CN113841615A - Tissue culture method of radix cynanchi wilfordii - Google Patents
Tissue culture method of radix cynanchi wilfordii Download PDFInfo
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- CN113841615A CN113841615A CN202111318831.XA CN202111318831A CN113841615A CN 113841615 A CN113841615 A CN 113841615A CN 202111318831 A CN202111318831 A CN 202111318831A CN 113841615 A CN113841615 A CN 113841615A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/002—Culture media for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method of Pistacia lentiscus. The method comprises the following steps: sterilizing the top bud of Dioscorea opposita Thunb, placing in a differentiation culture medium, and performing induction culture to obtain cluster buds; the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 2-4 mg/L of 6-benzylaminopurine and 0.1mg/L of naphthylacetic acid; transferring the cluster buds into a rooting culture medium, and performing rooting culture to obtain a complete plant; the rooting culture medium takes 1/2MS culture medium as a basic culture medium and also comprises 0.4-0.6 mg/L of naphthylacetic acid. The tissue culture method provided by the invention can differentiate the terminal bud of the cynanchum wilfordii plant into a complete individual through a proper differentiation culture medium and a proper rooting culture medium, so that the cynanchum wilfordii seedlings are propagated in batches.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture method of Pistacia lentiscus.
Background
Cynanchum wilfordii (ostrericum citriodorum), common name: radix Stephaniae Tetrandrae and radix Angelicae Dahuricae are plants of Apium of Umbelliferae. Produced in provinces such as Hunan, Jiangxi, Zhejiang, Guangxi, Guangdong, Fujian, etc. of China. Growing under the mountain slope shrubs or at the forest edge and in the grass clusters. The root can be used as medicine, has the functions of dispelling wind and clearing heat, promoting blood circulation by removing blood stasis, promoting qi circulation and relieving pain, and is commonly used for treating cough due to wind-heat, angina pectoris, stomach ache, malaria, dysentery, amenorrhea, leucorrhea, traumatic injury, etc.
The Pistacia lentiscus has a long medicine taking history in folks, but belongs to cold-back medicinal materials, and is not paid attention by operators because of infrequent use and small use amount, and the Pistacia lentiscus supplied in the market of the medicinal materials at present mainly comes from field artificial collection, and reports of introduction domestication and artificial propagation cultivation are not seen. In recent years, with the enhancement of health care consciousness of people in medicine use, the market demand of a plurality of cold-back medicinal materials is gradually increased, and wide market demand cannot be met only by depending on wild resources.
Disclosure of Invention
In order to solve the problems, the invention provides a tissue culture method of Pistacia lentiscus. The tissue culture method provided by the invention can obtain complete individuals by utilizing the terminal buds of the Dioscorea alata L.var.glauca plants, so that Dioscorea alata L.var.glauca seedlings can be bred in batches.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a tissue culture method of Pistacia lentiscus, which comprises the following steps:
sterilizing the top bud of Dioscorea opposita Thunb, placing in a differentiation culture medium, and performing induction culture to obtain cluster buds; the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 2-4 mg/L of 6-benzylaminopurine and 0.1mg/L of naphthylacetic acid;
transferring the cluster buds into a rooting culture medium, and performing rooting culture to obtain a complete plant; the rooting culture medium takes 1/2MS culture medium as a basic culture medium and also comprises 0.4-0.6 mg/L of naphthylacetic acid.
Preferably, the pH values of the differentiation medium and the rooting medium are respectively 5.6-5.8.
Preferably, the conditions of the induction culture include: the temperature is 22-25 ℃, the illumination time is 12h/d, and the illumination intensity is 1800-2500 lx.
Preferably, the time for induction culture is 28-30 d.
Preferably, the rooting culture conditions comprise: the temperature is 22-25 ℃, the illumination time is 12h/d, and the illumination intensity is 1800-2500 lx.
Preferably, the rooting culture time is 20-25 days.
Preferably, the method of sterilization comprises: sterilizing with 75% ethanol for 25-30 s, sterilizing with 0.1 wt.% mercuric chloride for 25-40 min, and washing with sterile water for 5 times.
