CN109220795B - Tissue culture medium and culture method for valeriana jatamansi jones - Google Patents
Tissue culture medium and culture method for valeriana jatamansi jones Download PDFInfo
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- 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
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- A—HUMAN NECESSITIES
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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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
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Abstract
The invention discloses a tissue culture medium and a tissue culture method for valeriana jatamansi jones, wherein the tissue culture medium has the advantages of simple components, low cost and easiness in obtaining, can obviously improve the induction rate, the multiplication coefficient and the rooting rate of valeriana jatamansi jones, can effectively improve the tissue culture efficiency, reduces the production cost, and has marketization application value.
Description
Technical Field
The invention relates to the field of traditional Chinese medicine tissue culture, in particular to a valeriana jatamansi jones tissue culture medium and a culture method.
Background
Valeriana jatamansi Jones, also known as calla Jones, is a perennial herb of valerian of the family valerianaceae, and is used as a medicine by its rhizome. Has the effects of regulating qi, relieving pain, promoting digestion, relieving diarrhea, dispelling wind, removing dampness, relieving convulsion and calming the nerves, is mainly used for treating symptoms such as abdominal distending pain, indigestion, diarrhea and dysentery, rheumatic arthralgia, soreness and weakness of waist and knees, insomnia and the like, and is a resource plant with important medicinal value. The volatile oil contained in the valeriana jatamansi jones has special fragrance, the main components of the volatile oil are isovaleric acid, 3-methylvaleric acid, camphor, borneol, myrcene, olivene and the like, the volatile oil is used as an additive of cigarettes, has antibacterial and antioxidant effects, is used as a functional additive of daily chemical products and food, and is also an important aromatic plant. The valeriana jatamansi jones are grown on grasslands, forests or stream sides of mountains with elevation of below 2500 m and are mainly distributed in Yunnan, Guizhou, Sichuan, Henan, Shanxi, Hunan, Hubei, Tibet and the like. Yunnan has distribution at elevation 2000 and 2800 meters.
With the continuous development and application of the pharmacological action of the valeriana jatamansi jones and the development and application of the valeriana jatamansi jones in the aspects of daily chemical products and food, the market demand of the valeriana jatamansi jones is gradually expanded, the valeriana jatamansi jones are supplied to the market by mainly digging wild resources at present, limited wild resources are dug excessively, and the ecological environment is damaged, so that the valeriana jatamansi jones resources are deficient, the application of the valeriana jatamansi jones function is limited, a small amount of valeriana jatamansi jones are cultivated only in Yunnan, Guizhou and the like at present, and the main propagation mode is to dig the wild resources for plant division propagation at present. On one hand, the total amount of wild resources and organisms is limited, on the other hand, the propagation coefficient of the plant division propagation is 2-5, the propagation rate is low, the propagation period is generally 2-3 years, the propagation period is long, and the rapid large-scale artificial cultivation in a short period is limited.
Aiming at the defects, the invention provides the valeriana jatamansi tissue culture medium and the culture method, provides the most core key technology for valeriana jatamansi tissue culture, fully utilizes the modern biotechnology, provides sufficient high-quality seedlings for valeriana jatamansi planting in a short time, and effectively protects the wild resources of valeriana jatamansi from being excessively harvested and the biological diversity of the ecological environment.
Disclosure of Invention
Aiming at the defects of the prior art, the valeriana jatamansi tissue culture medium and the culture method have high production efficiency and low production cost.
