A rooting culture medium and tissue culture method for Rhizophora stylosa
By using sugar-free culture medium and optimized culture conditions, the problem of difficult propagation of Rhizophora stylosa was solved, achieving efficient artificial cultivation and improving the rooting rate and seedling rate.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- JIANGMEN XINHUI FORESTRY SCIENCES RESEARCH INSTITUTE
- Filing Date
- 2024-07-19
- Publication Date
- 2026-07-03
AI Technical Summary
The propagation of *Rhizophora stylosa* is difficult, with high requirements for its growth environment. Self-incompatibility leads to slow seed growth, poor results in artificial cultivation, high risk of contamination, and low seedling and transplant survival rates.
Sugar-free culture medium was used for seedling cultivation and rooting culture. Sugars in conventional culture media were removed, and inorganic materials vermiculite and perlite were added. Culture conditions such as temperature, humidity and carbon dioxide concentration were controlled. Sodium nitrophenolate and potassium humate were used to promote rooting, and light conditions were optimized.
It improved the rooting rate, seedling rate, and seedling transplant survival rate of Rhizophora stylosa, reduced the risk of pollution, and achieved efficient artificial cultivation.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of plant culture technology, and in particular to a rooting culture medium for Rhizoma Rhizoma Cypriniforme and a tissue culture method therefor. Background Technology
[0002] Mangroves are a vital coastal ecosystem, playing a crucial role in maintaining marine ecological balance and protecting coastlines. However, due to factors such as human activities and climate change, the number and area of mangroves are steadily declining. Artificial propagation of mangroves can increase their area and quantity, maintaining and protecting the integrity and stability of coastal ecosystems. However, some mangrove species, whether trees or shrubs, have specific propagation requirements and may face difficulties propagating naturally; the mangrove shrub is one such example.
[0003] *Bruguiera gymnorrhiza* is a tree or shrub belonging to the genus *Bruguiera* in the family Rhizophoraceae. It has unisexual flowers, is dioecious (with male and female flowers on separate plants), and requires cross-pollination for reproduction. Furthermore, some *Bruguiera gymnorrhiza* trees exhibit self-incompatibility, resulting in slow or even non-germinating seeds from self-pollination, making reproduction difficult. Secondly, *Bruguiera gymnorrhiza* has high requirements for its growth and reproduction environment. Living in the tidal environment of seawater, its seeds can only germinate in a stable environment with suitable temperature, humidity, and sufficient oxygen. Poor growing conditions negatively impact its reproductive success. Therefore, it is necessary to develop an artificial cultivation method for *Bruguiera gymnorrhiza*. Summary of the Invention
[0004] To address the above problems, this invention provides a tissue culture method for *Rhizophora stylosa*, which enables the artificial culture of *Rhizophora stylosa* with high rooting rate, high seedling rate, and high survival rate of transplanted *Rhizophora stylosa* seedlings.
[0005] To achieve the above objectives, the present invention provides a tissue culture method for Rhizoma Cypripedium, comprising the following steps: obtaining explants, disinfecting them, transplanting them to a seedling culture medium for primary culture, subculturing them, transplanting the subcultured explants to a rooting culture medium for rooting culture, and obtaining Rhizoma Cypripedium seedlings.
[0006] The seedling culture medium and rooting culture medium are sugar-free culture media.
