Tea tree tissue culture efficient rapid propagation method based on stem explants

Abstract

The invention provides a stem explant-based tea tree tissue culture high-efficiency rapid propagation method, which comprises the following steps: disinfecting twigs with plump axillary buds by using alcohol, disinfecting by using a NaClO solution, washing by using sterile water, absorbing surface moisture by using filter paper, and selecting stems; the stem segments are inoculated into a fixed bud induction culture medium, and 0.5 mg / L to 3 mg / L of 6-BA and 0.05 mg / L to 0.3 mg / L of NAA are added; the induced fixed buds are placed in an MS culture medium for adventitious bud induction, and 1-3 mg / L of 6-BA and 0.1-0.3 mg / L of NAA are added; the induced adventitious buds are placed in an MS culture medium for adventitious bud induction, and 1 mg / L of 6-BA, 0.1-0.5 mg / L of NAA and 2 mg / L of TDZ are added; the induced adventitious buds are placed in an elongation culture medium, and 0.2 mg / L to 1 mg / L of 6-BA and 0.02 mg / L to 0.1 mg / L of NAA are added; bud seedlings with a good induced growth state are selected and placed in a 1 / 2 MS culture medium for adventitious root induction, and 1-5 mg / L of IBA is added. According to the method, two-step adventitious bud induction is adopted, so that the production efficiency of the tea seedlings is greatly improved, the bottleneck of a tissue culture technology in the aspect of tea seedling productive seedling raising is broken through, and the application of an in-vitro regeneration technology in tea seedling production is liberated.

Description

Technical Field

[0001] The invention belongs to the field of plant tissue culture, and in particular relates to a method for efficient and rapid propagation of tea tree tissue culture based on stem segment explants. Background Art

[0002] Tea (Camellia sinensis) is one of the three major non-alcoholic beverages in the world. It is deeply loved by people for its unique flavor and natural health functions. The sexual reproduction of tea trees has disadvantages such as self-incompatibility, self-depression, and low fruiting rate. In particular, it is easy to separate traits and lose excellent traits, which has brought great obstacles to the long-term development of tea gardens (Dong Lijuan. Research Progress on Hybrid Breeding of Tea Trees [J]. Tea Science, 2001, 21(1):7-10.). Asexual reproduction is mainly based on cuttings, but this method has certain defects in seedling cycle, reproduction coefficient, and requirements for the natural environment, which greatly limits the breeding and promotion speed of new tea varieties (Cheng Hao, Zeng Jianming, Zhou Jian, Wang Liyuan, et al. Factory-based Rapid Propagation Technology of Tea Seedlings [J]. Tea Science, 2007, 27(3):231-235.).

[0003] Plant tissue culture and in vitro rapid propagation technology is a technology that relies on the totipotent characteristics of cells, and through aseptic operation and suitable culture conditions, it can induce plant protoplasts, cells, tissues or organs to start, proliferate and root in a short time, and finally obtain complete plants with stable and uniform genetic properties (Li Xue, Li Guoze, Yang Ling, et al. Research Progress on Tissue Culture of Vaccinium Plants [J]. Acta Physiologica Sinica, 2020, 56(5): 921-930.). It has the advantages of short culture cycle, high reproduction coefficient and no seasonal restrictions. Especially in recent years, with the rapid development of transgenic and gene editing technologies, plant tissue culture and in vitro rapid propagation technology have provided an indispensable technical foundation for these scientific frontiers (Arndell T., Sharma N., Langridge P., et al. gRNA validation for wheat genome editing with the CRISPR-Cas9 system [J]. BMC Biotechnology, 2019, 19(1): 71.).

