Tissue culture method for rapidly propagating dandelion, culture medium used by tissue culture method and application of tissue culture method

Abstract

The invention relates to the field of plant tissue culture, and particularly discloses a tissue culture method for rapidly propagating dandelion, which comprises the following steps: performing callus induction by taking dandelion leaves as explants to obtain calluses, performing bud induction on the calluses to obtain cluster buds, and rooting the cluster buds to obtain dandelion tissue culture seedlings. According to the tissue culture method for rapidly propagating the dandelion, disclosed by the invention, a high-quality and efficient tissue culture propagation method is established by taking dandelion leaves as explants, inducing calluses through dedifferentiation, re-differentiating the calluses to germinate and rooting a single bud.

Description

Technical Field

[0001] The invention relates to the field of plant tissue culture, in particular to a tissue culture method for rapidly propagating dandelions and a culture medium and application thereof. Background Art

[0002] Dandelion Taraxacum mongolicum Handel-Mazzetti) is a plant of the genus Taraxacum Weber in the family Compositae. The plants of this genus are collectively referred to as dandelions. There are about 2,000 species of dandelions, which are mainly produced in the temperate to subtropical regions of the northern hemisphere, and a few are produced in tropical South America. Dandelions are sweet, bitter, cold, non-toxic, and belong to the liver and stomach meridians. They are traditional Chinese medicinal materials with the effects of clearing away heat and detoxifying, reducing swelling and dispersing knots, and promoting diuresis and relieving stranguria. Modern pharmacological studies have shown that they have good antibacterial and anti-inflammatory, gallbladder-protecting and immune-promoting effects. At present, the market demand is mainly supplied by field collection. Dandelions are plants with medicinal and edible properties, containing a variety of nutrients and chemical substances with health-care effects. With the continuous research and product development of dandelions, there will inevitably be a phenomenon of supply exceeding demand. The present invention utilizes plant tissue culture to culture dandelion seedlings, explores a method for rapid propagation of dandelion tissue culture, and provides technical support for the research and seedling production of dandelions. Summary of the invention

[0003] In order to achieve the goal of rapid propagation of dandelion tissue culture, dandelion leaves were used as explants for callus induction, bud induction and rooting culture, which greatly improved the induction rate, rooting rate and propagation efficiency, achieved rapid rooting, shortened the seedling cycle, and established a high-quality and efficient tissue culture propagation method.

[0004] In order to solve the above technical problems, the present invention provides a tissue culture method for rapid propagation of dandelion, wherein dandelion leaves are used as explants to perform callus induction to obtain callus tissue, bud induction is performed on the callus tissue to obtain clustered buds, and the clustered buds are rooted to obtain dandelion tissue culture seedlings; The types and contents of the plant growth regulators in the culture medium used for callus induction are: 1.0-2 mg / L of 6-benzylaminopurine, 0.3-0.7 mg / L of naphthylacetic acid and 0.2-0.4 mg / L of thidiazuron; preferably 1.5 mg / L of 6-benzylaminopurine, 0.5 mg / L of naphthylacetic acid and 0.2 mg / L of thidiazuron.

[0005] 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.

[0006] In the above method, the solidifying agent of the culture medium used for callus induction is agar.

[0007] 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.

[0008] In the above method, the callus induction is cultured for 15-30 days.

[0009] In the above method, the culture conditions for callus induction are temperature 22-25°C, light intensity 1500-3000 lx, and daily light exposure for 12-15 h.

[0010] In the above method, the type and content of the plant growth regulator in the culture medium used for bud induction are 2 mg / L 6-benzylaminopurine and 0.3-0.5 mg / L indolebutyric acid.

[0011] 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.

[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 solidifying agent of the culture medium used for bud induction is agar.

[0014] 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.

[0015] In the above method, the bud induction is cultured for 30 days.

[0016] In the above method, the culture conditions for bud induction are a temperature of 22-25°C, a light intensity of 1500-3000 lx, and a daily light intensity of 12-15 h.

[0017] In the above method, the type and content of the plant growth regulator in the culture medium used for rooting is 0.5-2 mg / L indolebutyric acid.

[0018] In the above method, 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.

[0019] In the above method, the solidifying agent of the culture medium used for rooting is agar.

