Tea tree callus induction culture medium and induction propagation method thereof
By adding Drynaria fortunei extract and other substances to the tea tree callus induction medium and employing plant tissue culture technology, the problem of low propagation efficiency of Guire No. 2 tea tree was solved, and a rapid and efficient seedling cultivation method was achieved.
Patent Information
- Application Number
- CN202511077578.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-01
- Publication Date
- 2025-10-31
AI Technical Summary
The Gui Re No. 2 tea tree is mainly propagated by asexual cuttings, which results in low seedling efficiency, long cycle, and large land area, making it difficult to meet the needs of large-scale planting.
Using plant tissue culture methods, MS medium was used as the base, with the addition of Drynaria fortunei extract and other plant extracts to prepare a tea tree callus induction medium. Callus tissue was obtained by induction through anthers or leaves.
It significantly improved the propagation speed of Guire No. 2 tea trees, shortened the seedling cycle, and met the needs of large-scale planting.
Smart Images
Figure CN120858874A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of rapid propagation technology of tea tree tissue, specifically relating to a method for rapidly inducing callus tissue propagation in Guangxi Baihao tea tree. Background Technology
[0002] With the continuous development of society and the economy, the number of tea drinkers is increasing, and consequently, the scale of the tea tree planting industry is also expanding. Guangxi, as an important tea-producing area in China, is rich in local specialty tea tree materials. The applicant's institution collected more than 1,400 germplasm resources of Guangxi Baihao tea, from which 289 core germplasm samples were selected. From these core germplasm samples, three national-level varieties—Gui Re No. 2, Ling Long No. 1, and Long Rui No. 2—were selected and bred, demonstrating strong resistance to adverse conditions, high yield, and wide adaptability. The finished tea produced from these varieties has a rich aroma and mellow taste. Among them, Gui Re No. 2 was selected as one of the "2023 Guangxi Agricultural Leading Varieties". Its registration number is GPD Tea Tree (2023) 450004. It has a yield increase of 42.9% compared with the national-level superior variety Lingyun Baihao Tea. It has the characteristics of early budding, strong bud cultivation ability, plump buds, abundant downy hairs, prominent downy hairs, wide adaptability, high amino acid content, and excellent dry tea quality. Its red, green, white and yellow tea series products have won awards in national tea competitions such as "China Tea Cup" and "National Drink Cup" many times. It has great potential in the tea tree planting industry.
[0003] Due to the difficulty in propagating Gui Re No. 2, the main propagation method is asexual cutting. This method requires a large number of cuttings and occupies a large space, necessitating the establishment of sufficiently large cutting nurseries and seedling nurseries to meet the demand for seedlings on a certain scale. Low seedling efficiency, long cycle, and high cost have become key issues hindering the expansion of Gui Re No. 2's planting scale. Summary of the Invention
[0004] To address the aforementioned issues, this invention aims to rapidly obtain callus tissue from Guire No. 2 tea trees through plant tissue culture, providing new insights for the rapid tissue propagation technology and genetic transformation cultivation of Guangxi Baihao tea trees.
[0005] The technical solution adopted in this invention is as follows:
[0006] A tea tree callus induction culture medium, using plant MS medium as the base medium and adding 0.2% to 1% Drynaria fortunei extract; the Drynaria fortunei extract is prepared by the following method: fresh Drynaria fortunei is dried to constant weight and pulverized; Drynaria fortunei powder is weighed and ethanol with a concentration of 60% to 90% is added at a mass-volume ratio of 1:10 to 20; the mixture is ultrasonicated at a temperature of 45 to 55°C for 20 to 30 minutes, and the filtrate obtained is the Drynaria fortunei extract.
[0007] When the callus is anther callus, the induction medium also contains 1.3–1.7 mg / L 6-BA, 0.2–0.4 mg / L 2,4-D, 29–31 g / L sucrose, and 7.4–7.6 g / L agar.
[0008] The pH value of the anther callus induction medium is 5.8–6.0.
[0009] The anther callus induction culture medium also contains 2% potato extract. The potato extract is prepared by cutting fresh potatoes into strips, boiling them in water at a weight ratio of 1:5 for at least 10 minutes, and then filtering. Alternatively, 5% wild chrysanthemum extract can be added. The wild chrysanthemum extract is prepared by adding dried wild chrysanthemum flowers to water at a weight ratio of 1:10, boiling them for at least 10 minutes, and then filtering.
[0010] When the callus is leaf callus, the induction medium also contains 0.4–0.8 mg / L 6-BA, 0.2–0.4 mg / L 2,4-D, 0.2–0.4 mg / L NAA, 4–6 g / L activated carbon, and 7–9 g / L agar.
[0011] The pH value of the leaf callus induction medium is 5.6-5.8.