[0004] Given the outstanding advantages of plant tissue culture and in vitro rapid propagation technology in plant breeding, the use of this technology to produce tea seedlings can not only maintain excellent traits, shorten the breeding period, and achieve rapid propagation, but also avoid environmental restrictions on breeding time and achieve batch and large-scale production (Liu Jing. Research on tissue culture of tea stem segments with axillary buds [J]. Shaanxi Agricultural Sciences, 2020, 66 (10): 43-45.). In 1969, Forrest conducted the first tissue culture study on tea trees (Forrest GIStudies on the polyphenol metabolism of tissue cultures derived from the tea plant (Camellia sinensis L.) [J]. Biochemical Journal, 1969, 113 (5): 765-781.), which was a milestone; Kato subsequently conducted a systematic and in-depth study on the in vitro regeneration of tea trees (Kato M. Regeneration of plantlets from tea stem callus [J]. Japanese Journal of Breeding, 1985, 35 (3): 317-322.). Most of the research on tea plant tissue culture focuses on the establishment of sterile in vitro culture systems, optimization of explant types and nutrient components of culture media. Xie Enjun et al. (Xie Enjun, Tian Weili, Chen Zhengwu, et al. Establishment of tissue culture rapid propagation system of stem segments with axillary buds of 'Zhonghuang No. 1' tea plants [J]. Molecular Plant Breeding, 2020, 18(15): 5071-5080.) obtained axillary bud germination medium MS+2mg / L 6-BA+1mg / LGA3, proliferation medium MS+4mg / L 6-BA+1mg / L IBA, and rooting medium 1 / 2MS+1.5mg / L IBA suitable for the 'Zhonghuang No. 1' tea variety through a large number of experiments. Chen Lijie et al. established a rapid propagation system for stem segments of Huaxi ancient tea trees, with an optimal proliferation coefficient of 1.5 and a rooting rate of 56.7% (Chen Lijie, Yang Xia, Gao Qian, et al. Preliminary report on research on in vitro propagation technology of Huaxi ancient tea trees [J]. Seed Industry Guide, 2019, 6(8):10-13.).

[0005] At present, the in vitro regeneration system of tea trees mainly uses two pathways: organ regeneration and somatic embryogenesis. Organ regeneration is induced by explants such as anthers, stem segments, and leaves of tea trees. Most of them can induce callus tissue, and very few can differentiate from callus tissue to form complete plants. There are many reports on inducing buds from tea tree stem segments as starting explants, but the efficiency is not high, and the reproduction coefficient is about 3 (Chen Zexiong, Liu Yiqing, Huang Dengyan. In vitro culture and plant regeneration of 'Yucha No. 1' [J]. Journal of Southwest University (Natural Science Edition), 2009, 31 (12): 67-70.). Summary of the invention

[0006] The technical problem to be solved by the present invention is how to improve the reproduction coefficient of inducing budding by using tea tree stem segments as starting explants.

[0007] The present invention solves the above technical problems through the following technical means:

[0008] The present invention provides a method for efficient and rapid propagation of tea tree tissue culture based on stem segment explants, comprising the following steps:

[0009] A. Preparation and disinfection of explant materials: Twigs with full axillary buds were first disinfected with alcohol, then with NaClO solution, then rinsed with sterile water, and finally the surface moisture was absorbed with filter paper, and the stem segments were selected;

[0010] B. Bud induction: inoculate the stem segments into bud induction medium, the basic medium is MS medium, supplemented with 0.5-3 mg / L 6-BA and 0.05-0.3 mg / L NAA;

[0011] C. Adventitious bud induction: Place the adventitious buds induced in step B in MS medium for adventitious bud induction, and add 1-3 mg / L 6-BA and 0.1-0.3 mg / L NAA;

[0012] D. Induction and proliferation of adventitious buds: The adventitious buds induced in step C were placed in MS medium for adventitious bud induction, and 1 mg / L 6-BA, 0.1-0.5 mg / L NAA and 2 mg / L TDZ were added;

[0013] E. Adventitious bud elongation: the adventitious buds induced in step D are placed in an elongation medium, wherein the elongation medium is an MS medium supplemented with 0.2-1 mg / L 6-BA and 0.02-0.1 mg / L NAA;

[0014] F. Adventitious root induction: Select the seedlings with good growth status induced in step E and place them in 1 / 2MS medium for adventitious root induction, and add 1-5 mg / L IBA.