The invention is realized by the following technical scheme:
a tissue culture method of valeriana jatamansi jones is characterized by comprising the following steps: (1) establishing a sterile system, (2) carrying out induction culture on the cluster buds, (3) carrying out enrichment culture on the cluster buds, (4) carrying out strong seedling culture on the cluster buds, and (5) carrying out rooting culture on the cluster buds;
preferably, the establishment of the sterile system in step (1) comprises the following steps: taking underground rhizome of valeriana jatamansi jones, cleaning, transferring to a super clean bench, disinfecting for 15-30 s by using 75% alcohol, rinsing for 2-3 times by using sterile water, disinfecting for 25-30 min by using 100mg/L chlorine dioxide solution, rinsing for 3-4 times by using sterile water, disinfecting for 10-15 min by using 0.1% mercuric chloride solution and 2 drops of Tween-80, and rinsing for 6-8 times by using sterile water;
preferably, the cluster bud induction culture of step (2) comprises the following steps: absorbing surface moisture of sterilized underground jatamans valeriana rhizome with sterile paper, cutting off the lower part of the sterilized underground jatamans valeriana rhizome, reserving the end part with bud heads, inoculating the sterilized underground jatamans valeriana rhizome into an induced proliferation culture medium according to polarity, and culturing under light-dark alternation at the temperature of 25 +/-2 ℃ and with the illumination intensity of 2000-3000 lx for 12h per day;
preferably, the propagation culture of the multiple shoots in step (3) comprises the following steps: cutting the induced cluster buds into single buds, inoculating the single buds into an induced proliferation culture medium, and culturing under light-dark alternation at the temperature of 25 +/-2 ℃ under the illumination intensity of 2000-3000 lx for a single day of illumination time of 12 h;
preferably, the culturing of strong seedlings of the cluster buds in the step (4) comprises the following steps: inoculating the induced or proliferated cluster buds into a strong seedling culture medium, and culturing under light-dark alternation at the temperature of 25 +/-2 ℃ and the illumination intensity of 2000-3000 lx for 12h in a single day;
preferably, the rooting culture of the cluster buds in the step (5) comprises the following steps: inoculating the strong single buds into a rooting culture medium, and culturing under light and dark alternation at the temperature of 25 +/-2 ℃ and with the illumination intensity of 2000-3000 lx and the single-day illumination time of 12 h;
more preferably, the proliferation-inducing medium is: modified MS + KT 5.0-10.0 mg/L + IBA 0.01-0.2 mg/L + activated carbon 0.5g/L + white sugar 30g/L + agar 3.8g/L, pH 5.8;
more preferably, the strong seedling culture medium is: modified MS + PAC 0.1-1.0 mg/L + activated carbon 0.5g/L + white sugar 50-60 g/L + agar 4.0g/L, pH 5.8;
more preferably, the rooting medium is: modified MS + NAA 0.2-0.5 mg/L + IBA 0.2-1.0 mg/L + active carbon 0.5g/L + white sugar 30-40 g/L + agar 4.0g/L, pH 5.8;
more preferably, the modified MS medium is: NH (NH)4NO3：1.65g/L，KH2PO4：0.34g/L，MgSO4·7H2O：0.55g/L，KNO3：2.28g/L，CaCl2·2H2O：0.33g/L，EDTA-Na2：74.60mg/L，FeSO4·7H2O: 55.60mg/L, inositol: 100.00mg/L, niacin: 0.50mg/L, glycine: 2.00mg/L, VB 6: 0.50mg/L, VB 1: 0.10mg/L, KI: 0.83mg/L, H3BO3：6.20mg/L，MnSO4·H2O：16.90mg/L，ZnSO4·7H2O：8.60mg/L，NaMoO4·2H2O：0.25mg/L，CuSO4·5H2O：0.025mg/L，CoCl2·6H2O：0.25mg/L。
The invention also provides a valeriana jatamansi tissue culture medium, which is used for callus induction or callus proliferation and comprises the following components in parts by weight: modified MS + KT 5.0-10.0 mg/L + IBA 0.01-0.2 mg/L + activated carbon 0.5g/L + white sugar 30g/L + agar 3.8g/L, pH 5.8;
the invention also provides a valeriana jatamansi tissue culture medium, which is used for strengthening seedlings and comprises the following components in parts by weight: modified MS + PAC 0.1-1.0 mg/L + activated carbon 0.5g/L + white sugar 50-60 g/L + agar 4.0g/L, pH 5.8;
the invention also provides a valeriana jatamansi tissue culture medium, which is used for rooting and comprises the following components: modified MS + NAA 0.2-0.5 mg/L + IBA 0.2-1.0 mg/L + active carbon 0.5g/L + white sugar 30-40 g/L + agar 4.0g/L, pH 5.8;
preferably, the modified MS medium is: NH (NH)4NO3：1.65g/L，KH2PO4：0.34g/L，MgSO4·7H2O：0.55g/L，KNO3：2.28g/L，CaCl2·2H2O：0.33g/L，EDTA-Na2：74.60mg/L，FeSO4·7H2O: 55.60mg/L, inositol: 100.00mg/L, niacin: 0.50mg/L, glycine: 2.00mg/L, VB 6: 0.50mg/L, VB 1: 0.10mg/L, KI: 0.83mg/L, H3BO3：6.20mg/L，MnSO4·H2O：16.90mg/L，ZnSO4·7H2O：8.60mg/L，NaMoO4·2H2O：0.25mg/L，CuSO4·5H2O：0.025mg/L，CoCl2·6H2O：0.25mg/L。
The invention has the beneficial effects that:
(1) the tissue culture method of the valeriana jatamansi jones, disclosed by the invention, has the advantages that the multiplication is geometrically increased by taking a month as a unit, a large number of seedlings can be produced in a short period, and the problem of the seedlings can be solved for the large-scale production of the valeriana jatamansi jones.
