Rapid propagation method of large-leaf tea tree tissue culture seedlings

Abstract

The application discloses a rapid propagation method of Camellia sinensis var. assamica tissue culture seedlings, and belongs to the field of plant tissue culture technology.The method comprises the following steps: 1) disinfecting and rinsing tender stem segments or current-year branches of Camellia sinensis var. assamica seedlings to obtain explants; 2) inoculating the explants into an axillary bud induction culture medium to induce culture to obtain axillary buds; 3) inoculating the axillary buds into an axillary bud proliferation induction culture medium to induce culture to obtain multiple shoots; and 4) inoculating the regenerated shoots into a rooting culture medium to induce culture of the multiple shoots to root to obtain Camellia sinensis var. assamica tissue culture seedlings.The rapid propagation method of Camellia sinensis var. assamica tissue culture seedlings can quickly induce culture of Camellia sinensis var. assamica tissue culture seedlings through selection of explants, specific disinfection methods and determination of culture media, and the method has good induction effect and good rooting effect of the tissue culture seedlings, and can be applied to Camellia sinensis var. assamica of different resources and has universality.

Description

Technical Field

[0001] This invention belongs to the field of plant tissue culture technology, specifically relating to a rapid propagation method for tissue culture seedlings of large-leaf tea trees. Background Technology

[0002] Large-leaf tea trees are tea resources or varieties with unique genetic backgrounds that have propagated under special ecological conditions. For example, the Yunnan large-leaf tea tree is characterized by strong growth, early budding, long harvesting period, and plump buds and leaves. Furthermore, its tea leaves are rich in various internal substances, far exceeding those of small- and medium-leaf varieties. The content of tea polyphenols and caffeine in large-leaf tea is 1.5 times and 1.8 times that of small-leaf varieties, respectively, and the content of catechins is 1.1 times that of small-leaf varieties. In production, large-leaf tea trees generally face the technical challenge of low propagation efficiency, posing significant difficulties for resource utilization, especially for the protection of rare and endangered resources.

[0003] Plant tissue culture and in vitro rapid propagation techniques, through aseptic operation and suitable culture conditions, induce germination, proliferation, and rooting of cells, tissues, and organs in a short period of time, ultimately obtaining complete plants with stable genetic traits. Tissue culture rapid propagation has advantages such as short culture cycles, high proliferation efficiency, and is not limited by environmental or seasonal factors. Through appropriate explant sterilization and other induction methods, coupled with the addition of suitable plant growth regulators and culture conditions, rare and endangered tea trees can be preserved. Among these, axillary bud stem segment induction has a high propagation coefficient in subculture, continuously producing clustered buds, and exhibiting stable maternal genetic traits, making it the preferred material for in vitro rapid propagation of tea trees. Currently, there are no reports on obtaining test-tube seedlings from large-leaf tea trees using sterilized explants. Therefore, this application establishes a method for rapid propagation of large-leaf tea tree test-tube seedlings, providing a technical reference for establishing a tea tree genetic transformation system and rapidly propagating test-tube seedlings of rare and endangered tea tree germplasm resources. Summary of the Invention

[0004] To address the problems existing in the prior art, the purpose of this invention is to provide a method for rapid propagation of tissue culture seedlings of large-leaf tea trees, which is achieved through the following technical solution:

[0005] A rapid propagation method for tissue culture seedlings of large-leaf tea trees includes the following steps:

[0006] 1) Disinfect and rinse the tender stem segments or current-year branches of the seedlings of the large-leaf tea tree to obtain explants;

[0007] 2) The explants obtained in step 1) are inoculated into axillary bud induction medium and induced to obtain axillary buds; the axillary bud induction medium is MS medium containing 0.1-2 mg / L of 6-BA and 0.1-2 mg / L of GA3;

[0008] 3) The axillary buds obtained in step 2) are inoculated into the axillary bud proliferation induction medium to induce the growth of clustered buds. The axillary bud proliferation induction medium is WPM medium, which contains 0.5 mg / L 6-BA, 0.1 mg / L IBA and 0.5 mg / L GA3.

[0009] 4) The regenerated buds obtained in step 3) are inoculated into the rooting medium to induce the bud clusters to root, and tissue culture seedlings of large-leaf tea trees are obtained. The rooting medium is a 1 / 8 MS basal medium containing 3 mg / L IBA and 0.5 mg / L NAA.