Preferably, the sterilized top bud of the radix cynanchi wilfordii further comprises: removing the damaged part of the terminal bud of the kadsura longipedunculata in the disinfection process.
Has the advantages that:
the invention provides a tissue culture method of Pistacia lentiscus, which comprises the following steps: sterilizing the top bud of Dioscorea opposita Thunb, placing in a differentiation culture medium, and performing induction culture to obtain cluster buds; the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 2-4 mg/L of 6-benzylaminopurine and 0.1mg/L of naphthylacetic acid; transferring the cluster buds into a rooting culture medium, and performing rooting culture to obtain a complete plant; the rooting culture medium takes 1/2MS culture medium as a basic culture medium and also comprises 0.4-0.6 mg/L of naphthylacetic acid. The tissue culture method provided by the invention can differentiate the terminal bud of the cynanchum wilfordii plant into a complete individual through a proper differentiation culture medium and a proper rooting culture medium, so that the cynanchum wilfordii seedlings can be propagated in batches.
Drawings
FIG. 1 is a diagram showing the rooting of the bush buds of the Pistacia lentiscus in example 1;
FIG. 2 is a diagram of tissue culture and propagation of seedlings of the Pistacia lentiscus in example 1;
FIG. 3 is the rooting map of the bush buds of the Pistacia lentiscus in example 2;
FIG. 4 is a diagram of tissue culture and propagation of seedlings of the Pistacia lentiscus in example 2.
Detailed Description
The invention provides a tissue culture method of Pistacia lentiscus, which comprises the following steps:
sterilizing the top bud of Dioscorea opposita Thunb, placing in a differentiation culture medium, and performing induction culture to obtain cluster buds; the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 2-4 mg/L of 6-benzylaminopurine and 0.1mg/L of naphthylacetic acid;
transferring the cluster buds into a rooting culture medium, and performing rooting culture to obtain a complete plant; the rooting culture medium takes 1/2MS culture medium as a basic culture medium and also comprises 0.4-0.6 mg/L of naphthylacetic acid.
The invention puts the sterilized top bud of Dioscorea alata into a differentiation culture medium for induction culture to obtain cluster buds. In the present invention, the method of sterilization preferably includes: sterilizing with 75% ethanol by volume for 25-30 s, sterilizing with 0.1 wt.% mercuric chloride for 25-40 min, and washing with sterile water for 5 times; more preferably, it comprises: sterilizing with 75% ethanol for 30s, sterilizing with 0.1 wt.% mercuric chloride for 25min, and washing with sterile water for 5 times; the sterilized top bud of the Pistacia lentiscus preferably further comprises: removing the damaged part of the top bud of the kadsura longipedunculata in the disinfection process; the removal operation is preferably performed on a clean bench; the means of removal preferably comprises cutting. In the invention, the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 2-4 mg/L of 6-benzylaminopurine and 0.1mg/L of naphthylacetic acid; more preferably, the MS culture medium is taken as a basic culture medium, and the MS culture medium also comprises 2mg/L of 6-benzylaminopurine and 0.1mg/L of naphthylacetic acid. In the present invention, the differentiation medium preferably comprises the following components: MS culture medium +2 mg/L6-benzylaminopurine +0.1mg/L naphthylacetic acid; the pH value of the differentiation medium is preferably 5.6-5.8, and more preferably 5.8. The sources of the components of the differentiation medium are not particularly limited in the present invention, and commercially available products well known to those skilled in the art may be used. The invention can differentiate the apical bud of the Dioscorea opposita into cluster buds through a proper differentiation culture medium.
In the present invention, the conditions for the induction culture preferably include: the temperature is 22-25 ℃, the illumination time is 12h/d, and the illumination intensity is 1800-2500 lx; more preferably, the temperature is 22 ℃, the illumination time is 12h/d, and the illumination intensity is 2000 lx; the time for induction culture is preferably 28-30 d.
After the cluster buds are obtained, the cluster buds are transferred into a rooting culture medium for rooting culture to obtain a complete plant.
In the invention, the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and preferably further comprises 0.6mg/L of naphthylacetic acid. The invention has no special requirements on the sources of all components of the rooting medium, and can adopt commercial products well known by the technical personnel in the field. The invention can induce the callus of the cynanchum wilfordii to generate new roots through a proper rooting culture medium, thereby being capable of propagating the cynanchum wilfordii seedlings in batches.
