Induction Method of Callus from Clivia Leaves
By using a specific proportion of hormone culture medium on Clivia leaves, the problem of callus induction in Clivia leaves is solved, rapid and efficient callus induction is achieved, and the efficiency of Clivia reproduction is improved.
Patent Information
- Application Number
- CN202310991064.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-08
- Publication Date
- 2025-07-25
- Estimated Expiration
- 2043-08-08
AI Technical Summary
The prior art is difficult to induce callus quickly and effectively induce callus in Clivia leaf, limiting the reproduction efficiency and application scope of Clivia.
The hormone culture medium was prepared using MS medium with a pH of 5.8 and different proportions of hormone solution (indoleacetic acid, zein, naphthaleneacetic acid, 6-benzylaminopurine) for the cultivation of Clivia leaves to induce callus.
Rapidly induce Clivia leaves to generate callus tissue, simple operation and easy to obtain raw materials, suitable for large-scale industrial operations, improving the efficiency of Clivia reproduction.
Smart Images
Figure CN117136844B_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of tissue culture, and particularly relates to a method for inducing callus of Clivia leaves. Background Art
[0002] Clivia is a perennial herbaceous plant, native to South Africa, and was introduced into China at the beginning of this century. It is named because of its elegant and handsome appearance and the demeanor of a gentleman. At the same time, it also has the functions of purifying the air and medicinal value, and is often used as an indoor potted plant display, with relatively high economic value.
[0003] The traditional propagation methods of Clivia mainly include sowing propagation and division propagation. After a healthy plant is pollinated within the same plant, only a few seeds are obtained, and after cross-pollination, generally only about 10 seeds are obtained; for an adult Clivia plant, at most a dozen offsets can be produced in a year; thus, it can be seen that there are great limitations in propagation by sowing and division.
[0004] In order to improve the propagation coefficient of Clivia, more and more technical personnel have begun to explore the induction culture of explant callus of Clivia. At present, callus has been successfully induced through explants such as bulbs and flower stalks, but there is still no report on the induction of callus using Clivia leaves. And leaves are easier to obtain than bulbs and flower stalks, and cause less damage to the Clivia mother plant. If callus can be successfully induced using Clivia leaves, it will have a broad market prospect.
[0005] Therefore, there is an urgent need for a method for inducing callus of Clivia leaves that can conveniently and quickly induce leaf callus. Summary of the Invention
[0006] The purpose of the present invention is to solve at least the above-mentioned defects and provide the advantages to be described later.
[0007] To achieve these purposes and other advantages of the present invention, there is provided a method for inducing callus of Clivia leaves, including:
[0008] Taking sterile Clivia leaves and culturing them in a hormone medium to obtain Clivia leaf callus.
[0009] The hormone medium includes an MS medium with a pH value of 5.8 and a hormone solution.
[0010] The hormone solution is indoleacetic acid and zeatin with a volume ratio of 2:1, or naphthaleneacetic acid and zeatin with a volume ratio of 2:1, or naphthaleneacetic acid and 6-benzylaminopurine with a volume ratio of 1:1, or naphthaleneacetic acid and zeatin with a volume ratio of 1:6; wherein, the concentrations of indoleacetic acid, zeatin, naphthaleneacetic acid, and 6-benzylaminopurine are all 1 g / L.
[0011] In the above solution, the method for inducing callus of Clivia leaves in the present invention provides suitable conditions for inducing callus of Clivia leaves by preparing a suitable culture medium, and can quickly induce the formation of callus on Clivia leaves.
[0012] The method for inducing callus of Clivia leaves in the present invention is simple to operate. The culture medium for inducing callus of Clivia leaves is easy to prepare, and the required raw materials are simple and easy to obtain, and it can be applied to large-scale industrial operations.
[0013] Preferably, the method for inducing callus of Clivia leaves includes the following steps: preparing the first culture medium: according to the ratio of 2.37 g / L of MS medium (Solarbio) and 30 g / L of sucrose, make up to 3 L with distilled water to obtain the MS medium, and use 1 M NaOH solution to adjust the pH value of the MS medium to 5.8 to obtain the first culture medium;
[0014] Preparing the second culture medium: mixing according to the ratio of 1.5 g of agar and 200 mL of the first culture medium.
[0015] Preparing the third culture medium: adding the hormone solution to 200 mL of the second culture medium and mixing well.
[0016] Inducing callus of leaves: Obtain sterile Clivia leaves and place them in the third culture medium, and culture them in a constant temperature incubator at 25 °C to obtain callus of Clivia leaves.