(2) The valeriana jatamansi tissue culture medium can obviously improve the induction rate, the multiplication coefficient and the rooting rate, thereby improving the tissue culture efficiency, reducing the tissue culture cost and having extremely high industrial popularization value.
Detailed Description
The present invention will be further described with reference to the following examples, but the present invention is not limited thereto.
Example 1
1. Establishing an aseptic system: cleaning underground rhizome of Valeriana jatamansi Jones with tap water, rinsing with detergent solution for 30min for 3 times, scraping epidermis, rinsing with tap water for 80min, transferring to super clean bench, sterilizing with 75% alcohol for 20s, rinsing with sterile water for 2 times, sterilizing with 100mg/L chlorine dioxide solution for 30min, rinsing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride solution and 2 drops of Tween-80 for 10min, and rinsing with sterile water for 7 times.
2. And (3) inducing and culturing cluster buds: inoculating sterilized underground rhizome of Valeriana jatamansi jones with bud in an induced proliferation culture medium of modified MS + KT5.0 mg/L + IBA 0.01mg/L + activated carbon 0.5g/L + white sugar 30g/L + agar 3.8g/L, adjusting pH to 5.8, culturing at 25 + -1 deg.C under illumination intensity of 2500lx for 12h per day for 60 days under light-dark alternation, and counting induction conditions.
3. And (3) carrying out multiplication culture on cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into an induced proliferation culture medium of modified MS + KT5.0 mg/L + IBA 0.01mg/L + active carbon 0.5g/L + white sugar 30g/L + agar 3.8g/L, adjusting the pH to 5.8, culturing at 25 +/-1 ℃ under the illumination intensity of 2500lx for 12h in a single day for 40 days under light-dark alternation, and counting the proliferation conditions.
4. Culturing strong seedlings of cluster buds: cutting the cluster buds after induced proliferation into single buds, inoculating the single buds into a sound seedling culture medium of improved MS + PAC 0.5mg/L + activated carbon 0.5g/L + white sugar 60g/L + agar 4.0g/L, adjusting the pH to 5.8, and culturing for 30 days under the condition of light-dark alternation at the temperature of 25 +/-1 ℃ and with the illumination intensity of 2500lx and the single-day illumination time of 12 h.
5. And (3) rooting culture of cluster buds: the bud after strong seedling is inoculated in a rooting culture medium of improved MS, NAA0.2mg/L, IBA 1.0mg/L, activated carbon 0.5g/L, white sugar 30g/L and agar 4.0g/L, the pH is adjusted to 5.8, the cultivation is carried out under the condition that the temperature is 25 +/-1 ℃, the illumination intensity is 2500lx and the single-day illumination time is 12 hours under the condition of light-dark alternation, and the rooting condition is counted.
6. The improved MS culture medium in the steps 1-5 is as follows: NH (NH)4NO3：1.65g/L，KH2PO4：0.34g/L，MgSO4·7H2O：0.55g/L，KNO3：2.28g/L，CaCl2·2H2O：0.33g/L，EDTA-Na2：74.60mg/L，FeSO4·7H2O: 55.60mg/L, inositol: 100.00mg/L, niacin: 0.50mg/L, glycine: 2.00mg/L, VB 6: 0.50mg/L, VB 1: 0.10mg/L, KI: 0.83mg/L, H3BO3：6.20mg/L，MnSO4·H2O：16.90mg/L，ZnSO4·7H2O：8.60mg/L，NaMoO4·2H2O：0.25mg/L，CuSO4·5H2O：0.025mg/L，CoCl2·6H2O：0.25mg/L。
Example 2
1. Establishing an aseptic system: washing underground rhizome of valeriana jatamansi jones with tap water for 30min, washing with detergent solution for 3 times, scraping epidermis, washing with tap water for 60min, transferring to a super clean bench, sterilizing with 75% alcohol for 30s, washing with sterile water for 3 times, sterilizing with 100mg/L chlorine dioxide solution for 30min, washing with sterile water for 4 times, sterilizing with 0.1% mercuric chloride solution and 2 drops of Tween-80 for 15min, and washing with sterile water for 6-8 times.