[0010] Furthermore, in steps 2), 3), and 4), the conditions for induction culture are: 16 hours of light per day and a light intensity of 4000-4500 lux.

[0011] Further, in step 1), the specific steps for disinfecting and rinsing the tender stem segments of the large-leaf tea seedlings are as follows: rinse the tender stem segments with running water overnight or for more than 4 hours, then soak them in polyvinylpyrrolidone solution. After the treatment, perform the following steps in sequence: alcohol disinfection for 60 seconds, rinsing with sterile water 3-4 times, mercuric chloride solution disinfection for 9-15 minutes, rinsing with sterile water 4-5 times, and then absorbing the moisture with filter paper.

[0012] Furthermore, the concentration of the mercuric chloride solution was 0.1%, and the disinfection time was 9 minutes. During the disinfection process, the solution was stirred every 2-3 minutes to ensure thorough disinfection.

[0013] Furthermore, in step 1), when the current year's shoots are used as explants, the specific steps for disinfection and rinsing are as follows: after the current year's shoots are picked, place their bottoms in water, and after the axillary buds sprout, rinse them in turn with running water containing detergent, rinse them with running water, repeat twice, and then continue to disinfect them with mercuric chloride solution, rinse them with sterile water, and absorb the water with filter paper.

[0014] Furthermore, the concentration of detergent in the running water is 10-20%, the detergent rinsing time is 3 minutes, the first running water rinsing time is 1 hour, and the second running water rinsing time is more than 3 hours.

[0015] Furthermore, the concentration of the mercuric chloride solution is 0.1%, the disinfection time is 5-7 minutes, and the solution is rinsed with sterile water 3-4 times, each time for 3 minutes.

[0016] The present invention provides a rapid propagation method for large-leaf tea tree tissue culture seedlings. Through the selection of explants, specific disinfection methods, and determination of culture medium, large-leaf tea tree tissue culture seedlings can be rapidly induced and cultured. The induction effect of the method of the present invention is good, and the rooting effect of the tissue culture seedlings is good. At the same time, the method of the present invention can be applied to large-leaf tea trees of different resources and has universality. Attached Figure Description

[0017] Figure 1 The growth performance after two subcultures under different WPM medium formulations;

[0018] Figure 2 A statistical chart of the propagation rate of different genotypes of large-leaf tea tree resources during propagation culture;

[0019] Figure 3 A graph showing the propagation rate of different genotypes of large-leaf tea tree resources.

[0020] Figure 4 Graphs showing the rooting induction effects of different genotypes of large-leaf tea tree resources. Detailed Implementation

[0021] The present invention will be further described below with reference to specific embodiments in order to better understand the technical solution.

[0022] Plant materials: The experimental materials were stem segments of seedlings of large-leaf tea trees (seedlings were germinated from tea seeds in a 1:1 ratio of vermiculite and perlite, watered every 3-5 days, and grew to one or two true leaves in 18-21 days, with the roots and leaves removed but the petioles retained) or new shoots of the current year.

[0023] Example

[0024] 1) Preparation and disinfection of explants

[0025] Preparation and disinfection of tender stem segments from seedlings: Using tender stem segments from seedlings as explants, remove the roots and leaves, leaving the petioles. Gently wash with detergent, then soak in detergent for about 10 minutes, stirring constantly. Rinse thoroughly with tap water and rinse under running water overnight or for at least 4 hours. Next, soak in a 2 g / L polyvinylpyrrolidone (PVPP) solution for 30 minutes and place in a clean bench for later use. After washing, rinse the stem segments with 75% alcohol for 60 seconds and then with sterile water 3-4 times. Then, disinfect with 0.1% mercuric chloride at three times (9 min, 12 min, 15 min), stirring every 2-3 minutes to ensure thorough disinfection. Rinse with sterile water 4-5 times and blot dry with filter paper. Cut stem segments about 1 cm long with axillary buds using sterilized scissors and tweezers.