[0020] 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.

[0021] In the above method, the rooting is cultured for 10-21 days.

[0022] In the above method, the rooting culture conditions are a temperature of 22-25°C, a light intensity of 1500-3000 lx, and a daily light intensity of 15 h.

[0023] In the above method, the dandelion leaves are sterile leaves.

[0024] The sterile leaves are from A1 or A2: A1. After sterilizing dandelion seeds, the seeds are cultured in MS solid culture medium to obtain sterile seedlings, and larger and tenderer leaves on the sterile seedlings are collected to obtain sterile leaves; A2. Collecting larger and tenderer leaves from the dandelion tissue culture seedlings to obtain sterile leaves.

[0025] The tissue culture method for rapid propagation of dandelions of the present invention also comprises the step of hardening the dandelion tissue culture seedlings and then transplanting and planting them.

[0026] The present invention also protects the culture medium used for the above-mentioned bud induction.

[0027] The present invention also protects the use of the method or the culture medium in dandelion production.

[0028] The tissue culture method for rapid propagation of dandelions of the present invention has the following beneficial effects: (1) It has the characteristics of strong production capacity, short cultivation cycle, high proliferation coefficient and strong seedling resistance.

[0029] (2) By adopting tissue culture asexual propagation technology, not only can the excellent traits of the mother plant be maintained, but also virus-free seedlings can be obtained.

[0030] (3) The culture medium is simple and easy to prepare. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0033] The dandelion variety used in this embodiment is Lushou No. 1, and the seeds were purchased from Shouguang Xinxinran Horticulture Co., Ltd.

[0034] Example 1 This embodiment carries out tissue culture of dandelion, and the specific steps are as follows: S1. Acquisition of Sterile Materials Disinfection: The seeds were sieved to remove impurities, washed and soaked in tap water, the seeds floating on the water surface were discarded, the seeds sunk to the bottom were rinsed with running water for 3-5 min (specifically 2 min in this embodiment), first soaked in 75% alcohol by volume for 30-40 s (specifically 30 s in this embodiment) in a clean bench, rinsed with sterile water for 5-6 times (specifically 5 times in this embodiment), and then soaked in a sodium hypochlorite aqueous solution with a solute mass percentage of 2%-3% (specifically 2% in this embodiment) for 3-5 min (specifically 5 min in this embodiment), rinsed with sterile water for 5-6 times (specifically 5 times in this embodiment) to obtain disinfected seeds.

[0035] Place the sterilized seeds on sterile filter paper to absorb surface moisture, and place them in MS solid culture medium. After 5-7 days, if sprouts grow in the MS culture medium, they will become sterile seedlings.

[0036] In this embodiment, the MS basic medium is prepared by dissolving MS basic medium salt in sterile water (product of Phytotech, item number: M519), and the amount of MS basic medium salt used is 4.43 g / L. In this embodiment, the 1 / 2 MS basic medium is prepared by dissolving 1 / 2 MS basic medium salt in sterile water, and the amount of 1 / 2 MS basic medium salt used is 2.22 g / L.

[0037] The MS solid medium in this example is a solid medium obtained by adding a carbon source (sucrose 30 g / L) and a coagulant (agar 7-9 g / L) to the MS basic medium. The 1 / 2 MS solid medium in this example is a solid medium obtained by adding a carbon source (sucrose 30 g / L) and a coagulant (agar 7-9 g / L) to the 1 / 2 MS basic medium. S2. Selection of explants Select the larger and tenderer leaves from the sterile seedlings as sterile leaves and cut them into 1-2 cm thick leaves in a clean bench. 2 (Specifically 1 cm in this embodiment 2 ) and keep small pieces of sterile leaves for later use.

[0038] S3. Callus induction culture Seven callus induction media were set up, as follows: A1, MS + 1.5 mg / L 6-BA + 0.5 mg / L NAA + 0.2 mg / L TDZ, that is, the culture medium is based on MS solid medium, the content of 6-BA (6-benzylaminopurine) is 1.5 mg / L, the content of NAA (naphthaleneacetic acid) is 0.5 mg / L, and the content of TDZ (thidiazuron, CAS number: 51707-55-2) is 0.2 mg / L.