[0015] Beneficial effects: The present invention uses two-step adventitious bud induction to gradually establish and improve the stem segment rapid propagation system of 'Baochun No. 1' based on tissue culture; this system greatly improves the production efficiency of tea seedlings, breaks the bottleneck of tissue culture technology in productive tea seedling cultivation, and liberates the application of in vitro regeneration technology in tea seedling production.

[0016] The invention adopts a two-step adventitious bud induction method to improve the reproduction coefficient of tea tree stem segments as starting explants for inducing budding, and the reproduction coefficient of the invention reaches up to 10.43.

[0017] Preferably, in step A, the tender branch needs to retain two buds, be cut off from the third axillary bud of the bud, and remove the branches and part of the leaves of the lignified part.

[0018] Preferably, in step A, the alcohol concentration is 75%, and the disinfection time is 30 seconds; the NaClO solution concentration is 7.5%, and the disinfection time is 6 to 14 minutes; and the sterile water is rinsed 5 times.

[0019] Preferably, in step B, the concentration of 6-BA is 1.5 mg / L, and the concentration of NAA is 0.15 mg / L.

[0020] Preferably, in step C, the concentration of 6-BA is 2 mg / L, and the concentration of NAA is 0.2 mg / L.

[0021] Preferably, in step D, the concentration of 6-BA is 1 mg / L, the concentration of NAA is 0.3 mg / L, and the concentration of TDZ is 2 mg / L.

[0022] Preferably, in step E, the concentration of 6-BA is 0.8 mg / L, and the concentration of NAA is 0.08 mg / L.

[0023] Preferably, in step F, the concentration of IBA is 2-3 mg / L.

[0024] Preferably, the basic culture medium in steps B to F contains 30 g / L sucrose, 0.1 g / L inositol, 6.5 g / L agar powder, and a pH value of 5.8±0.1.

[0025] Preferably, the culture conditions in steps B to F are 24±1° C., light intensity 1500-2500 lx, light 16 h / d, and darkness 8 h / d.

[0026] The advantages of the present invention are:

[0027] The present invention uses two-step adventitious bud induction to gradually establish and improve the stem segment rapid propagation system of 'Baochun No. 1' based on tissue culture; this system greatly improves the production efficiency of tea seedlings, breaks the bottleneck of tissue culture technology in productive tea seedling cultivation, and liberates the application of in vitro regeneration technology in tea seedling production.

[0028] The invention adopts a two-step adventitious bud induction method to improve the reproduction coefficient of tea tree stem segments as starting explants for inducing budding, and the reproduction coefficient of the invention reaches up to 10.43. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Explants selected for the embodiments;

[0030] Figure 2 The diagram is a diagram of the bud induction process of the tea tree stem segment of 'Baochun No. 1' in the embodiment, A, B, and C represent the state of the stem segment growing on the bud induction medium (MS+1.5mg / L 6-BA+0.15mg / L NAA) for 7d, 14d, and 21d (bar=1cm);

[0031] Figure 3 The diagram is of the adventitious bud proliferation stage I in the example, A and B represent the state of growing on adventitious bud induction medium (MS+2mg / L6-BA+0.2mg / L NAA) for 28d and 45d (bar=1cm);

[0032] Figure 4 The diagram is of the adventitious bud proliferation phase II in the example, A and B represent the state of growing on adventitious bud induction medium (MS+1mg / L6-BA+0.3mg / L NAA+2mg / L TDZ) for 30d and 45d (bar=1cm);

[0033] Figure 5 This is a state diagram of the plant growing on adventitious bud elongation medium (MS+0.8mg / L 6-BA+0.08mg / L NAA) for 30 days in the example (bar=0.5cm);

[0034] Figure 6 Figure 1 shows the adventitious root induction in the examples, where A and B represent the state of growing on adventitious root induction medium (1 / 2MS+3mg / L IBA) for 15d and 30d (bar=0.5cm);

[0035] Figure 7 Figures of adventitious root induction in the examples, A and B represent the state of growth for 50 days in adventitious root induction medium (1 / 2MS+3 mg / L IBA) (bar=0.5 cm). DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the test materials and reagents used in the following examples can be obtained from commercial sources.

