Tissue culture method for rapid propagation of taraxacum mongolicum and culture medium and application thereof

Abstract

This invention relates to the field of plant tissue culture, specifically disclosing a method for rapidly propagating dandelion through tissue culture. The method includes the following steps: inducing callus tissue using dandelion leaves as explants; inducing shoot formation from the callus tissue; and rooting the shoots to obtain dandelion tissue culture seedlings. This invention provides a high-quality and efficient tissue culture propagation method for dandelion by utilizing dandelion leaves as explants, inducing callus tissue through dedifferentiation, allowing the callus tissue to redifferentiate and produce shoots, and then rooting individual shoots.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture, and more particularly to a method for rapidly propagating dandelion through tissue culture, the culture medium used, and its applications. Background Technology

[0002] Dandelion ( Taraxacum mongolicum Taraxacum Weber (Handel-Mazzetti) is a plant belonging to the genus Taraxacum Weber in the family Compositae. Plants in this genus are collectively known as dandelions. The genus Taraxacum contains approximately 2,000 species, mainly produced in temperate to subtropical regions of the Northern Hemisphere, with a few found in tropical South America. Dandelion is sweet, bitter, cold in nature, and non-toxic. It enters the liver and stomach meridians and is a traditional Chinese medicine with the effects of clearing heat and detoxifying, reducing swelling and dissipating nodules, and promoting urination. Modern pharmacological research shows that it has good antibacterial, anti-inflammatory, choleretic, hepatoprotective, and immune-boosting effects. Currently, it is mainly supplied to the market through wild collection. Dandelion is a plant with both medicinal and edible uses, containing various nutrients and chemical substances with health benefits. With continuous research and product development of dandelion, a supply shortage is inevitable. This invention utilizes plant tissue culture for dandelion seedling cultivation, exploring a method for rapid propagation of dandelion through tissue culture, providing technical support for dandelion research and seedling production. Summary of the Invention

[0003] To achieve rapid propagation of dandelion through tissue culture, callus induction, bud induction, and rooting culture were conducted using dandelion leaves as explants. This significantly improved the induction rate, rooting rate, and propagation efficiency, resulting in rapid rooting and a shorter seedling cycle. This established a high-quality and efficient tissue culture propagation method.

[0004] To solve the above-mentioned technical problems, the present invention provides a method for rapid propagation of dandelion tissue culture, which involves using dandelion leaves as explants to induce callus tissue, inducing buds in the callus tissue to obtain clustered buds, and rooting the clustered buds to obtain dandelion tissue culture seedlings.

[0005] The plant growth regulators used in the callus induction culture medium contain the following types and contents: 1.0-2 mg / L of 6-benzylaminopurine, 0.3-0.7 mg / L of naphthaleneacetic acid, and 0.2-0.4 mg / L of thidiazuron; preferably 1.5 mg / L of 6-benzylaminopurine, 0.5 mg / L of naphthaleneacetic acid, and 0.2 mg / L of thidiazuron.

[0006] In the above method, the culture medium used for callus induction is a solid culture medium obtained by adding sucrose, a coagulant and a plant growth regulator to MS basic culture medium.

[0007] In the above method, the solidifying agent of the culture medium used for callus induction is agar.

[0008] In the above method, the sucrose content in the culture medium used for callus induction is 30 g / L, the agar content is 7-9 g / L, and the pH value is 5.8-6.0.

[0009] In the above method, the callus induction is performed by culturing for 15-30 days.

[0010] In the above method, the culture conditions for callus induction are a temperature of 22-25℃, a light intensity of 1500-3000 lx, and a daily light exposure of 12-15 h.

[0011] In the above method, the plant growth regulators in the culture medium used for bud induction are 6-benzylaminopurine 2 mg / L and indolebutyric acid 0.3-0.5 mg / L.

[0012] In the above method, the culture medium used for bud induction is a solid culture medium obtained by adding sucrose, a coagulant and a plant growth regulator to MS basic culture medium.

[0013] In the above method, the culture medium used for bud induction is a solid culture medium obtained by adding sucrose, a coagulant and a plant growth regulator to MS basic culture medium.

[0014] In the above method, the solidifying agent of the culture medium used for bud induction is agar.

[0015] In the above method, the sucrose content in the culture medium used for bud induction is 30 g / L, the agar content is 7-9 g / L, and the pH value is 5.8-6.0.

