature of 26-30℃ and a light intensity of 300-500 Lx for 5-10 days, followed by culturing under a light intensity of 2000-3000 Lx and a light duration of 8-12 h.

5. The method for tissue culture regeneration of *Tea spp.* as described in claim 1, characterized in that, The disinfection and drying steps are as follows: Soak the stem segments with axillary buds in 75% alcohol for 10-30 seconds with stirring, then rinse with sterile water; then soak in 0.1% mercuric chloride solution for 6-12 minutes, stirring every 2-3 minutes during soaking, rinse with sterile water, blot dry the surface moisture of the stem segments with axillary buds with sterile filter paper, and then cut off 0.3-0.5 cm from both ends of the stem segments with axillary buds to obtain stem segments with 1-2 axillary buds.

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