[0038] If no specific techniques or conditions are specified in the examples, they can be carried out according to the techniques or conditions described in the literature in the art or according to the product instructions.

[0039] The main reagents, formulas and materials used in the embodiments of the present invention are:

[0040] (1) 6-benzyladenine (6-BA), naphthylacetic acid (NAA), indolebutyric acid (IBA), and MS medium (Phyto Tech);

[0041] (2) Glycine (Gly), thiamine hydrochloride (VB1), niacin (VB3), pyridoxine hydrochloride (VB6), glucose, sucrose, anhydrous ethanol, and inositol (Sinopharm Chemical Reagent Co., Ltd.);

[0042] (3) Hydrochloric acid (Xilong Scientific Co., Ltd.);

[0043] (4) Sodium hydroxide (Beijing Solebow Technology Co., Ltd.);

[0044] (5) Agar powder, sodium hypochlorite (Shanghai Exploration Platform Co., Ltd.).

[0045] (6) The experimental materials were stem segments of the tea tree 'Baochun No. 1', which came from the Baojia Laozhuang asexual tea garden in Sanchong Village, Shucheng County, Lu'an City, Anhui Province. 'Baochun No. 1' was harvested in early June on a sunny day with no rain in the previous week, when it was in the one-bud and two-leaf stage, and the buds and leaves were clean and strong.

[0046] Culture conditions: All culture media were added with 30 g / L sucrose, 0.1 g / L inositol, and 6.5 g / L agar powder, and the pH value of the culture medium was adjusted to 5.8±0.1; the temperature was 24±1°C, the light intensity was 1500-2500 lx, the light was 16 h / d, and the darkness was 8 h / d.

[0047] Example 1

[0048] This embodiment provides a method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants, comprising the following steps:

[0049] 1. Preparation and Sterilization of Stem Segment Explant Materials

[0050] From the Baojia Laozhuang asexual tea garden in Sanchong Village, Shucheng County, Lu'an City, Anhui Province, we selected the No. 1 Primrose Branches that were strong and less polluted when they were in the one bud and two leaf period. We first wrapped the upper part of the branch with clean wet gauze, and then put the bottom of the branch into a plastic bag filled with pure water, and then put it into a foam box, and put an ice bag in the foam box, and finally sealed the foam box to ensure the cleanliness and freshness of the branch.

[0051] Keep two buds, cut off the third axillary bud from the bud, and cut off the lignified branches and most of the leaves.

[0052] First, rinse with running water for 2 hours outside the clean bench, add an appropriate amount of detergent to clean; vortex oscillate with 5‰ potassium permanganate for 2 to 3 minutes; rinse the axillary buds with a syringe of 75% alcohol; rinse with water 3 times.

[0053] Then put it in the clean bench for disinfection. First, use 75% alcohol to disinfect for 30 seconds, then use 7.5% NaClO solution to disinfect for 6-14 minutes, then rinse with sterile water for 5 times, and finally use filter paper to absorb the surface moisture. Select the second stem segment from top to bottom. This part of the stem segment with axillary buds is the cleanest and most vigorous. Cut the stem segment with axillary buds into about 1 cm cross-sections, cut off the excess leaves, and leave only 2-3 mm petioles to protect the axillary buds.

[0054] The stem segments were inoculated in the culture medium, the basic culture medium was MS culture medium, supplemented with 0.5mg / L 6-BA and 0.05mg / L NAA. One plant was inoculated in each bottle, 50 bottles were used for each treatment, and repeated 3 times. From the first day after inoculation to the seventh day, the contamination was observed and recorded at any time, and the cleanliness rate and survival rate of each treatment were calculated.

[0055] 2. Bud Induction

[0056] The stem segments were inoculated into the bud induction medium, the basic medium was MS medium, supplemented with 0.5-3 mg / L 6-BA and 0.05-0.3 mg / L NAA. Ten bottles were inoculated for each treatment, with 3 explants in each bottle, repeated 3 times, cultured for 3 weeks, and the bud induction rate was calculated.

