CN104082138A - Tissue-culture rapid propagation method of Aristolochia fordiana Hemsl - Google Patents
Tissue-culture rapid propagation method of Aristolochia fordiana Hemsl Download PDFInfo
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Abstract
The invention provides a tissue-culture rapid propagation method of Aristolochia fordiana Hemsl and relates to a seedling growing method for obtaining stable-strain seedlings of the Aristolochia fordiana Hemsl by use of a tissue culture technique. The method is used for realizing tissue-culture rapid propagation of the seedlings of the Aristolochia fordiana Hemsl by taking the node rattan of the Aristolochia fordiana Hemsl as the explant and by virtue of the stages such as adventitious bud induction, multiplication culture of multiple shoots, test-tube seedling rooting, and seedling tempering and transplanting. The tissue-culture rapid propagation method of the Aristolochia fordiana Hemsl is used for satisfying large-scale supply of the Aristolochia fordiana Hemsl seedlings and also providing raw material guarantee for pharmaceutical production with the Aristolochia fordiana Hemsl medicinal material as the raw material.
Description
Technical field
The present invention relates to the method for Plant Tissue Breeding in agricultural biotechnologies, specifically, relate to a kind of tissue cultivation rapid breeding method of ford dutchmanspipe root or herb.
Background technology
Ford dutchmanspipe root or herb (
aristolochia fordianahemsl) five brave Tongcheng, centering grass, natural grass, blood wither again, blood rattan secretly disappears, Wilson Begonia Rhizome, for Aristolochiaceae Aristolochia (
aristolochia) liana medicinal plant, be Guangxi famous herbal medicine among the people, there is good analgesic activity, the treatment for diseases such as pain wound, snakebite, high heat among the people.Ford dutchmanspipe root or herb is rich in aristolochic acid (aristolochia acid), its rhizome aristolochic acid content is up to 0.6%, pharmacology at home and abroad of aristolochic acid and clinical research all prove the effect of antibacterial, anti-inflammatory reliably, analgesia and enhanced machine body immunity function, and commercially produced, as " Tardolyt " of external clinical practice and the domestic power of biting acid sheet, be aristolochic acid total acid.
Ford dutchmanspipe root or herb is mainly distributed in the ground such as Guangxi, Yunnan, because eucalyptus plantation is widelyd popularize in various places in recent years, and the reasons such as the frequent and weed killer herbicide use of refining mountain afforestation, wild resource sharply reduces, and cannot meet medical needs.Development artificial planting is one of approach solving ford dutchmanspipe root or herb imbalance between supply and demand; ford dutchmanspipe root or herb sapling multiplication mainly relies on seminal propagation and root division, layer of vine and stays root and stem of certain plants breeding at present; these modes of reproduction speed are slow, efficiency is low; cannot meet the demand of ford dutchmanspipe root or herb large-scale planting to high quality seedling; therefore, be necessary very much to set up a set of complete ford dutchmanspipe root or herb tissue culture quick breeding system.At present, ford dutchmanspipe root or herb tissue culture technique there is not yet report.
Summary of the invention
The object of the present invention is to provide a kind of tissue cultivation rapid breeding method of ford dutchmanspipe root or herb, by stages such as evoking adventive bud, adventitious buds proliferation, rooting of vitro seedling, acclimatization and transplantses, the rapid propagation in vitro mode that makes ford dutchmanspipe root or herb pass through Multiple Buds obtains a large amount of test-tube plantlets, thereby has realized object of the present invention.
The tissue cultivation rapid breeding method of a kind of ford dutchmanspipe root or herb of the present invention, comprises following processing step:
(1) evoking adventive bud: choosing ford dutchmanspipe root or herb band joint rattan is explant, after 5～15 seconds kinds of 75%～80% alcohol disinfecting, with aseptic water washing, be placed on for 2～5 times in 0.1%～0.3% mercuric chloride solution and sterilize 10～30 minutes, then use aseptic water washing 3～6 times, after aseptic filter paper suck dry moisture, be inoculated on inducing culture, complete dark cultivation under 25～28 ℃ of conditions, until induction forms indefinite bud.