[0026] Preparation and disinfection of explant materials from new shoots: Place the bottom of the harvested new shoots from the current year in water and leave them in a relatively clean environment for about 2 weeks. After the axillary buds sprout, first pour 10-20% detergent into a running water rinsing device, shake and rinse for 3 minutes until there is no more foam, then rinse with running water for 1 hour, and continue to shake and rinse for 3 minutes, and finally continue to rinse with running water for more than 3 hours; disinfect the rinsed axillary buds with 0.1% mercuric chloride in a laminar flow hood for 5-7 minutes, wash with sterile water 3-4 times, 3 minutes each time, and blot dry with filter paper.

[0027] 2) Axillary bud induction

[0028] Axillary buds were induced by horizontally attaching the cut stem segments to axillary bud induction medium. Each stem segment from a seedling was placed independently, or axillary buds with xylem (biological growth direction downward) were inserted obliquely into the medium, one axillary bud per bottle. The basic medium was MS medium supplemented with 0.1-2 mg / L 6-BA and 0.1-2 mg / L GA3.

[0029] 3) The induced axillary buds were inoculated into axillary bud proliferation induction medium to induce the growth of clustered buds. The axillary bud proliferation induction medium was WPM medium supplemented with 0.5 mg / L 6-BA, 0.1 mg / L IBA and 0.5 mg / L GA3.

[0030] 4) The obtained regenerated buds were inoculated into rooting medium to induce rooting of the clustered buds, and tissue culture seedlings of large-leaf tea trees were obtained. The rooting medium was 1 / 8 MS basal medium containing 3 mg / L IBA and 0.5 mg / L NAA.

[0031] Verification Example 1

[0032] In this embodiment, during the axillary bud induction stage, for the seedling stem segments, two seedling stem segments were inoculated on each plate, each seedling had 3-5 stem segments, and each treatment had 48 seedlings. The contamination rate and germination rate were counted after 20 days.

[0033] Contamination rate (%) = (Number of contaminated explants / Total number of explants) × 100%

[0034] Germination rate (%) = (Number of germinated explants / Total number of explants) × 100%

[0035] In this embodiment, different disinfectants were used to disinfect the explants, while other disinfection steps were the same. The effects of different disinfectants on the disinfection effect of stem segments are shown in Table 1.

[0036] Table 1

[0037]

[0038]

[0039] As can be seen from Table 1, disinfection using 0.1% mercuric chloride of the present invention results in a low contamination rate and a high germination rate.

[0040] Explant viability and axillary bud germination rate gradually decrease with prolonged disinfection time. The contamination rate and germination rate results obtained according to the disinfection method and axillary bud induction method in this embodiment are shown in Table 2.

[0041] Table 2: Effect of different treatment times with 0.1% mercuric chloride on the disinfection effect of stem segments

[0042]

[0043] Note: Data represent mean ± standard error. Different lowercase letters in the same column indicate significant differences in the Duncan test (P ≤ 0.05).

[0044] As shown in Table 2, 0.1% mercuric chloride disinfection takes 9 minutes to achieve the best effect.

[0045] During the proliferation induction phase, different culture media and hormone formulations have a significant impact on axillary bud proliferation. In this example, a comparative experiment was conducted on different culture media and formulations, and the results are shown in 3.

[0046] Table 3

[0047]

[0048] Note: Data represent mean ± standard error. Different lowercase letters in the same column indicate significant differences in the Duncan test (P≤0.05).

[0049] As shown in Table 3, the test-tube seedlings exhibited low proliferation and differentiation capacity on MS medium, while the average number of shoots induced on WPM medium was higher than that induced on MS medium.

[0050] The growth performance after two subcultures under different WPM medium formulations is shown in the figure. Figure 1 As shown, Figure 1 Medium, A: WPM+0.5mg / L6-BA, 0.1mg / LIBA and 0.5mg / L GA3; B: WPM+0.2mg / L6-BA, 0.1mg / LIBA and 0.5mg / L GA3; C: WPM+1.0mg / L6-BA, 0.1mg / LIBA and 0.5mg / L GA3. Depend on Figure 1It can be seen that a comparative experiment with different hormone formulations was set up during the proliferation culture stage, and the growth and proliferation effects were compared after two consecutive subcultures. The results showed that when the concentration of 6-BA was 0.5 mg / L, the tissue culture seedlings had the best proliferation and growth effects. The plants showed good growth vitality and strong proliferation ability. At other concentrations, the tea plant tissue culture seedlings gradually aged or grew abnormally. Therefore, the addition of 0.5 mg / L 6-BA, 0.1 mg / L 6-BA, and 0.5 mg / L GA3 WPM induced tender green leaves in the clustered buds, with the highest average number of buds and strong differentiation ability.