[0057] The bud induction rate = (the number of explants induced to bud / the total number of explants) × 100%.

[0058] 3. Adventitious bud induction

[0059] The induced fixed buds were cut off and the base was transferred to MS medium containing different combinations of 1-3 mg / L 6-BA and 0.1-0.3 mg / L NAA for adventitious bud induction. After 8 weeks, the adventitious bud induction rate and the average bud number were counted.

[0060] In order to induce more adventitious buds, the induced adventitious buds were further cut off, and the bottom stump was transferred to MS culture medium containing different combinations of 1 mg / L6-BA, 0.1-0.5 mg / L NAA and 2 mg / L TDZ. After 4 weeks of continuous culture, the average number of adventitious buds was counted.

[0061] Ten bottles were inoculated with three explants per bottle for each hormone combination, and the results were repeated three times. The cells were transferred every four weeks.

[0062] Adventitious bud induction rate = (number of explants induced with adventitious buds / total number of explants) × 100%.

[0063] 4. Elongation of adventitious buds

[0064] The induced adventitious buds were transferred to the elongation medium, the basic medium was MS medium, and 0.2-1 mg / L 6-BA and 0.02-0.1 mg / L NAA were added. Ten bottles were inoculated with each hormone combination, and 3 explants were inoculated in each bottle, and repeated 3 times. After 4 weeks of light culture, the elongation rate of the adventitious buds of the stem segments was counted, and the growth state of the adventitious buds was observed to screen out the appropriate concentration and combination of plant growth regulators.

[0065] Adventitious bud elongation rate = (number of explants with elongated adventitious buds / total number of explants) × 100%.

[0066] 5. Adventitious root induction

[0067] Select the 3-4 cm long seedlings with good and consistent growth status obtained by induction and inoculate them into 1 / 2MS medium containing different concentrations of IBA for adventitious root induction. Inoculate 10 bottles per group, 3 explants per bottle, and repeat 3 times. After 6 weeks, the rooting rate was counted to screen the appropriate concentration of plant growth regulator.

[0068] Adventitious root induction rate = (number of explants induced with adventitious roots / total number of explants) × 100%.

[0069] Statistical analysis: Each experiment was repeated 3 to 10 times, and the relevant data were analyzed using Microsoft Excel, and the significance level was analyzed using IBM SPSS Statistics 26. Sigmaplot 12.5 was used for drawing and correlation analysis, and Adobe Illustrator 2021 software was used for image editing.

[0070] (1) Preparation and disinfection of explant materials

[0071] The explant stem segments that have been treated in the early stage, such as Figure 1As shown in the figure, after being treated with 75% alcohol for 30s, 7.5% NaClO was used for different treatment times to calculate the cleanliness rate and survival rate. As shown in Table 1, with the extension of NaClO disinfection time, the number of contaminated explants gradually decreased, the cleanliness rate gradually decreased, but the survival rate of the explants showed a trend of first rising and then falling, indicating that the disinfection time should be appropriate. Too long disinfection time will cause some explants to be inactivated or passivated, thereby affecting the survival rate. When 7.5% NaClO was treated for 12 minutes, the comprehensive disinfection effect was the best, the cleanliness rate was 86.67%, and the survival rate was the highest, at 80%.

[0072] Table 1 Effects of 7.5% NaClO treatment at different times on the disinfection of stem segments

[0073]

[0074]

[0075] Note: Data represent mean ± standard error. Different lowercase letters in the same column indicate significant differences in Duncan's test (P≤0.05).

[0076] (2) Bud induction

[0077] The disinfected stem segments with axillary buds were inoculated on MS medium containing different concentrations of 6-BA and NAA for bud induction. After 3 weeks of culture, the bud induction rate was counted and the growth of the stem segments was observed. The results are shown in Table 2. As the concentrations of 6-BA and NAA increased, the bud induction rate increased first and then decreased. The bud induction rate of the stem segments in 1.5 mg / L 6-BA and 0.15 mg / L NAA was the highest, reaching 82.2%. The induced apical buds grew well and grew fast (e.g. Figure 2 This laid a good foundation for the subsequent adventitious bud induction.