[0016] In the above method, the bud induction is carried out by culturing for 30 days.

[0017] In the above method, the culture conditions for bud induction are a temperature of 22-25℃, a light intensity of 1500-3000 lx, and a daily light exposure of 12-15 h.

[0018] In the above method, the plant growth regulator in the rooting culture medium is 0.5-2 mg / L indolebutyric acid.

[0019] In the above method, the rooting medium is a solid medium obtained by adding sucrose, a coagulant and a plant growth regulator to 1 / 2 MS basic medium.

[0020] In the above method, the solidifying agent of the rooting culture medium is agar.

[0021] In the above method, the sucrose content in the rooting medium is 30 g / L, the agar content is 7-9 g / L, and the pH value is 5.8-6.0.

[0022] In the above method, the rooting process takes 10-21 days.

[0023] In the above method, the rooting culture conditions are a temperature of 22-25℃, a light intensity of 1500-3000 lx, and a daily light exposure of 15 h.

[0024] In the above method, the dandelion leaves are sterile leaves.

[0025] The sterile leaflet is from A1 or A2:

[0026] A1. After disinfecting dandelion seeds, sterile seedlings were obtained by culturing them in MS solid medium. Larger and tender leaves from the sterile seedlings were collected to obtain sterile leaves.

[0027] A2. Collect larger and tender leaves from the dandelion tissue culture seedlings to obtain sterile leaves.

[0028] The tissue culture method for rapid propagation of dandelion of the present invention further includes the step of transplanting and planting dandelion tissue culture seedlings after hardening them off.

[0029] This invention also protects the culture medium used for the above-mentioned bud induction.

[0030] The present invention also protects the application of the method or the culture medium in dandelion production.

[0031] The tissue culture method for rapid propagation of dandelion of the present invention has the following beneficial effects:

[0032] (1) It has the characteristics of strong production capacity, short cultivation cycle, high proliferation coefficient and strong seedling resistance.

[0033] (2) Due to the use of tissue culture asexual reproduction technology, not only can the excellent traits of the mother be maintained, but virus-free seedlings can also be obtained.

[0034] (3) The culture medium is simple to prepare and easy to obtain. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0037] The dandelion variety used in this embodiment is Lu Shou No. 1, and the seeds were purchased from Shouguang Xinxinran Horticulture Co., Ltd.

[0038] Example 1

[0039] This embodiment involves tissue culture of dandelion, specifically following these steps:

[0040] S1. Obtaining sterile materials

[0041] Disinfection: The seeds are sieved to remove impurities, rinsed and soaked in tap water, and the seeds floating on the surface are discarded. The seeds that sink to the bottom are rinsed with running water for 3-5 minutes (2 minutes in this example). In a clean bench, the seeds are first soaked in 75% alcohol by volume for 30-40 seconds (30 seconds in this example), and then rinsed with sterile water 5-6 times (5 times in this example). Then, the seeds are soaked in a sodium hypochlorite aqueous solution with a solute mass percentage of 2%-3% (2% in this example) for 3-5 minutes (5 minutes in this example), and then rinsed with sterile water 5-6 times (5 times in this example) to obtain disinfected seeds.

[0042] After sterilization, place the seeds on sterile filter paper to absorb surface moisture, then place them in MS solid medium. After 5-7 days, the germination in MS medium will result in sterile seedlings.

[0043] In this embodiment, the MS basal medium was prepared by dissolving MS basal medium salt in sterile water (Phytotech product, catalog number: M519), and the amount of MS basal medium salt was 4.43 g / L. In this embodiment, the 1 / 2 MS basal medium was prepared by dissolving 1 / 2 MS basal medium salt in sterile water, and the amount of 1 / 2 MS basal medium salt was 2.22 g / L.

[0044] In this embodiment, the MS solid medium is obtained by adding a carbon source (30 g / L sucrose) and a solidifying agent (7-9 g / L agar) to the MS basal medium. In this embodiment, the 1 / 2 MS solid medium is obtained by adding a carbon source (30 g / L sucrose) and a solidifying agent (7-9 g / L agar) to 1 / 2 MS basal medium.

[0045] S2. Selection of explants

[0046] Select larger, tender leaves from sterile seedlings as sterile leaves, and cut them into 1-2 cm pieces in a clean bench.2 (Specifically, 1cm in this embodiment) 2 Prepare small pieces of sterile leaf material for later use.