(2) adventitious buds proliferation is cultivated: indefinite bud is inoculated into and on proliferated culture medium, breeds cultivation, inoculation is placed on illumination every day 12～15 hours, intensity of illumination is 1000～1500lx, and cultivation temperature is to cultivate under the condition of 25～28 ℃, and switching in 20～30 days once.
(3) rooting of vitro seedling: the Multiple Buds that grows to 2～4cm is cut to be inoculated into and carry out root induction on root media, after inoculation, first under 25～28 ℃ of conditions, entirely secretly cultivate 7～14 days, then be placed in illumination every day 12～15 hours, intensity of illumination is 2000～3000lx, and cultivation temperature is to be cultured to and to take root under the condition of 25～28 ℃.
(4) acclimatization and transplants: natural lighting lower refining seedling was opened and be placed in to the blake bottle bottle cap that grows to the rooting tube plantlet of 6～8cm after 5～7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant in the matrix being mixed into by peat soil and river sand (1:1) and be colonizated in large Tanaka.
The described ford dutchmanspipe root or herb band rattan explant of above-mentioned steps (1) needed to carry out pretreatment before carrying out adventitious bud inducing, the method of processing is: with running water, rinse the ford dutchmanspipe root or herb band rattan from field acquisition well silt and be placed on 0.01%～0.05% liquor potassic permanganate and soak 4～8 hours, then after rinsing 3～5 times with running water glue plastic bag sealing to be placed on 3～5 ℃ of refrigerator overnight standby.
The described inducing culture of above-mentioned steps (1) is: take GS as minimal medium; additional 2.0%～3.5% sucrose, 0.35%～0.5% agar, 0.05%～0.1% active carbon, 0.05%～0.5% acidic hydrolysis casein and 0.5～2.0mg/L KT, 0.1～2.0mg/L IBA and 0.1～1.0mg/L 2; 4-D, pH value is 5.4～5.8;
The described proliferated culture medium of above-mentioned steps (2) is: take MS as minimal medium, additional 0.2～3.0mg/L 6-BA, 0.1～1.5mg/L NAA, 1.0～6.0mg/L vitamin b3,0.1～0.5mg/L D-VB5 calcium, 1.0～6.0mg/L ascorbic acid, 1.0～5.0mg/L Cys and 2.5%～3.5% sucrose, 0.35%～0.5% agar, 0.05%～0.1% active carbon, pH value is 5.4～5.8;
The described root media of above-mentioned steps (3) is: take 1/2MS as minimal medium, additional 0.1～0.8mg/L IBA, 0.1～1.0mg/L GGR(note: two gill-GGR, purchased from Beijing Ai Bidi research and development centre), 0.5～2.5mg/L Cys and 2.0%～2.5% sucrose, 0.4%～0.6% agar, 0.05%～0.1% active carbon, pH value is 5.4～5.8;
The feature that the present invention has, the present invention utilizes plant tissue culture technique to carry out the large-scale production of ford dutchmanspipe root or herb seedling, has set up the tissue cultivation rapid breeding method of ford dutchmanspipe root or herb, and the present invention has simply, easy capable, economic feature.The ford dutchmanspipe root or herb test-tube seedling transplanting survival rate being bred as by the present invention reaches more than 93%, can provide seedling guarantee for ford dutchmanspipe root or herb large-scale planting.
Embodiment
Following examples are to further illustrate of the present invention, are not limitations of the present invention.
Embodiment mono-
1, explant pretreatment
With running water, rinse the ford dutchmanspipe root or herb band rattan from field acquisition well silt and be placed on 0.03% liquor potassic permanganate and soak 5 hours, then after rinsing 5 times with running water glue plastic bag sealing to be placed on 3 ℃ of refrigerator overnight standby.