[0078] Table 2 Effects of plant growth regulators on bud induction

[0079]

[0080] Note: Data represent mean ± standard error. Different lowercase letters in the same column indicate significant differences in Duncan's test (P≤0.05).

[0081] (3) Adventitious bud induction is divided into two stages:

[0082] Adventitious bud proliferation stage I

[0083] The fixed buds grown during the fixed bud induction stage were cut off and inoculated into MS medium containing 1-3 mg / L 6-BA and 0.1-0.3 mg / L NAA to induce adventitious buds. During this period, the callus continued to expand. After 4 weeks of culture, adventitious buds gradually grew out. The results are as follows: Figure 3 As shown in Table 3, after 8 weeks of cultivation, the induction rate of adventitious buds and the average number of adventitious buds were statistically analyzed. As the concentrations of 6-BA and NAA increased, the induction rate of adventitious buds and the average number of adventitious buds first increased and then decreased. When 6-BA and NAA were added too much or too little, the stem segment had only 1 to 2 adventitious buds, and the growth was poor. When 2 mg / L 6-BA and 0.2 mg / L NAA were added, the induction effect of adventitious buds was the best, the average number of buds was the largest, the induction rate was 77.78%, the average number of buds was 5.4, the buds were green, and the leaves were stretched.

[0084] Table 3 Effects of plant growth regulators on adventitious bud proliferation induction phase I

[0085]

[0086] Note: Data represent mean ± standard error. Different lowercase letters in the same column indicate significant differences in Duncan's test (P≤0.05).

[0087] Adventitious bud proliferation phase II

[0088] In order to further induce more adventitious buds, the adventitious buds induced in the adventitious bud proliferation phase I were cut off and inoculated in MS medium containing 1 mg / L 6-BA, 0.1-0.5 mg / L NAA and 2 mg / L TDZ. After 4 weeks, a large number of adventitious buds grew out. The results are shown in the figure. Figure 4 As shown in the figure, the number of adventitious buds induced in the adventitious bud proliferation phase II was significantly higher than that in the adventitious bud proliferation phase I. The statistical results are shown in Table 4. With the increase of NAA concentration, the number of induced adventitious buds first increased and then decreased. When 1 mg / L 6-BA, 0.3 mg / L NAA and 2 mg / LTDZ were added, the induction effect of adventitious buds was the best, and the average number of buds was the largest, which was 10.43.

[0089] Table 4 Effects of plant growth regulators on adventitious bud proliferation induction phase II

[0090]

[0091] Note: Data represent mean ± standard error. Different lowercase letters in the same column indicate significant differences in Duncan's test (P≤0.05).

[0092] (4) Adventitious bud elongation

[0093] The adventitious buds induced in the adventitious bud proliferation phase II were inoculated into MS medium containing 0.2-1 mg / L 6-BA and 0.02-0.1 mg / L NAA to induce the elongation of the adventitious buds. The elongation rate of the adventitious buds was calculated after 4 weeks. As shown in Table 5, different concentrations of hormone combinations all induced the elongation of the adventitious buds, but there were great differences in the elongation effects of the adventitious buds. With the increase of the concentrations of 6-BA and NAA, the elongation rate of the adventitious buds showed a trend of first increasing and then decreasing. When 0.8 mg / L 6-BA and 0.08 mg / L NAA were used in combination, the effect of inducing the elongation of the adventitious buds was the best, with an elongation rate of 63.33%, buds and leaves stretched, stem segments thick and internodes long, such as Figure 5 As shown, suitable for rooting.

[0094] Table 5 Effects of plant growth regulators on adventitious bud elongation

[0095]

[0096] Note: Data represent mean ± standard error. Different lowercase letters in the same column indicate significant differences in Duncan's test (P≤0.05).