[0012] A method for inducing and propagating tea tree callus includes the following steps:
[0013] (1) Select unopened tea buds, wash them, and place them in the refrigerator's crisper drawer for 6-8 hours at a low temperature.
[0014] (2) Disinfect and clean the flower buds in sequence with alcohol, 84 disinfectant, and sterile water;
[0015] (3) Cut open the flower buds and use sterile forceps to pick up the anthers and place them into the prepared anther callus induction medium. The formulation of the induction medium is as follows: plant MS medium as the base medium, with the addition of 1.3-1.7 mg / L 6-BA, 0.2-0.4 mg / L 2,4-D, 29-31 g / L sucrose, 7.4-7.6 g / L agar, and 0.2%-1% Drynaria fortunei extract. The Drynaria fortunei extract is prepared by the following method: fresh Drynaria fortunei is dried to constant weight and pulverized; Drynaria fortunei powder is weighed and ethanol with a concentration of 60%-90% is added at a mass-volume ratio of 1:10-20. The mixture is ultrasonicated at a temperature of 45-55℃ for 20-30 min, and the filtrate is the Drynaria fortunei extract. The pH value of the induction medium is 5.8-6.0.
[0016] (4) Culture in the dark for 2 to 3 days, then transfer to 25 to 26°C and continue to culture under 8 hours of darkness and 16 hours of light until callus tissue appears.
[0017] The aforementioned method for inducing and propagating tea tree callus also includes adding 2% potato extract or 5% wild chrysanthemum extract to the anther callus induction culture medium.
[0018] The tea tree variety described in this invention is Gui Re No. 2.
[0019] Experiments have verified that the callus induction culture medium and its induction propagation method for Guire No. 2 tea trees described in this invention can induce callus tissue using anthers or leaves, effectively improving the propagation speed of Guire No. 2. Attached Figure Description
[0020] Figure 1 Photographs showing the effect of adding Drynaria fortunei extract on callus induction in Example 1 of this invention.
[0021] Figure 2 Photographs showing the effect of adding Drynaria fortunei extract on leaf callus induction in Example 2 of this invention.
[0022] Figure 3 These are photographs showing the effects of different treatments in Examples 1, 3-7 of the present invention on the induction of callus tissue in anthers. Detailed Implementation
[0023] To make the technical solution and beneficial effects of the present invention more apparent and understandable, the technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the embodiments described below are merely exemplary embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0024] The reagents used in the following examples and their suppliers were as follows: MS plant culture medium, naphthaleneacetic acid (NAA), 6-benzyladenine (6-BA), 2,4-dichlorophenoxyacetic acid (2,4-D), sucrose, agar, 6-furfuryladenine (KT), MgCl2, etc., were purchased from Shanghai Jizhi Biochemical Technology Co., Ltd., and fresh materials such as Drynaria fortunei, potatoes, bananas, wild chrysanthemums, and apples were purchased from local farmers' markets. Unless otherwise specified, the percentages mentioned in this invention are mass percentages.
[0025] The callus induction and propagation methods in the following embodiments are all implemented according to the following methods:
[0026] (1) Select Gui Re No. 2 tea tree propagation material, clean the surface stains with laundry detergent, rinse with running water for 3 hours, and then put it in a 4℃ refrigerator for 6 hours.
[0027] (2) Disinfect the breeding material with 75% alcohol for 30 seconds, rinse with sterile water 3 times, disinfect with 50% 84 disinfectant for 12 minutes, and then rinse with sterile water 5 times until the 84 disinfectant is completely removed.
[0028] (3) Cut open the sterilized propagation material and take the anthers or the center of the leaves, and put them into the prepared induction medium.
[0029] (4) After culturing in the dark for 2 days, the tissue was transferred to 26°C and kept in the dark for 8 hours and then in the light for 16 hours. Callus tissue appeared in about 2 weeks.
[0030] Example 1
[0031] Propagation material: Unopened flower buds of Guire No. 2 tea tree.
[0032] Configure the following three processes:
[0033] Example 1: Culture medium formulation: Based on plant MS medium, 1.5 mg / L 6-BA, 0.3 mg / L 2,4-D, 30 g / L sucrose, 7.5 g / L agar, and 1% *Drynaria fortunei* extract were added. The *Drynaria fortunei* extract was prepared as follows: Fresh *Drynaria fortunei* was cleaned and dried in an oven to constant weight, then ground into a powder and passed through an 80-mesh sieve. 2.0 g of *Drynaria fortunei* powder was accurately weighed, and 30 mL of 70% ethanol was added. The mixture was sonicated at 50°C for 30 min, and the filtrate was used as the *Drynaria fortunei* extract for the experiment. The pH of the anther callus induction medium was 5.8–6.0.
[0034] Culture medium formulation for Experiment Example 2: Unlike Experiment Example 1, the concentration of Drynaria fortunei extract added was 0.2%, while the rest was the same as Experiment Example 1.