[0047] S3, callus induction culture

[0048] Seven callus induction culture media were prepared, as follows:

[0049] A1, MS + 1.5 mg / L 6-BA + 0.5 mg / L NAA + 0.2 mg / L TDZ, is a medium based on MS solid medium with 1.5 mg / L 6-BA (6-benzylaminopurine), 0.5 mg / L NAA (naphthaleneacetic acid), and 0.2 mg / L TDZ (thidiazuron, CAS No.: 51707-55-2).

[0050] A2, MS + 2.0 mg / L 6-BA + 0.2 mg / L NAA, is a medium based on MS solid medium with 2.0 mg / L 6-BA and 0.2 mg / L NAA.

[0051] A3, MS + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 0.5 mg / L 2,4-D, is a medium based on MS solid medium with 2.0 mg / L 6-BA, 0.2 mg / L NAA, and 0.5 mg / L 2,4-D.

[0052] A4, MS + 2.0 mg / L 6-BA + 0.5 mg / L 2,4-D, is a medium based on MS solid medium with 2.0 mg / L 6-BA and 0.5 mg / L 2,4-D.

[0053] A5, MS + 1.0 mg / L 6-BA + 0.5 mg / L 2,4-D, is a medium based on MS solid medium with 1.0 mg / L 6-BA and 0.5 mg / L 2,4-D.

[0054] A6, MS + 2.0 mg / L 6-BA, is a medium based on MS solid medium with a 6-BA concentration of 2.0 mg / L.

[0055] A7, MS + 1.5 mg / L 6-BA + 0.5 mg / L 2,4-D, is a medium based on MS solid medium with 1.5 mg / L 6-BA and 0.5 mg / L 2,4-D.

[0056] The specific preparation methods for the callus induction media of A1-A7 mentioned above are as follows: add plant growth regulators and sucrose to MS basic medium according to the concentration, adjust the volume to 1L, set the pH to 5.8, add solidifying agent, autoclave at 121℃ for 20min and then cool.

[0057] Sterile leaf fragments were inoculated into callus induction media A1, A2, A3, A4, A5, A6, and A7, with three culture dishes for each medium and six sterile leaf fragments inoculated into each dish. The culture dishes were placed in a tissue culture room (temperature 22-25℃, light intensity 3000 lx, 15h light per day). Callus formation occurred after approximately 15 days, and callus induction was completed after approximately 25 days. The callus induction rate was calculated at 25 days of culture, and the results are shown in Table 1.

[0058] Table 1. Effects of different culture media on callus induction rate

[0059]

[0060]

[0061] Table 1 shows that the callus induction rates of callus induction media A1 (MS + 1.5 mg / L 6-BA + 0.5 mg / L NAA + 0.2 mg / LTDZ) and A7 (MS + 1.5 mg / L 6-BA + 0.5 mg / L 2,4-D) were all higher than 85%, with A5 showing the best callus induction effect at 97.7%. Therefore, A1 was selected as the callus induction medium for subsequent experiments.

[0062] S4, Adventitious bud induction culture

[0063] Eight adventitious bud induction media, B1, B2, B3, B4, B5, B6, B7, and B8, were prepared using the following methods: Add plant growth regulators and sucrose to MS basal medium according to their respective concentrations, bring the volume to 1 L, adjust the pH to 5.8, add a solidifying agent, autoclave at 121°C for 20 min, and then cool. The specific types and concentrations of plant growth regulators in each adventitious bud induction medium are shown in Table 2.

[0064] Callus induced on callus induction medium A1 was cut into small pieces and inoculated into eight different shoot induction media, with three culture dishes per type of shoot induction medium and six callus pieces inoculated into each culture dish. The media were then placed in a tissue culture room (temperature 22-25℃, light intensity 3000 lx, 15 h light per day). Shoot formation occurred after approximately 15 days of culture, and shoot induction was completed after approximately 30 days. The shoot induction rate was calculated at 30 days of culture. The results are shown in Table 2.

[0065] Table 2 Effects of different culture media on budding

[0066]

[0067] Table 2 shows that B3 bud induction medium (MS + 2 mg / L 6-BA + 0.5 mg / L IBA) has the best induction effect. Therefore, B3 was selected as the bud induction medium for subsequent experiments.