[0017] Among them, the hormone solution is obtained by mixing 400 μL of indoleacetic acid and 200 μL of zeatin; or 400 μL of naphthylacetic acid and 200 μL of zeatin; or 100 μL of naphthylacetic acid and 100 μL of 6-benzylaminopurine; or 100 μL of naphthylacetic acid and 600 μL of zeatin; the concentrations of indoleacetic acid, zeatin, naphthylacetic acid, and 6-benzylaminopurine are all 1 g / L.
[0018] In the above solution, adjusting the pH value of the MS medium to 5.8 with 1 M NaOH solution specifically includes: sequentially using pH buffers with pH = 4, pH = 6.86, and pH = 9.18 to calibrate the pH meter; using 1 M NaOH solution to adjust the pH value of the MS medium to 5.8.
[0019] Preferably, the preparation of the third culture medium specifically includes: filtering the hormone solution through a 0.22 μm filter membrane, and adding the filtered hormone solution to 200 mL of the second culture medium and mixing well to obtain the third culture medium.
[0020] Preferably, the method for inducing callus of Clivia miniata leaves further includes: after the second culture medium is prepared, it is sealed and sterilized by high temperature and high pressure. The sterilization conditions are a pressure of 103 kPa, a temperature of 120 °C, and a sterilization time of 20 min. After the temperature of the sterilized second culture medium drops to 50 - 60 °C, the hormone solution is added to 200 mL of the second culture medium and mixed thoroughly to obtain the third culture medium. After the third culture medium has completely cooled and solidified, the sterile Clivia miniata leaves are placed in the solidified third culture medium for cultivation.
[0021] Preferably, the first culture medium, the second culture medium, and the third culture medium are all prepared on a superclean workbench, and the required instruments need to be irradiated with ultraviolet light for at least 30 min before operation.
[0022] Preferably, obtaining sterile Clivia miniata leaves specifically includes: preparing a mercuric chloride solution: dissolving 1 g of mercuric chloride in distilled water and making up the volume to 1 L with distilled water; obtaining clean Clivia miniata leaves and trimming them into Clivia segments with a length of 7 - 10 cm; the Clivia segments are successively soaked in 75% alcohol for at least 30 s, soaked and washed with sterile water 3 times (1 min each time), soaked in the mercuric chloride solution for 8 min, and soaked and washed with sterile water 5 times (1 min each time) to obtain the treated Clivia segments; the four sides of the treated Clivia segments that come into contact with the sterilizing solution are cut off, and then cut into a size of 1 cm × 1 cm to 2 cm × 2 cm, and sterile Clivia miniata leaves can be obtained.
[0023] Preferably, the induction of leaf callus specifically includes: placing the third culture medium in a culture bottle, and putting 3 - 4 sterile Clivia miniata leaves in one culture bottle; tying up the mouth of the culture bottle containing the leaves and transferring it to a constant temperature culture room at 25 °C for cultivation;
[0024] Among them, the sterile Clivia miniata leaves are transferred to the culture bottle with forceps. After picking up each sterile Clivia miniata leaf, the forceps need to be sterilized by high temperature. After the forceps have cooled down, the next leaf can be picked up.
[0025] Advantages of the present invention:
[0026] First of all, the method for inducing callus of Clivia miniata leaves of the present invention provides suitable conditions for inducing callus of Clivia miniata leaves by preparing a suitable culture medium, and can quickly induce the formation of callus on Clivia miniata leaves.
[0027] Secondly, the method for inducing callus of Clivia miniata leaves of the present invention is simple to operate. The culture medium for inducing the formation of callus on Clivia miniata leaves is easy to prepare, and the required raw materials are simple and easy to obtain, and it can be applied to large-scale industrial operations. Description of the drawings
[0028] Figure 1 Partial result diagrams for inducing callus from Clivia miniata leaves using the method of Example 1 of the present invention;
[0029] Figure 2 Partial result diagrams for inducing callus from Clivia miniata leaves using the method of Example 2 of the present invention;
[0030] Figure 3 Partial result diagrams for inducing callus from Clivia miniata leaves using the method of Example 3 of the present invention;
[0031] Figure 4 Partial result diagrams for inducing callus from Clivia miniata leaves using the method of Example 4 of the present invention;
[0032] Figure 5 Partial result diagrams for inducing callus from Clivia miniata leaves using the method of Comparative Example 1 of the present invention;
[0033] Figure 6 Partial result diagrams for inducing callus from Clivia miniata leaves using the method of Comparative Example 2 of the present invention;
[0034] Figure 7 Partial result diagrams for inducing callus from Clivia miniata leaves using the method of Comparative Example 3 of the present invention;
[0035] Figure 8 Partial result diagrams for inducing callus from Clivia miniata leaves using the method of Comparative Example 6 of the present invention. Detailed implementation manners
[0036] The following further elaborates on the present invention to enable those skilled in the art to implement it with reference to the text of the specification.