[0051] Verification Example 2

[0052] The propagation and cultivation of different genotypes of large-leaf tea tree resources were carried out according to the method of this invention, and the statistical results of the propagation rate are as follows: Figure 2 As shown, the proliferation effect is as follows Figure 3 As shown. From Figure 2 It can be seen that the propagation rate of different genotype large-leaf tea tree resources using the method of the present invention is much higher than the reported propagation rate of tea tree tissue culture, indicating that the method of the present invention has universality.

[0053] Verification Example 3

[0054] Rooting induction was performed according to the method of this invention, and the rooting induction performance of different genotypes of large-leaf tea tree resources is shown in the figure. Figure 4 ,Depend on Figure 4 It can be seen that tea tree resources of different genotypes with large leaves can all take root quickly, with a rooting rate of over 95%.

Claims

1. A method for rapid propagation of tissue culture seedlings of large-leaf tea trees, characterized in that, Includes the following steps: 1) Disinfect and rinse the tender stem segments or current-year branches of the seedlings of the large-leaf tea tree to obtain explants; 2) The explants obtained in step 1) are inoculated into axillary bud induction medium to induce axillary buds; the axillary bud induction medium is MS medium containing 0.1-2 mg / L of 6-BA and 0.1-2 mg / L of GA3; 3) The axillary buds obtained in step 2) are inoculated into the axillary bud proliferation induction medium to induce the growth of clustered buds. The axillary bud proliferation induction medium is WPM medium, which contains 0.5 mg / L 6-BA, 0.1 mg / L IBA and 0.5 mg / L GA3. 4) The clustered buds obtained in step 3) are inoculated into the rooting medium to induce rooting of the clustered buds and obtain tissue culture seedlings of large-leaf tea tree. The rooting medium is 1 / 8MS basal medium containing 3 mg / L IBA and 0.5 mg / L NAA. In steps 2), 3), and 4), the conditions for induction culture are: 16 hours of light per day and light intensity of 4000-4500 lux.

2. The method for rapid propagation of large-leaf tea tree tissue culture seedlings as described in claim 1, characterized in that... In step 1), the specific steps for disinfecting and rinsing the tender stem segments of the large-leaf tea seedlings are as follows: rinse the tender stem segments with running water overnight or for more than 4 hours, then soak them in polyvinylpyrrolidone solution. After the treatment, disinfect them with alcohol for 60 seconds, rinse them with sterile water 3-4 times, disinfect them with mercuric chloride solution for 9-15 minutes, rinse them with sterile water 4-5 times, and then blot them dry with filter paper.

3. The method for rapid propagation of large-leaf tea tree tissue culture seedlings as described in claim 2, characterized in that... The mercuric chloride solution concentration is 0.1%, and the disinfection time is 9 minutes. During the disinfection process, the solution is stirred every 2-3 minutes to ensure thorough disinfection.

4. The method for rapid propagation of large-leaf tea tree tissue culture seedlings as described in claim 1, characterized in that... In step 1), when using current-year shoots as explants, the specific steps for disinfection and rinsing are as follows: After harvesting the current-year shoots, place their bottoms in water. After the axillary buds sprout, rinse them with running water containing detergent, rinse them with running water, repeat twice, and then continue to disinfect them with mercuric chloride solution, rinse them with sterile water, and absorb the water with filter paper.

5. The method for rapid propagation of large-leaf tea tree tissue culture seedlings as described in claim 4, characterized in that... The concentration of detergent in the running water is 10-20%, the detergent rinsing time is 3 minutes, the first rinsing time is 1 hour, and the second rinsing time is more than 3 hours.

6. The method for rapid propagation of large-leaf tea tree tissue culture seedlings as described in claim 4, characterized in that... The concentration of the mercuric chloride solution is 0.1%, the disinfection time is 5-7 minutes, and the solution is rinsed with sterile water 3-4 times, each time for 3 minutes.

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