[0007] When the inventors studied the cultivation of *Rhizophora stylosa*, they found that the poor results of artificial cultivation were mainly due to the following technical difficulties: 1. High material and labor costs: the raw materials for the culture medium suitable for *Rhizophora stylosa* are expensive and the process is complex; 2. In order to provide sufficient nutrients, sugars are usually added to the culture medium used to cultivate *Rhizophora stylosa*, but this also makes the culture medium susceptible to contamination by bacteria, resulting in a high risk of contamination; 3. Due to its specific growth and reproduction habits, *Rhizophora stylosa* has a low seed germination rate and a low rooting rate. Slight changes in the composition ratio of the culture medium, temperature, humidity, light intensity, and other conditions can affect rooting; 4. Difficulty in hardening off seedlings and low survival rate after transplanting. Therefore, the inventors propose using a sugar-free culture medium for the seedling and rooting culture of *Rhizophora stylosa*. The sugars in the medium are eliminated, reducing the risk of contamination. Furthermore, in natural propagation, *Rhizophora stylosa* mostly reproduces through the hypocotyl. During the germination stage, with sufficient nutrients, the plants grow rapidly. However, once the hypocotyl's nutrients are depleted, many seedlings cannot adapt to the natural environment and are prone to death. In artificial cultivation, using sugar-free tissue culture seedlings allows the *Rhizophora stylosa* to adapt to the microenvironment from the beginning, making the transition easier and more adaptable. Simultaneously, the above cultivation method requires less manual labor, allows for large-scale container cultivation, results in a large and uniform seedling output, and achieves better cultivation results.
[0008] In one embodiment, the seedling culture medium includes a seedling culture solution, vermiculite, and perlite; the volume ratio of vermiculite to the seedling culture solution is 1.5L:(600-1000)mL, and the volume ratio of vermiculite to perlite is (2-4):1.
[0009] The above-mentioned seedling culture medium is a sugar-free medium. Sugars have been removed, which reduces the risk of contamination. Inorganic materials are used as the culture medium, which is low in material cost. Copper welding wire is used because the seedlings of *Rhizophora stylosa* require a lot of water. Perlite is relatively light, which can greatly reduce the weight of the substrate and absorb a certain amount of water, which helps to regulate soil moisture and avoid the soil being too wet or too dry. It also has better air permeability.
[0010] In one embodiment, the seedling culture medium comprises raw materials of the following concentrations:
[0011]
[0012] In one embodiment, the rooting medium comprises raw materials at the following concentrations:
[0013]
[0014]
[0015] During the tissue culture process of *Rhizophora stylosa*, the inventors discovered that the root growth of *Rhizophora stylosa* seedlings was inhibited due to obstructed water and nutrient absorption. After repeated experiments, the inventors found that this was caused by excessively high concentrations of solution in the soil. Therefore, they proposed removing NaCl, a common component in conventional mangrove culture media, to reduce the concentration of the culture medium solution. At the same time, sodium nitrophenolate was added to promote protoplasmic streaming, improve cell viability, and promote callus formation at the cut, thus achieving rapid rooting. Potassium humate was added to promote the synthesis of auxin enzymes, improve plant resistance, and help the plant adapt to the environment quickly.
[0016] In one embodiment, the conditions for the initial culture include: a temperature of 25-26°C, humidity of 80-85%, light intensity of 2000-2500 LX, a light duration of 10 h / d, and the introduction of carbon dioxide at a concentration of 1000-1500 μmol / mol during light exposure; the initial culture time is 3.5-4.5 weeks.
[0017] In one embodiment, the subculture conditions include: a temperature of 25-27°C, a humidity of 80-85%, a light intensity of 3000-4000 LX, a light duration of 10 h / d, and the introduction of carbon dioxide at a concentration of 1800-2500 μmol / mol during light exposure.
[0018] In one embodiment, the rooting culture conditions include: temperature 26-27℃, humidity 65-80%, light intensity 3000-4000 LX, light duration 10 h / d, and carbon dioxide concentration of 3000-3500 μmol / mol during light exposure; the rooting culture time is 3.5-4.5 weeks.
[0019] In the above-mentioned primary culture, subculture, and rooting culture steps, carbon dioxide is artificially introduced to promote the conversion of plants from heterotrophic to autotrophic, resulting in a high seedling rate.
[0020] In one embodiment, obtaining the explant includes: pruning the target branch, spraying the target branch with potassium dihydrogen phosphate solution 2.5-3.5 days after pruning, and obtaining the explant 1.5-2.5 weeks after pruning.