In the present invention, the rooting culture conditions preferably include: the temperature is 22-25 ℃, the illumination time is 12h/d, and the illumination intensity is 1800-2500 lx; more preferably, the temperature is 22 ℃, the illumination time is 12h/d, and the illumination intensity is 2000 lx; the rooting culture time is preferably 20-25 days.
In order to further illustrate the present invention, the following examples are provided to describe the tissue culture method of Pistacia lentiscus in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
A tissue culture method of radix cynanchi wilfordii comprises the following steps:
(1) selecting terminal buds of the cynanchum wilfordii plants as explants, and disinfecting the explants; the specific disinfection method comprises the following steps: sterilizing with 75% ethanol for 30s, sterilizing with 0.1 wt.% mercuric chloride for 25min, and washing with sterile water for 5 times;
(2) placing the explant treated in the step (1) on a super clean bench to cut off the damaged part of the top bud of the kadsura longipedunculata in the disinfection process, and then placing the explant in a differentiation culture medium; the formula of the differentiation medium is as follows: based on MS culture medium, adding 2 mg/L6-benzylpurine (6-BA) and 0.1mg/L naphthylacetic acid (NAA), and adjusting pH to 5.8;
(3) the explants are subjected to induction culture in a differentiation medium (the lobules begin to differentiate after about one week, and the cluster buds differentiate from the basal part about 4 weeks), and the specific culture conditions are as follows: the temperature is 22 ℃, the illumination time is 12h/d, and the illumination intensity is 2000 lx;
(4) transferring the cluster buds generated in the step (3) into a rooting culture medium (the formula of the rooting culture medium is that 1/2MS culture medium is taken as a base, 0.6mg/L of naphthylacetic acid (NAA) is added, and the pH value is adjusted to 5.8) for continuous culture, wherein the specific culture conditions are as follows: the temperature was 22 ℃, the illumination time was 12h/d, the illumination intensity was 2000lx, and the whole plants were obtained after about 3 weeks, as shown in FIG. 1 and FIG. 2.
Example 2
A tissue culture method of radix cynanchi wilfordii comprises the following steps:
(1) selecting terminal buds of the cynanchum wilfordii plants as explants, and disinfecting the explants; the specific disinfection method comprises the following steps: sterilizing with 75% ethanol for 25s, sterilizing with 0.1 wt.% mercuric chloride for 40min, and washing with sterile water for 5 times;
(2) placing the explant treated in the step (1) on a super clean bench to cut off the damaged part of the top bud of the kadsura longipedunculata in the disinfection process, and then placing the explant in a differentiation culture medium; the formula of the differentiation medium is as follows: based on MS culture medium, adding 6-benzyl purine (6-BA)4mg/L and Naphthalene Acetic Acid (NAA)0.1mg/L, and adjusting pH to 5.6;
(3) the explants are subjected to induction culture in a differentiation medium (the lobules begin to differentiate after about one week, and the cluster buds differentiate from the basal part about 4 weeks), and the specific culture conditions are as follows: the temperature is 25 ℃, the illumination time is 12h/d, and the illumination intensity is 2500 lx;
(4) transferring the cluster buds generated in the step (3) into a rooting culture medium (the formula of the rooting culture medium is that 1/2MS culture medium is taken as a base, 0.4mg/L of naphthylacetic acid (NAA) is added, and the pH value is adjusted to 5.6) for continuous culture, wherein the specific culture conditions are as follows: the temperature was 25 ℃, the illumination time was 12h/d, the illumination intensity was 1800lx, and after about 25 days, complete plants were obtained, see fig. 3 and 4.
Comparative example 1
A tissue culture method similar to that of example 1, the only difference being that the formulation of the differentiation medium is: the pH was adjusted to 5.8 based on MS medium.
Comparative example 2
A tissue culture method similar to comparative example 1, the only difference being that the root of the acerola plant was selected as the explant.
Comparative example 3
A tissue culture method similar to comparative example 1, the only difference being that the stem of the acerola plant was selected as the explant.