[0037] Method for obtaining sterile Clivia miniata leaves:
[0038] Step A: Prepare a mercuric chloride solution: Dissolve 1 g of mercuric chloride in distilled water and make up the volume to 1 L with distilled water.
[0039] Step B: Obtain clean Clivia miniata leaves and trim them into leaf segments of 7 - 10 cm in length.
[0040] Step C: Immerse the Clivia miniata segments successively in 75% alcohol for at least 30 s, soak and wash them 3 times (1 min each time) with sterile water, immerse them in the mercuric chloride solution for 8 min, and soak and wash them 5 times (1 min each time) with sterile water to obtain processed Clivia miniata segments.
[0041] Step D: Cut off the parts of the processed Clivia miniata segments that come into contact with the sterilizing solution, and then cut them into specifications of 1 cm × 1 cm to 2 cm × 2 cm to obtain sterile Clivia miniata leaves.
[0042] Example 1
[0043] A method for inducing callus of Clivia leaves, comprising:
[0044] Step 1: Prepare the first medium: According to the ratio of 2.37 g / L MS medium (Solarbio) and 30 g / L sucrose, make up to 3 L with distilled water to obtain the MS medium, and adjust the pH value of the MS medium to 5.8 with 1 M NaOH solution to obtain the first medium.
[0045] Step 2: Prepare the second medium: Mix according to the ratio of 1.5 g agar and 200 mL of the first medium.
[0046] Step 3: Prepare stock solutions of indoleacetic acid and zeatin with a concentration of 1 g / L each.
[0047] Step 4: Prepare the third medium: Add 400 μL of the indoleacetic acid stock solution and 200 μL of the zeatin stock solution to 200 mL of the second medium and mix well.
[0048] Step 5: Induce callus of leaves: Obtain sterile Clivia leaves and place them in the third medium, and culture them in a constant temperature incubator at 25 °C to obtain callus of Clivia leaves.
[0049] Example 2
[0050] A method for inducing callus of Clivia leaves, comprising:
[0051] Step 1: Prepare the first medium: According to the ratio of 2.37 g / L MS medium (Solarbio) and 30 g / L sucrose, make up to 3 L with distilled water to obtain the MS medium, and adjust the pH value of the MS medium to 5.8 with 1 M NaOH solution to obtain the first medium.
[0052] Step 2: Prepare the second medium: Mix according to the ratio of 1.5 g agar and 200 mL of the first medium.
[0053] Step 3: Prepare stock solutions of naphthaleneacetic acid and zeatin with a concentration of 1 g / L each.
[0054] Step 4: Prepare the third medium: Add 400 μL of the naphthaleneacetic acid stock solution and 200 μL of the zeatin stock solution to 200 mL of the second medium and mix well.
[0055] Step 5: Induce callus of leaves: Obtain sterile Clivia leaves and place them in the third medium, and culture them in a constant temperature incubator at 25 °C to obtain callus of Clivia leaves.
[0056] Example 3
[0057] An induction method for callus of Clivia leaves, comprising:
[0058] Step 1, prepare the first medium: According to the ratio of 2.37 g / L of MS medium (Solarbio) and 30 g / L of sucrose, make up to 3 L with distilled water to obtain the MS medium, and use 1 M NaOH solution to adjust the pH value of the MS medium to 5.8 to obtain the first medium.
[0059] Step 2, prepare the second medium: Mix according to the ratio of 1.5 g of agar and 200 mL of the first medium.
[0060] Step 3, prepare stock solutions of naphthaleneacetic acid and 6-benzylaminopurine with a concentration of 1 g / L each.
[0061] Step 4, prepare the third medium: Add 100 μL of the naphthaleneacetic acid stock solution and 100 μL of the 6-benzylaminopurine stock solution to 200 mL of the second medium and mix well.
[0062] Step 5, induction of leaf callus: Obtain sterile Clivia leaves and place them in the third medium, and culture them in a constant temperature incubator at 25 °C to obtain Clivia leaf callus.
[0063] Example 4
[0064] An induction method for callus of Clivia leaves, comprising:
[0065] Step 1, prepare the first medium: According to the ratio of 2.37 g / L of MS medium (Solarbio) and 30 g / L of sucrose, make up to 3 L with distilled water to obtain the MS medium, and use 1 M NaOH solution to adjust the pH value of the MS medium to 5.8 to obtain the first medium.