2. And (3) inducing and culturing cluster buds: inoculating sterilized underground rhizome of Valeriana jatamansi jones with bud in an induced proliferation culture medium of modified MS + KT 10.0mg/L + IBA 0.2mg/L + active carbon 0.5g/L + white sugar 30g/L + agar 3.8g/L, adjusting pH to 5.8, culturing at 25 + -2 deg.C under illumination intensity of 2000lx for 12h per day for 60 days under light-dark alternation, and counting induction conditions.
3. And (3) carrying out multiplication culture on cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into an induced proliferation culture medium of improved MS + KT 10.0mg/L + IBA 0.2mg/L + active carbon 0.5g/L + white sugar 30g/L + agar 3.8g/L, adjusting the pH to 5.8, culturing at the temperature of 25 +/-2 ℃ under the illumination intensity of 2000lx for 12h in a single day for 40 days under light-dark alternation, and counting the proliferation conditions.
4. Culturing strong seedlings of cluster buds: cutting the cluster buds after induced proliferation into single buds, inoculating the single buds into a sound seedling culture medium of improved MS + PAC 1.0mg/L + activated carbon 0.5g/L + white sugar 50g/L + agar 4.0g/L, adjusting the pH to 5.8, and carrying out light-dark alternate cultivation for 30 days at the temperature of 25 +/-2 ℃ and the illumination intensity of 2000lx for a single day of illumination time of 12 h.
5. And (3) rooting culture of cluster buds: the bud after strong seedling is inoculated in a rooting culture medium of improved MS, NAA0.5mg/L, IBA 0.5mg/L, activated carbon 0.5g/L, white sugar 40g/L and agar 4.0g/L, the pH value is adjusted to 5.8, the bud is cultured for 30 days under the condition of light-dark alternation at the temperature of 25 +/-2 ℃ and the illumination intensity of 2000lx and the single-day illumination time of 12h, and the rooting condition is counted.
6. The improved MS culture medium in the steps 1-5 is as follows: NH (NH)4NO3：1.65g/L，KH2PO4：0.34g/L，MgSO4·7H2O：0.55g/L，KNO3：2.28g/L，CaCl2·2H2O：0.33g/L，EDTA-Na2：74.60mg/L，FeSO4·7H2O: 55.60mg/L, inositol: 100.00mg/L, niacin: 0.50mg/L, glycine: 2.00mg/L, VB 6: 0.50mg/L, VB 1: 0.10mg/L, KI: 0.83mg/L, H3BO3：6.20mg/L，MnSO4·H2O：16.90mg/L，ZnSO4·7H2O：8.60mg/L，NaMoO4·2H2O：0.25mg/L，CuSO4·5H2O：0.025mg/L，CoCl2·6H2O：0.25mg/L。
Comparative example 1
The culture is carried out according to the tissue culture method in the specific embodiment of the specification of the patent CN 102668981.
The data of examples 1-2 and comparative example 1 were statistically analyzed, and the results are shown in the following table.
| Treatment of | Callus induction rate | Coefficient of proliferation | Rooting rate |
| Example 1 | 98.1％ | 7.3 | 98.2％ |
| Example 2 | 97.7％ | 7.1 | 98.1％ |
| Comparative example 1 | 91.8％ | 4.8 | 90.7％ |
Therefore, the valeriana jatamansi jones tissue culture method can obviously improve the induction rate, the multiplication coefficient and the rooting rate, and in the prior art, the tissue culture medium and the tissue culture method can obviously improve the tissue culture efficiency, reduce the cost and have market application value.