[0035] Comparative culture medium formulation: Unlike Example 1, no Drynaria fortunei extract was added; otherwise, it was the same as Example 1.
[0036] Each treatment was repeated with 5 culture flasks. Callus growth was photographed and recorded after 50 days. Results are shown below. Figure 1 .
[0037] from Figure 1It is evident that adding *Drynaria fortunei* extract to the culture medium significantly improved the callus induction effect. Specifically, in Experiment 1 with 1% *Drynaria fortunei* extract, the anther-induced callus was pale yellow, light in color, and transparent, with good callus size and virtually no browning, achieving an induction rate exceeding 60%. In Experiment 2 with 0.2% *Drynaria fortunei* extract, the anther-induced callus was bright green, slightly smaller, and showed some browning, but the induction rate was still over 60%. In contrast, the control group without *Drynaria fortunei* extract showed a significantly lower anther-induced callus induction rate (less than 30%), smaller callus size, and a higher proportion of browning.
[0038] Example 2
[0039] Propagation material: Select newly unfolded leaves from the Gui Re No. 2 tea tree in March or April each year, cut them open and take about 1cm from the center of the leaf. 2 The square part.
[0040] Induction medium formulation: Based on plant MS medium, supplemented with 0.6 mg / L 6-BA, 0.3 mg / L 2,4-D, 0.3 mg / L NAA, 5 g / L activated charcoal, 8 g / L agar, and 1% *Drynaria fortunei* extract. The pH of the leaf callus induction medium was 5.6–5.8. The preparation method of the *Drynaria fortunei* extract was the same as described in Experimental Example 1 of Example 1.
[0041] The callus growth was photographed and recorded after 50 days of culture. The results are shown below. Figure 2 .from Figure 2 It is evident that adding Drynaria fortunei extract can quickly and effectively obtain leaf callus tissue.
[0042] Example 3
[0043] Following the method and formulation described in Example 1 of Example 1, 2% potato extract was further added to the culture medium formulation. The potato extract was prepared as follows: 200g of fresh potatoes were cut into strips, boiled in 1L of water for 10 minutes, and the filtrate obtained by filtering through 8 layers of gauze was the potato extract used in the experiment. Other procedures were the same as described in Example 1 of Example 1 of Example 1.
[0044] Example 4
[0045] Following the method and formula described in Example 1 of Example 1, 5% wild chrysanthemum extract was further added to the culture medium formula. The wild chrysanthemum extract was prepared as follows: 10g of dried wild chrysanthemum was boiled in 100ml of water for 10 minutes, and the filtrate obtained by filtering through two layers of gauze was the chrysanthemum extract used in the experiment. Other procedures were the same as described in Example 1 of Example 1 of Example 1.
[0046] Example 5
[0047] Following the method and formulation described in Example 1 of Example 1, 5% apple juice was added to the culture medium formulation. The apple juice was prepared as follows: 250g of fresh apples were crushed in a high-speed blender for 2 minutes. The crushed liquid was then filtered through eight layers of gauze, and the filtrate was the apple juice used in the experiment. Other procedures were the same as described in Example 1 of Example 1 of Example 1.
[0048] Example 6
[0049] Following the method and formulation described in Example 1 of Example 1, 3% banana juice was further added to the culture medium formulation. The banana juice was prepared as follows: 100g of fresh bananas were added to 50mL of water, and the mixture was blended for 2 minutes using a high-speed blender. The blended liquid was then filtered through eight layers of gauze, and the filtrate was the banana juice used in the experiment. Other procedures were the same as described in Example 1 of Example 1 of Example 1.
[0050] Example 7
[0051] Following the method and formulation described in Example 1 of Example 1, 0.2 mg / L KT and 0.1 g / L MgCl2 were further added to the culture medium formulation. All other aspects were the same as described in Example 1 of Example 1.
[0052] The anthers were inoculated and cultured according to the methods described in Examples 1, 3-7. Five culture bottles were inoculated for each treatment, with three anthers inoculated in each bottle, and the callus induction rate was recorded.
[0053] The results of callus growth are shown in Figure 3 The induction results are shown in the table below.
[0054] Example Induction rate (%) Callus color Callus size Example 1, Test Case 1 60 Emerald green smaller Example 3 80 Pale yellow, light in color and transparent Larger Example 4 94 Emerald green big Example 5 53 reddish-brown maximum Example 6 94 Emerald green Minimum Example 7 80 Pale yellow, light in color smaller
[0055] The results in the table above show that adding *Drynaria fortunei* to the culture medium in Example 1 (Experiment 1) resulted in a higher induction rate, but the callus growth was smaller. Example 3 showed that adding potato extract to the culture medium in Example 1 (Experiment 1) increased the induction rate and resulted in larger callus growth. Example 4 showed that adding *Chrysanthemum indicum* extract to the culture medium in Example 1 (Experiment 1) resulted in the highest induction rate and good callus growth. Example 5 showed that adding apple juice to the culture medium in Example 1 (Experiment 1) did not improve the induction rate and resulted in an unhealthy color, but resulted in the largest callus growth. Example 6 showed that adding banana juice to the culture medium in Example 1 (Experiment 1) improved the induction rate, but resulted in the smallest callus growth. Example 7 showed that adding KT and MgCl2 to the culture medium in Example 1 (Experiment 1) improved the induction rate, but resulted in smaller callus growth.