Comparative example 4
A tissue culture method similar to comparative example 1, the only difference being that the leaves of the acerola plant were selected as the explant.
Comparative example 5
A tissue culture method similar to that of example 1, the only difference being that the formulation of the differentiation medium is: based on MS culture medium, 2mg/L of 6-benzylpurine (6-BA) and 0.1mg/L of dichlorophenoxyacetic acid (2,4-D) are added, and pH value is adjusted to 5.8.
Comparative example 6
A tissue culture method similar to that of comparative example 5, the only difference being that the root of the plant of Gekko Swinhonis was selected as the explant.
Comparative example 7
A tissue culture method similar to that of comparative example 5, the only difference being that the stem of the plant of Dioscorea septemata was selected as the explant.
Comparative example 8
A tissue culture method similar to that of comparative example 5, the only difference being that the leaves of the plant of Gekko Swinhonis were selected as explants.
Comparative example 9
A tissue culture method similar to that of example 1, the only difference being that the root of the acerola plant was selected as the explant.
Comparative example 10
A tissue culture method similar to that of example 1, the only difference being that the stem of the acerola plant was selected as the explant.
Comparative example 11
A tissue culture method similar to that of example 1, the only difference being that leaves of the acerola plant were selected as explants.
Comparative example 12
A tissue culture method similar to that of example 1, the only difference being that the rooting medium has the formula: based on 1/2MS culture medium, 0.4mg/L of 6-benzylpurine (6-BA), 20g/L of sucrose and 7g/L of carrageenan are added, and the pH value is adjusted to 5.8.
Comparative application example 1
The differentiation condition of the explants and the rooting condition of the callus of example 1 and comparative examples 1-12 are observed and counted, the rooting rate is counted, and the results are shown in table 1.
TABLE 1 test results for different groups
Note: the "/" in the table indicates that intact callus or clumpy shoots were not differentiated and subsequent rooting culture was not performed.
As can be seen from Table 1, the terminal bud of the Pistacia lentiscus is suitable for being used as an explant for tissue culture, and the differentiation medium and the rooting medium provided by the invention are the most suitable media for tissue culture of the Pistacia lentiscus, so that the terminal bud of the Pistacia lentiscus can be differentiated into a complete individual, and the Pistacia lentiscus seedlings can be propagated in batches.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A tissue culture method of radix cynanchi wilfordii is characterized by comprising the following steps:
sterilizing the top bud of Dioscorea opposita Thunb, placing in a differentiation culture medium, and performing induction culture to obtain cluster buds; the differentiation culture medium takes an MS culture medium as a basic culture medium, and also comprises 2-4 mg/L of 6-benzylaminopurine and 0.1mg/L of naphthylacetic acid;
transferring the cluster buds into a rooting culture medium, and performing rooting culture to obtain a complete plant; the rooting culture medium takes 1/2MS culture medium as a basic culture medium and also comprises 0.4-0.6 mg/L of naphthylacetic acid.
2. The tissue culture method according to claim 1, wherein the differentiation medium and the rooting medium have respective pH values of 5.6 to 5.8.
3. The tissue culture method of claim 1, wherein the conditions for inducing culture comprise: the temperature is 22-25 ℃, the illumination time is 12h/d, and the illumination intensity is 1800-2500 lx.
4. The tissue culture method according to claim 1 or 3, wherein the time for the induction culture is 28 to 30 days.
5. The tissue culture method of claim 1, wherein the conditions of the rooting culture comprise: the temperature is 22-25 ℃, the illumination time is 12h/d, and the illumination intensity is 1800-2500 lx.
6. The tissue culture method according to claim 1 or 5, wherein the rooting culture time is 20-25 days.
7. The tissue culture method of claim 1, wherein the sterilizing method comprises: sterilizing with 75% ethanol for 25-30 s, sterilizing with 0.1 wt.% mercuric chloride for 25-40 min, and washing with sterile water for 5 times.
8. The tissue culture method according to claim 1 or 7, further comprising, after the sterilizing of the top bud of the Pistacia lentiscus: removing the damaged part of the terminal bud of the kadsura longipedunculata in the disinfection process.
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