[0021] In one embodiment, during the explant acquisition step, the target branch is pruned down to the remaining 2-3 leaves at the top.
[0022] In one embodiment, the disinfection includes: cleaning with a benzalkonium bromide solution with a volume concentration of 8-12%, and soaking in a sodium hypochlorite solution with a volume concentration of 4-6% for 15-25 minutes.
[0023] The present invention also provides a rooting culture medium for the tissue culture method.
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] The present invention provides a rooting culture medium and tissue culture method for Rhizoma Cypripedium, which enables artificial culture of Rhizoma Cypripedium with high rooting rate, high seedling rate, and high survival rate of transplanted seedlings. Attached Figure Description
[0026] Figure 1 This is a schematic diagram of the *Rhizophora stylosa* seedlings cultivated in Example 1;
[0027] Figure 2 This is a schematic diagram of the *Rhizophora stylosa* seedlings cultivated in Example 2;
[0028] Figure 3 This is a schematic diagram of the *Rhizophora stylosa* seedlings cultivated in Example 3;
[0029] Figure 4 This is a schematic diagram of the *Rhizophora stylosa* seedlings cultivated in Example 4. Detailed Implementation
[0030] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.
[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
[0032] source:
[0033] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all experimental methods are conventional experimental methods in this field.
[0034] Example 1
[0035] A tissue culture method for Rhizophora stylosa.
[0036] I. Obtaining explants.
[0037] Two weeks before obtaining the explants, the target branches were pruned, leaving only the top two leaves. Three days after pruning, the branches were sprayed with a 0.3% potassium dihydrogen phosphate solution to obtain the explants.
[0038] II. Disinfection.
[0039] After washing the explants with a 10% benzalkonium bromide solution, soak them in a 5% sodium hypochlorite solution for 20 minutes, rinse them thoroughly with clean water, and then transplant them into the seedling culture medium.
[0040] Third, conduct initial generation training and subsequent generation training.
[0041] The explants were first cultured, and then subcultured.
[0042] The seedling culture medium consists of vermiculite + perlite and culture solution A (i.e., seedling culture solution). For every 1.5L of vermiculite + perlite, add 800mL of culture solution A. The volume ratio of vermiculite to perlite is 3:1.
[0043] The formula for culture medium A is shown below:
[0044]
[0045]
[0046] In this embodiment, culture medium A is composed of raw materials with the following concentrations:
[0047]
[0048] Initial culture conditions: temperature 25-26℃, humidity 80-85%, light intensity 2000-2500 LX, 10h / d, carbon dioxide is introduced simultaneously with light and maintained at a concentration of 1000-1500 μmol / mol. After 4 weeks, subculture once. The branches are transferred to a sterilized workbench, cut into stem segments of about 5cm with leaves, and transplanted into a new culture medium. Repeat this step until sufficient material is obtained.
[0049] Subculture conditions: temperature 25-27℃, humidity 80-85%, light intensity 3000-4000 LX, 10 h / d, carbon dioxide is introduced simultaneously with light and maintained at a concentration of 1800-2500 μmol / mol.
[0050] IV. Rooting culture.
[0051] The formula for the rooting medium is as follows:
[0052]
[0053] In this embodiment, the rooting medium consists of the following concentrations of raw materials:
[0054]
[0055]
[0056] Rooting culture conditions: temperature 26-27℃, humidity 65-80%, light intensity 3000-4000 LX, 10 h / d, carbon dioxide concentration maintained at 3000-3500 μmol / mol. -1 After maintaining the soil for 4 weeks, the rooting rate reached 99%, yielding *Rhizophora stylosa* seedlings, which can then be transplanted. Example 1's cultivation method has a wider humidity control range and requires a higher carbon dioxide concentration.
[0057] Example 1 shows the cultivation of *Rhizophora stylosa* seedlings. Figure 1 As shown.