[0066] Step 2, prepare the second medium: Mix according to the ratio of 1.5 g of agar and 200 mL of the first medium.
[0067] Step 3, prepare stock solutions of naphthaleneacetic acid and zeatin with a concentration of 1 g / L each.
[0068] Step 4, prepare the third medium: Add 100 μL of the naphthaleneacetic acid stock solution and 600 μL of the zeatin stock solution to 200 mL of the second medium and mix well.
[0069] Step 5, induction of leaf callus: Obtain sterile Clivia leaves and place them in the third medium, and culture them in a constant temperature incubator at 25 °C to obtain Clivia leaf callus.
[0070] Comparative Example 1
[0071] Step 1. Prepare the first medium: According to the ratio of 2.37 g / L MS medium (Solarbio) and 30 g / L sucrose, make up to 3 L with distilled water to obtain the MS medium. Adjust the pH value of the MS medium to 5.8 with 1 M NaOH solution to obtain the first medium.
[0072] Step 2. Prepare the second medium: Mix according to the ratio of 1.5 g agar and 200 mL of the first medium.
[0073] Step 3. Take 200 mL of the second medium, obtain sterile Clivia leaves and place them in the second medium, and culture them in a constant temperature incubator at 25°C.
[0074] Comparative Example 2
[0075] Step 1. Prepare the first medium: According to the ratio of 2.37 g / L MS medium (Solarbio) and 30 g / L sucrose, make up to 3 L with distilled water to obtain the MS medium. Adjust the pH value of the MS medium to 5.8 with 1 M NaOH solution to obtain the first medium.
[0076] Step 2. Prepare the second medium: Mix according to the ratio of 1.5 g agar and 200 mL of the first medium.
[0077] Step 3. Prepare stock solutions of indoleacetic acid and zeatin with a concentration of 1 g / L each.
[0078] Step 4. Prepare the third medium: Add 200 μL of the indoleacetic acid stock solution and 200 μL of the zeatin stock solution to 200 mL of the second medium and mix well.
[0079] Step 5. Induce leaf callus: Obtain sterile Clivia leaves and place them in the third medium, and culture them in a constant temperature incubator at 25°C.
[0080] Comparative Example 3
[0081] Based on Comparative Example 2, replace Steps 3 and 4 with:
[0082] Step 3. Prepare stock solutions of indoleacetic acid and zeatin with a concentration of 1 g / L each.
[0083] Step 4. Prepare the third medium: Add 400 μL of the indoleacetic acid stock solution and 400 μL of the zeatin stock solution to 200 mL of the second medium and mix well.
[0084] Comparative Example 4
[0085] Based on Comparative Example 2, replace Steps 3 and 4 with:
[0086] Step 3: Prepare naphthaleneacetic acid and zeatin with a mother liquor concentration of 1 g / L each.
[0087] Step 4: Prepare the third culture medium: Add 200 μL of the naphthaleneacetic acid mother liquor and 200 μL of the zeatin mother liquor to 200 mL of the second culture medium and mix well.
[0088] Comparative Example 5
[0089] Based on Comparative Example 2, replace Step 3 and Step 4 with:
[0090] Step 3: Prepare naphthaleneacetic acid and zeatin with a mother liquor concentration of 1 g / L each.
[0091] Step 4: Prepare the third culture medium: Add 400 μL of the naphthaleneacetic acid mother liquor and 400 μL of the zeatin mother liquor to 200 mL of the second culture medium and mix well.
[0092] Comparative Example 6
[0093] Based on Comparative Example 2, replace Step 3 and Step 4 with:
[0094] Step 3: Prepare naphthaleneacetic acid and 2,4-dichlorophenoxyacetic acid with a mother liquor concentration of 1 g / L each.
[0095] Step 4: Prepare the third culture medium: Add 100 μL of the naphthaleneacetic acid mother liquor and 100 μL of the 2,4-dichlorophenoxyacetic acid mother liquor to 200 mL of the second culture medium and mix well.
[0096] Comparative Example 7
[0097] Based on Comparative Example 2, replace Step 3 and Step 4 with:
[0098] Step 3: Prepare 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine with a mother liquor concentration of 1 g / L each.
[0099] Step 4: Prepare the third culture medium: Add 100 μL of the 2,4-dichlorophenoxyacetic acid mother liquor and 100 μL of the 6-benzylaminopurine mother liquor to 200 mL of the second culture medium and mix well.