[0056] The above embodiments are only used to help illustrate the present invention. The implementation of the present invention is not limited to the above embodiments. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. All modifications, substitutions, improvements, etc., made within the basic principles of the present invention should be included within the protection scope of the present invention.
Claims
1. A tea plant callus induction culture medium, based on plant MS medium, characterized in that, The plant MS culture medium contains 0.2% to 1% Drynaria fortunei extract. The Drynaria fortunei extract is prepared by the following method: fresh Drynaria fortunei is dried to constant weight and pulverized; Drynaria fortunei powder is weighed and ethanol with a concentration of 60% to 90% is added at a mass-volume ratio of 1:10 to 20; the mixture is ultrasonicated for 20 to 30 minutes at a temperature of 45 to 55°C, and the filtrate is the Drynaria fortunei extract.
2. The tea tree callus induction culture medium according to claim 1, characterized in that, When the callus is anther callus, the plant MS medium also contains 1.3–1.7 mg / L 6-BA, 0.2–0.4 mg / L 2,4-D, 29–31 g / L sucrose, and 7.4–7.6 g / L agar.
3. The tea tree callus induction culture medium according to claim 2, characterized in that, The pH value of the induction culture medium for the anther callus was 5.8–6.
0.
4. The tea tree callus induction culture medium according to claim 2 or 3, characterized in that, The anther callus induction culture medium also contains 2% potato extract. The potato extract is prepared by cutting fresh potatoes into strips, boiling them in water at a weight ratio of 1:5 for more than 10 minutes, and then filtering.
5. The tea tree callus induction culture medium according to claim 2 or 3, characterized in that, The induction culture medium for the anther callus is also supplemented with 5% wild chrysanthemum extract. The wild chrysanthemum extract is prepared by adding dried wild chrysanthemums to water at a weight ratio of 1:10, boiling for more than 10 minutes, and then filtering.
6. The tea tree callus induction culture medium according to claim 1, characterized in that, When the callus is leaf callus, the plant MS medium also contains 0.4–0.8 mg / L 6-BA, 0.2–0.4 mg / L 2,4-D, 0.2–0.4 mg / L NAA, 4–6 g / L activated carbon, and 7–9 g / L agar.
7. The tea tree callus induction culture medium according to claim 6, characterized in that, The pH value of the induction medium for leaf callus is 5.6 to 5.
8.
8. A method for inducing and propagating tea tree callus, characterized in that, Includes the following steps: (1) Select unopened tea buds, wash them, and place them in the refrigerator's crisper drawer for 6-8 hours at a low temperature. (2) Disinfect and clean the flower buds in sequence with alcohol, 84 disinfectant, and sterile water; (3) Cut open the flower buds and use sterile forceps to pick up the anthers and place them into the prepared anther callus induction medium. The formulation of the induction medium is as follows: plant MS medium as the base medium, with the addition of 1.3-1.7 mg / L 6-BA, 0.2-0.4 mg / L 2,4-D, 29-31 g / L sucrose, 7.4-7.6 g / L agar, and 0.2%-1% Drynaria fortunei extract. The Drynaria fortunei extract is prepared by the following method: fresh Drynaria fortunei is dried to constant weight and pulverized; Drynaria fortunei powder is weighed and ethanol with a concentration of 60%-90% is added at a mass-volume ratio of 1:10-20. The mixture is ultrasonicated at a temperature of 45-55℃ for 20-30 min, and the filtrate is the Drynaria fortunei extract. The pH value of the induction medium is 5.8-6.
0. (4) Culture in the dark for 2 to 3 days, then transfer to 25 to 26°C and continue to culture under 8 hours of darkness and 16 hours of light until callus tissue appears.
9. The method for inducing and propagating tea tree callus tissue according to claim 8, characterized in that, In step (3), 2% potato extract is added to the induction culture medium. The potato extract is prepared by cutting fresh potatoes into strips, boiling them in water at a weight ratio of 1:5 for more than 10 minutes, and then filtering.
10. The method for inducing and propagating tea tree callus tissue according to claim 8, characterized in that, In step (3), 5% wild chrysanthemum extract is added to the induction culture medium. The wild chrysanthemum extract is prepared by adding dried wild chrysanthemums to water at a weight ratio of 1:10 and boiling for more than 10 minutes, then filtering.