[0058] Example 2
[0059] A tissue culture method for Rhizoma Cylindrica is basically the same as in Example 1, except that sodium nitrophenolate and potassium humate are not used in the rooting medium. The resulting Rhizoma Cylindrica seedlings are shown below. Figure 2 As shown.
[0060] Example 3
[0061] A tissue culture method for Rhizoma Cylindrica is basically the same as in Example 1, except that the rooting medium does not contain potassium humate. The resulting Rhizoma Cylindrica seedlings are shown below. Figure 3 As shown.
[0062] Example 4
[0063] A method for tissue culture of Rhizophora stylosa is basically the same as in Example 1, except that the rooting medium does not contain sodium nitrophenolate. The resulting Rhizophora stylosa seedlings are shown below. Figure 4 As shown.
[0064] Example 5
[0065] A tissue culture method for Rhizophora stylosa is basically the same as that in Example 1, except that the concentration of sodium nitrophenolate in the rooting medium is 0.04 mg / L and the concentration of potassium humate is 1 mg / L.
[0066] Experimental Example
[0067] The *Rhizophora stylosa* seedlings cultivated in the above embodiments were tested, and the test results are shown below.
[0068] Table 1. Results of *Rhizophora stylosa* seedlings obtained in each embodiment.
[0069]
[0070] Note: The "Culture Solution A" item in the table above refers to the use of Culture Solution A in both the seedling cultivation and rooting cultivation steps as a control.
[0071] The data in the table above shows that Example 1 had the best cultivation effect. Although Example 5 also contained sodium nitrophenolate and potassium humate, the concentrations of these two components were higher than in Example 1. Conversely, this caused stress to the normal growth of the Rhizophora stylosa, resulting in a decrease in all indicators except for the average number of roots. The cultivation method in Example 1 had a wider range of humidity control and required a higher carbon dioxide concentration.
[0072] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0073] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. A method for tissue culture of Rhizophora stylosa, characterized in that, Includes the following steps: Prune the target branches. 2.5-3.5 days after pruning, spray the target branches with potassium dihydrogen phosphate solution. 1.5-2.5 weeks after pruning, obtain the explants. Disinfect the plants, transplant them to the seedling culture medium for primary culture, subculture, and then transplant the subcultured explants to the rooting culture medium for rooting culture to obtain Rhizophora stylosa seedlings. The seedling culture medium and rooting culture medium are sugar-free culture media; The seedling culture medium comprises a seedling culture solution, vermiculite, and perlite; the volume ratio of vermiculite to the seedling culture solution is 1.5L:(600-1000)mL, and the volume ratio of vermiculite to perlite is (2-4):1; the seedling culture solution comprises raw materials of the following concentrations: The rooting medium comprises the following concentrations of raw materials: 。 2. The tissue culture method according to claim 1, characterized in that, The conditions for the initial culture include: temperature of 25-26℃, humidity of 80-85%, light intensity of 2000-2500 LX, light duration of 10 h / d, and carbon dioxide concentration of 1000-1500 μmol / mol during light exposure; the initial culture time is 3.5-4.5 weeks.
3. The tissue culture method according to claim 2, characterized in that, The conditions for the subculture include: temperature of 25-27℃, humidity of 80-85%, light intensity of 3000-4000 LX, light duration of 10 h / d, and carbon dioxide concentration of 1800-2500 μmol / mol during light exposure.
4. The tissue culture method according to claim 2, characterized in that, The conditions for rooting culture include: temperature 26-27℃, humidity 65-80%, light intensity 3000-4000 LX, light duration 10 h / d, and carbon dioxide concentration of 3000-3500 μmol / mol during light exposure; the rooting culture time is 3.5-4.5 weeks.
5. The tissue culture method according to claim 2, characterized in that, The disinfection process includes: cleaning with a benzalkonium bromide solution with a volume concentration of 8-12%, and soaking in a sodium hypochlorite solution with a volume concentration of 4-6% for 15-25 minutes.
6. The rooting medium in the tissue culture method according to any one of claims 1-5.