[0100] Conduct a comparative experiment on the induction of callus from Clivia leaves using the methods of Examples 1-4 and Comparative Examples 1-7
[0101] Obtain Clivia leaves after the same aseptic treatment, randomly divide them into 11 groups with 9 leaves in each group, and under the same environmental conditions, use the methods of Examples 1-4 and Comparative Examples 1-7 to induce callus from Clivia leaves, record the phenomena during the induction process and the time to induce callus, and calculate the induction rate; the induction rate is the percentage of the number of leaves that successfully induced callus to the number of test samples.
[0102] Among them, the induction time starts to be counted after placing the sterile Clivia leaves in the third culture medium, and 24 hours is regarded as 1 day.
[0103] Table 1. Induction results of Clivia leaves
[0104]
[0105]
[0106] From Table 1, the culture conditions of Examples 1 to 4 of the present invention can effectively induce the formation of callus of Clivia leaves, while the culture conditions of Comparative Examples 1 to 7 cannot induce the formation of callus of Clivia leaves. Under unsuitable conditions, the leaves gradually turn yellow from the periphery to the middle over time, lose their vitality, and eventually die.
[0107] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and embodiments. It can be fully applied to various fields suitable for the present invention. For those skilled in the art, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details.
Claims
1. Method for inducing callus of Clivia leaves, characterized in that, Including: Step 1: Prepare the first medium: According to the ratio of 2.37 g / L MS medium and 30 g / L sucrose, make up to 3 L with distilled water to obtain the MS medium. Use 1 M NaOH solution to adjust the pH value of the MS medium to 5.8 to obtain the first medium; Step 2: Prepare the second medium: Mix according to the ratio of 1.5 g agar and 200 mL of the first medium; Step 3: Prepare the third medium: Add the hormone solution to 200 mL of the second medium and mix well; Step 4: Induce leaf callus: Obtain sterile Clivia leaves and place them in the third medium, and culture them in a constant temperature incubator at 25°C to obtain Clivia leaf callus; Among them, the hormone solution is obtained by mixing 400 μL of indoleacetic acid and 200 μL of zeatin; or 400 μL of naphthylacetic acid and 200 μL of zeatin; or 100 μL of naphthylacetic acid and 600 μL of zeatin; the concentrations of indoleacetic acid, zeatin, and naphthylacetic acid are all 1 g / L; Obtaining sterile Clivia leaves specifically includes: Prepare mercuric chloride solution: Dissolve 1 g of mercuric chloride in distilled water and make up to 1 L with distilled water; Obtain clean Clivia leaves and cut them into Clivia segments with a length of 7 - 10 cm; The Clivia segments are successively soaked in 75% alcohol for at least 30 s, soaked and washed 3 times with sterile water, soaked in mercuric chloride solution for 8 min, and soaked and washed 5 times with sterile water to obtain processed Clivia segments; Cut off the four sides of the processed Clivia segments in contact with the sterilizing solution, and then cut them into specifications of 1 cm×1 cm to 2 cm×2 cm to obtain sterile Clivia leaves; Among them, each soaking and washing with sterile water is for 1 min.
2. The method for inducing callus of Clivia leaves according to claim 1, characterized in that, The preparation of the third medium specifically includes: The hormone solution is filtered through a 0.22 μm filter membrane, and the filtered hormone solution is added to 200 mL of the second medium and mixed to obtain the third medium.
3. The method for inducing callus of Clivia leaves according to claim 1, characterized in that, Also included: After the second medium is prepared, seal it and perform high-temperature and high-pressure sterilization treatment. The sterilization conditions are a pressure of 103 kPa, a temperature of 120°C, and a sterilization time of 20 min; After the temperature of the sterilized second medium drops to 50 - 60°C, add the hormone solution to 200 mL of the second medium and mix well to obtain the third medium; After the third medium completely cools and solidifies, place the sterile Clivia leaves in the solidified third medium for culture.
4. The method for inducing callus of Clivia leaves according to claim 1, characterized in that, The preparation of the first medium, the second medium, and the third medium is all carried out in a laminar flow hood, and the required instruments need to be irradiated with ultraviolet light for at least 30 min before operation.
5. The method for inducing callus of Clivia leaves according to claim 1, characterized in that, The induction of leaf callus specifically includes: Take the third medium and place it in a culture bottle, and put 3 - 4 sterile Clivia leaves in one culture bottle; Wrap the mouth of the culture bottle with the leaves placed in it and transfer it to a constant temperature incubator at 25°C for culture. Among them, sterile Clivia leaves are transferred to a culture bottle using forceps. After picking up each sterile Clivia leaf, the forceps need to be sterilized at high temperature. Wait for the forceps to cool down before picking up the next leaf.