[0068] S5, Rooting Culture

[0069] Twelve adventitious bud induction media, C1, C2, C3, C4, C5, C6, C7, C8, C9, and C10, were prepared using the following methods: Add plant growth regulators and sucrose to 1 / 2 MS basal medium at the specified concentrations, bring the volume to 1 L, adjust the pH to 5.8, add a solidifying agent, autoclave at 121°C for 20 min, and then cool. The specific types and concentrations of plant growth regulators in each adventitious bud induction medium are shown in Table 3.

[0070] When the induced shoot clusters on B3 medium reached approximately 5 cm in length, individual shoots were cut off and transferred to rooting medium for rooting culture. Three Erlenmeyer flasks were inoculated with six individual shoots per flask. The flasks were placed in a tissue culture room under the following conditions: temperature 22-25℃, light intensity 3000 lx, 15 h light per day. Roots appeared after approximately 10 days of culture. The rooting rate was recorded on day 14 of rooting culture. The results are shown in Table 3.

[0071] Table 3 Effects of different culture media on shoot rooting

[0072]

[0073] Note: Different lowercase letters after the data in the same column indicate significant differences between treatments, P < 0.05.

[0074] Table 3 shows that the rooting medium can be 1 / 2 MS + 1-2 mg / L IBA (i.e., a medium with 1 / 2 MS solid medium as the base medium and an indolebutyric acid (IBA) content of 1-2 mg / L). In this embodiment, C3 (1 / 2 MS + 1.5 mg / L IBA, i.e., a medium with 1 / 2 MS solid medium as the base medium and an indolebutyric acid (IBA) content of 1.5 mg / L) was finally selected as the rooting medium.

[0075] S6. Seedling hardening and transplanting

[0076] First, prepare the mixed substrate for planting (mixed in a ratio of V vermiculite:V soil = 1:1), place it in a 121℃ autoclave for 30 minutes, and then let the mixed substrate cool before use.

[0077] Selected seedlings with abundant and healthy root growth are removed from the culture room. Loosen the bottle caps for 1-2 days and place them in the room to harden off. Then, partially open the caps for 1-2 days, and finally completely remove the caps. After 2-3 days, remove the seedlings from the bottles, wash off the culture medium, let them dry, and then transplant them into pots containing mixed substrate. Place them in a place with low light, cool and ventilated conditions and a humidity of 80%-90%.

[0078] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A tissue culture method for rapid propagation of Taraxacum officinale, characterized by, The process includes the following steps: using dandelion leaves as explants to induce callus tissue, inducing shoots in the callus tissue to obtain clustered shoots, and rooting the clustered shoots to obtain dandelion tissue culture seedlings. The plant growth regulators used in the callus induction culture medium contained the following: 1.5 mg / L of 6-benzylaminopurine, 0.5 mg / L of naphthaleneacetic acid, and 0.2 mg / L of thiamethoxam. The plant growth regulators used in the bud induction medium consist of 2 mg / L 6-benzylaminopurine and 0.5 mg / L indolebutyric acid. The types and contents of plant growth regulators in the rooting culture medium are as follows: indolebutyric acid 1.5 mg / L; The callus induction was carried out for 15-25 days; the bud induction for 30 days; and the rooting culture for 10-21 days. The culture was carried out at a temperature of 22-25℃, a light intensity of 1500-3000 lx, and a daily light exposure of 12-15 h.

2. The method of claim 1, wherein, The culture medium used for callus induction and the culture medium used for shoot induction are both solid culture media obtained by adding sucrose, a coagulant and a plant growth regulator to MS basic culture medium; the culture medium used for rooting is a solid culture medium obtained by adding sucrose, a coagulant and a plant growth regulator to 1 / 2 MS basic culture medium.

3. The method according to claim 2, characterized in that, The solidifying agent for the culture medium used for callus induction, the culture medium used for shoot induction, and the culture medium used for rooting is agar.

4. The method according to claim 3, characterized in that, The sucrose content in the culture medium used for callus induction, the culture medium used for shoot induction, and the culture medium used for rooting are all 30 g / L, the agar content is 7-9 g / L, and the pH is 5.8-6.

0.

5. The method according to any one of claims 1-4, characterized in that, The dandelion leaves mentioned are sterile leaves.

6. The method according to claim 1, characterized in that, The method also includes the step of transplanting and planting dandelion tissue culture seedlings after hardening them off.

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