[0039] A2, MS + 2.0 mg / L 6-BA + 0.2 mg / L NAA, that is, the culture medium is based on MS solid medium, the content of 6-BA is 2.0 mg / L, and the content of NAA is 0.2 mg / L.

[0040] A3, MS + 2.0 mg / L 6-BA + 0.2 mg / L NAA + 0.5 mg / L 2,4-D, that is, the culture medium is based on MS solid medium, the content of 6-BA is 2.0 mg / L, the content of NAA is 0.2 mg / L, and the content of 2,4-D is 0.5 mg / L.

[0041] A4, MS + 2.0 mg / L 6-BA + 0.5 mg / L 2,4-D, that is, the culture medium is based on MS solid medium, the content of 6-BA is 2.0 mg / L, and the content of 2,4-D is 0.5 mg / L.

[0042] A5, MS + 1.0 mg / L 6-BA + 0.5 mg / L 2,4-D, that is, the culture medium is based on MS solid medium, the content of 6-BA is 1.0 mg / L, and the content of 2,4-D is 0.5 mg / L.

[0043] A6, MS + 2.0 mg / L 6-BA, that is, the culture medium is based on MS solid medium and the 6-BA content is 2.0 mg / L.

[0044] A7, MS + 1.5 mg / L 6-BA + 0.5 mg / L 2,4-D, that is, the culture medium is based on MS solid medium, the content of 6-BA is 1.5 mg / L, and the content of 2,4-D is 0.5 mg / L.

[0045] The specific preparation methods of the callus induction medium of A1-A7 are as follows: add plant growth regulators and sucrose to MS basic medium according to the concentration, adjust the volume to 1L, pH to 5.8, add coagulant, sterilize at 121°C for 20 minutes and then cool.

[0046] The cut sterile leaf pieces were inoculated into callus induction medium A1, A2, A3, A4, A5, A6 and A7 respectively. There were 3 culture dishes for each callus induction medium, and 6 sterile leaf pieces were inoculated into each culture dish. They were placed in the tissue culture room for culture (temperature 22-25℃, light intensity 3000 lx, 15h of light per day). Callus was formed after about 15 days of culture, and callus induction was completed after about 25 days. The callus induction rate was calculated on the 25th day of culture, and the results are shown in Table 1: Table 1 Effects of different culture media on callus induction rate

[0047]

[0048] Table 1 shows that the callus induction rates of callus induction medium 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 both higher than 85%, among which A5 had the best callus induction effect, reaching 97.7%. Therefore, A1 was selected as the callus induction medium for subsequent experiments.

[0049] S4. Adventitious bud induction culture Eight adventitious bud induction mediums B1, B2, B3, B4, B5, B6, B7 and B8 were set up, and the specific preparation methods were as follows: add plant growth regulators and sucrose to MS basic medium according to the concentration, adjust the volume to 1L, pH to 5.8, add coagulant, sterilize at 121℃ for 20min and then cool. The specific types and concentrations of plant growth regulators in each adventitious bud induction medium are shown in Table 2.

[0050] The callus induced on callus induction medium A1 was cut into small pieces and inoculated into 8 kinds of bud induction mediums, 3 culture dishes for each bud induction medium, 6 pieces of callus were inoculated into each culture dish, and placed in a tissue culture room for culture (temperature 22-25°C, light intensity 3000lx, 15h of light per day). Buds were formed after about 15 days of culture, and bud induction was completed after about 30 days. The bud induction rate was counted on the 30th day of culture. The results are shown in Table 2: Table 2 Effects of different culture media on budding

[0051] Table 2 shows that B3 bud induction medium (MS+2 mg / L 6-BA + 0.5 mg / L IBA) had the best induction effect, so B3 was selected as the bud induction medium for subsequent experiments.

[0052] S5. Rooting culture Twelve adventitious bud induction mediums C1, C2, C3, C4, C5, C6, C7, C8, C9 and C10 were set up, and the specific preparation methods were as follows: add plant growth regulators and sucrose to 1 / 2 MS basic medium according to the concentration, adjust the volume to 1L, pH to 5.8, add coagulant, sterilize at 121℃ for 20min and then cool. The specific types and concentrations of plant growth regulators in each adventitious bud induction medium are shown in Table 3.