Claims (1)
1. A tissue culture method of valeriana jatamansi jones is characterized by comprising the following steps:
(1) establishing an aseptic system: taking underground rhizome of valeriana jatamansi jones, cleaning, transferring to a super clean bench, disinfecting for 15-30 s by using 75% alcohol, rinsing for 2-3 times by using sterile water, disinfecting for 25-30 min by using 100mg/L chlorine dioxide solution, rinsing for 3-4 times by using sterile water, disinfecting for 10-15 min by using 0.1% mercuric chloride solution and 2 drops of Tween-80, and rinsing for 6-8 times by using sterile water;
(2) and (3) inducing and culturing cluster buds: absorbing surface moisture of sterilized underground jatamans valeriana rhizome with sterile paper, cutting off the lower part of the sterilized underground jatamans valeriana rhizome, reserving the end part with bud heads, inoculating the sterilized underground jatamans valeriana rhizome into an induced proliferation culture medium according to polarity, and culturing under light-dark alternation at the temperature of 25-2 ℃ under the illumination intensity of 2000-30001 x and the single-day illumination time of 12 h; the culture medium for inducing proliferation comprises: modified MS + KT 5.0-10.0 mg/L + IBA 0.01-0.2 mg/L + activated carbon 0.5g/L + white sugar 30g/L + agar 3.8g/L, pH 5.8;
(3) and (3) carrying out multiplication culture on cluster buds: cutting the induced cluster buds into single buds, inoculating the single buds into an induced proliferation culture medium, and culturing under light-dark alternation at the temperature of 25 soil and 2 ℃ under the illumination intensity of 2000-30001 x for 12h under single sunlight; the culture medium for inducing proliferation comprises: modified MS + KT 5.0-10.0 mg/L + IBA 0.01-0.2 mg/L + activated carbon 0.5g/L + white sugar 30g/L + agar 3.8g/L, pH 5.8;
(4) culturing strong seedlings of cluster buds: inoculating the induced or proliferated cluster buds into a strong seedling culture medium, and culturing under light-dark alternation at the temperature of 25 ℃ and 2 ℃ under the illumination intensity of 2000-30001 x for 12h under single-day illumination; the strong seedling culture medium comprises: modified MS + PAC 0.1-1.0 mg/L + activated carbon 0.5g/L + white sugar 50-60 g/L + agar 4.0g/L, pH 5.8;
(5) and (3) rooting culture of cluster buds: inoculating the strong single buds into a rooting culture medium, and culturing under light-dark alternation at the temperature of 25 soil and 2 ℃ under the illumination intensity of 2000-30001 x and the single-day illumination time of 12 h; the rooting culture medium comprises: modified MS + NAA 0.2-0.5 mg/L + IBA 0.2-1.0 mg/L + active carbon 0.5g/L + white sugar 30-40 g/L + agar 4.0g/L, pH 5.8;
the improved MS culture medium involved in the above steps is: NH (NH)4NO3:1.65g/L，KH2P04：0.34g/L，MgS04•7H20：0.55g/L，KN03：2.28g/L，CaCl2•2H20：0.33g/L，EDTA-Na2：74.60mg/L，FeS04•7H255.60mg/L, inositol 100.00mg/L, niacin: 0.50mg/L, glycine: 2.00mg/L, VB 6: 0.50mg/L, VB 1: 0.10mg/L, KI: 0.83mg/L, H3B03:6.20mg/L，MnS04•H20：16.90mg/L，ZnS04•7H20：8. 60mg/L，NaMo04•2H20：0. 25mg/L，CuS04•5H20：0.025mg/L，CoCl2•6H20：0. 25mg/L。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102668981A (en) * | 2012-05-17 | 2012-09-19 | 云南自然谷生物开发有限公司 | Valeriana jatamansi jones breeding method |
| CN108566888A (en) * | 2017-11-23 | 2018-09-25 | 江苏农牧科技职业学院 | A kind of medicinal plant jatamans valeriana rhizome tissue culture mating system |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102668981A (en) * | 2012-05-17 | 2012-09-19 | 云南自然谷生物开发有限公司 | Valeriana jatamansi jones breeding method |
| CN108566888A (en) * | 2017-11-23 | 2018-09-25 | 江苏农牧科技职业学院 | A kind of medicinal plant jatamans valeriana rhizome tissue culture mating system |
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