[0097] (5) Adventitious root induction

[0098] The elongated adventitious bud clusters were divided into individual plants, and the individual plants were inoculated in 1 / 2MS medium supplemented with 1-5 mg / L IBA for root induction. As shown in Table 6, when the IBA concentration was 1 mg / L and 5 mg / L, there was no rooting reaction at the base, and the bottom absorption surface gradually turned black. When the IBA concentration was 4 mg / L, a convex part similar to the root was produced at the base, but it could not be further extended. Only when the IBA concentration was 2 mg / L and 3 mg / L, a small number of roots were induced, as shown in Table 6. Figure 6-7 As shown, when the concentration of IBA was 3 mg / L, no lateral roots were produced at 15d, 30d and 50d of growth for a single plant, and the growth cycle was longer, the rooting effect was much better than others.

[0099] Table 6 Effects of different concentrations of IBA on adventitious root induction

[0100]

[0101] In summary, the present invention uses two-step adventitious bud induction to gradually establish and improve the stem segment rapid propagation system of 'Baochun No. 1' based on tissue culture; this system greatly improves the production efficiency of tea seedlings, breaks the bottleneck of tissue culture technology in productive tea seedling cultivation, and liberates the application of in vitro regeneration technology in tea seedling production.

[0102] The invention adopts a two-step adventitious bud induction method to improve the reproduction coefficient of tea tree stem segments as starting explants for inducing budding, and the reproduction coefficient of the invention reaches up to 10.43.

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants, characterized in that: The following steps are involved: A. Preparation and disinfection of explant materials: Twigs with full axillary buds were first disinfected with alcohol, then with NaClO solution, then rinsed with sterile water, and finally the surface moisture was absorbed with filter paper, and the stem segments were selected; B. Bud induction: inoculate the stem segments into bud induction medium, the basic medium is MS medium, supplemented with 0.5-3 mg / L 6-BA and 0.05-0.3 mg / L NAA; C. Adventitious bud induction: Place the adventitious buds induced in step B in MS medium for adventitious bud induction, and add 1-3 mg / L 6-BA and 0.1-0.3 mg / L NAA; D. Induction and proliferation of adventitious buds: The adventitious buds induced in step C were placed in MS medium for adventitious bud induction, and 1 mg / L 6-BA, 0.1-0.5 mg / L NAA and 2 mg / L TDZ were added; E. Adventitious bud elongation: the adventitious buds induced in step D are placed in an elongation medium, wherein the elongation medium is an MS medium supplemented with 0.2-1 mg / L 6-BA and 0.02-0.1 mg / L NAA; F. Adventitious root induction: Select the seedlings with good growth status induced in step E and place them in 1 / 2MS medium for adventitious root induction, and add 1-5 mg / L IBA.

2. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: In step A, the tender branch needs to retain two buds, and be cut off from the third axillary bud of the bud, and the branches and part of the leaves of the lignified part are cut off.

3. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: In step A, the alcohol concentration is 75%, and the disinfection time is 30 seconds; the NaClO solution concentration is 7.5%, and the disinfection time is 6 to 14 minutes; and the sterile water is rinsed 5 times.

4. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: In step B, the concentration of 6-BA is 1.5 mg / L, and the concentration of NAA is 0.15 mg / L.

5. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: In step C, the concentration of 6-BA is 2 mg / L, and the concentration of NAA is 0.2 mg / L.

6. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: In step D, the concentration of 6-BA is 1 mg / L, the concentration of NAA is 0.3 mg / L, and the concentration of TDZ is 2 mg / L.

7. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: In step E, the concentration of 6-BA is 0.8 mg / L, and the concentration of NAA is 0.08 mg / L.

8. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: In step F, the concentration of IBA is 2-3 mg / L.

9. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: The basic culture medium in steps B to F contains 30 g / L sucrose, 0.1 g / L inositol, 6.5 g / L agar powder, and a pH value of 5.8±0.

1.

10. The method for efficient and rapid propagation of tea plant tissue culture based on stem segment explants according to claim 1, characterized in that: The culture conditions in steps B to F are 24±1°C, light intensity 1500-2500 lx, light 16 h / d, and darkness 8 h / d.

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