[0053] When the clustered buds induced on the B3 medium grow to about 5 cm, the single buds are cut off and transferred to the rooting medium for rooting culture. Three triangular bottles are inoculated, and each triangular bottle is inoculated with 6 single buds. They are placed in the tissue culture room for culture under the following culture conditions (temperature 22-25℃, light intensity 3000 lx, 15 h of light per day). Roots grow after about 10 days of culture. The rooting rate is counted on the 14th day of rooting culture. The results are shown in Table 3: Table 3 Effects of different culture media on shoot rooting

[0054] Note: Different lowercase letters after the data in the same column represent significant differences among treatments, P < 0.05.

[0055] Table 3 shows that the rooting medium can be a medium of 1 / 2 MS + 1-2 mg / L IBA (i.e., a medium with 1 / 2MS 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 / 2MS solid medium as the base medium and an indolebutyric acid IBA content of 1.5 mg / L) was finally screened out as the rooting medium.

[0056] S6. Hardening and transplanting First, prepare the mixed matrix for planting (mixed in a ratio of V vermiculite: V soil = 1:1), sterilize it in a 121℃ high pressure sterilizer for 30 minutes, and set it aside after the mixed matrix cools down.

[0057] Move the selected bottle seedlings with good root growth out of the culture room, loosen the bottle cap for 1-2 days, place them in the room for seedling hardening, then partially open the cap for 1-2 days, and finally remove the cap completely. After 2-3 days, take out the bottle seedlings from the bottle, wash the culture medium, dry it, and transplant it into a pot filled with mixed substrate, and plant it in a low-light, cool and ventilated place with a humidity of 80%-90%.

[0058] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. A tissue culture method for rapid propagation of dandelions, characterized in that: The method comprises the following steps: using dandelion leaves as explants to perform callus induction to obtain callus tissue, performing bud induction on the callus tissue to obtain clustered buds, and rooting the clustered buds to obtain dandelion tissue culture seedlings; The types and contents of the plant growth regulators in the culture medium used for callus induction are: 1.0-2 mg / L of 6-benzylaminopurine, 0.3-0.7 mg / L of naphthylacetic acid and 0.2-0.4 mg / L of thidiazuron.

2. The method according to claim 1, characterized in that The type and content of the plant growth regulator in the culture medium used for bud induction are preferably 2 mg / L of 6-benzylaminopurine and 0.3-0.5 mg / L of indolebutyric acid.

3. The method according to claim 1, characterized in that The type and content of the plant growth regulator in the culture medium used for rooting culture are: indolebutyric acid 0.5-2 mg / L.

4. The method according to claim 1, characterized in that The culture medium used for callus induction and the culture medium used for bud induction are both solid culture mediums obtained by adding medium sucrose, a coagulant and a plant growth regulator to an MS basic culture medium; the culture medium used for rooting induction is a solid culture medium obtained by adding medium sucrose, a coagulant and a plant growth regulator to 1 / 2 MS basic culture medium.

5. The method according to claim 4, characterized in that The solidifying agents of the culture medium for callus induction, the culture medium for bud induction and the culture medium for rooting are all agar.

6. The method according to claim 5, characterized in that The sucrose content in the culture medium for callus induction, the culture medium for bud induction and the culture medium for rooting is 30 g / L, the agar content is 7-9 g / L, and the natural pH value is 5.8-6.

0.

7. The method according to any one of claims 1 to 6, characterized in that: The dandelion leaves are sterile leaves.

8. The method according to claim 7, characterized in that The sterile leaves are from A1 or A2: A1. After sterilizing dandelion seeds, the seeds are cultured in MS solid culture medium to obtain sterile seedlings, and larger and tenderer leaves on the sterile seedlings are collected to obtain sterile leaves; A2. Collecting larger and tenderer leaves from the dandelion tissue culture seedlings to obtain sterile leaves.

9. The method according to claim 7, characterized in that: The callus induction is cultured for 15-25 days; the bud induction is cultured for 30 days; the rooting culture is cultured for 10-21 days; the culture is carried out under the conditions of a temperature of 22-25°C, a light intensity of 1500-3000 lx, and a daily light intensity of 12-15 hours.

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

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