2, incubation
(1) evoking adventive bud: ford dutchmanspipe root or herb band joint rattan is after 8 seconds kinds of 75% alcohol disinfecting, with aseptic water washing, be placed on for 3 times in 0.1% mercuric chloride solution and sterilize 15 minutes, then use aseptic water washing 4 times, after aseptic filter paper suck dry moisture, be inoculated on inducing culture, under 28 ℃ of conditions, complete dark cultivation, cultivates and forms indefinite bud after 45 days.The inducing culture adopting is: GS+1.5mg/L KT+0.3mg/L IBA and 0.4mg/L 2, and 4-D+3.5% sucrose+0.35% agar+0.08% active carbon+0.3% acidic hydrolysis casein, pH value is 5.8;
(2) adventitious buds proliferation is cultivated: indefinite bud is inoculated on proliferated culture medium and breed cultivations, inoculate and be placed on illumination every day 15 hours, intensity of illumination is 1000lx, and cultivation temperature is to cultivate under the condition of 28 ℃, within 25 days, transfers once, and growth coefficient is 5～7.The proliferated culture medium adopting is: MS+1.5mg/L 6-BA+0.5mg/L NAA+1.0mg/L vitamin b3+0.5mg/L D-VB5 calcium+1.0mg/L ascorbic acid+1.0mg/L Cys+3.0% sucrose+0.35% agar+0.1% active carbon, and pH value is 5.8;
(3) rooting of vitro seedling: the Multiple Buds that grows to 2～4cm is cut to be inoculated into and carry out root induction on root media, after inoculation, first under 28 ℃ of conditions, entirely secretly cultivate 7 days, then be placed in illumination every day 15 hours, intensity of illumination is 3000lx, cultivation temperature is to cultivate and take root for 20 days under the condition of 28 ℃, and rooting rate reaches 95%.The root media adopting is: 1/2MS+0.3mg/L IBA+0.6mg/L GGR+1.0mg/L Cys+2.0%% sucrose+0.4% agar+0.1% active carbon, and pH value is 5.8;
(4) acclimatization and transplants: natural lighting lower refining seedling was opened and be placed in to the blake bottle bottle cap that grows to the rooting tube plantlet of 6～8cm after 5 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant in the matrix being mixed into by peat soil and river sand (1:1) and be colonizated in large Tanaka, survival rate reaches 92%.
Embodiment bis-
1, explant pretreatment
With running water, rinse the ford dutchmanspipe root or herb band rattan from field acquisition well silt and be placed on 0.01% liquor potassic permanganate and soak 8 hours, then after rinsing 3 times with running water glue plastic bag sealing to be placed on 5 ℃ of refrigerator overnight standby.
2, incubation
(1) evoking adventive bud: ford dutchmanspipe root or herb band joint rattan is after 5 seconds kinds of 78% alcohol disinfecting, with aseptic water washing, be placed on for 5 times in 0.2% mercuric chloride solution and sterilize 10 minutes, then use aseptic water washing 5 times, after aseptic filter paper suck dry moisture, be inoculated on inducing culture, under 25 ℃ of conditions, complete dark cultivation, cultivates and forms indefinite bud after 42 days.The inducing culture adopting is: GS+0.8mg/L KT+0.2mg/L IBA and 0.2mg/L 2, and 4-D+3.0% sucrose+0.4% agar+0.1% active carbon+0.5% acidic hydrolysis casein, pH value is 5.6;
(2) adventitious buds proliferation is cultivated: indefinite bud is inoculated on proliferated culture medium and breed cultivations, inoculate and be placed on illumination every day 12 hours, intensity of illumination is 1300lx, and cultivation temperature is to cultivate under the condition of 25 ℃, within 28 days, transfers once, and growth coefficient is 4～6.The proliferated culture medium adopting is: MS+0.8mg/L 6-BA+0.3mg/L NAA+3.0mg/L vitamin b3+0.3mg/L D-VB5 calcium+3.5mg/L ascorbic acid+4.5mg/L Cys+3.5% sucrose+0.5% agar+0.08% active carbon, and pH value is 5.6;
(3) rooting of vitro seedling: the Multiple Buds that grows to 2～4cm is cut to be inoculated into and carry out root induction on root media, after inoculation, first under 25 ℃ of conditions, entirely secretly cultivate 10 days, then be placed in illumination every day 12 hours, intensity of illumination is 2500lx, cultivation temperature is to cultivate and take root for 28 days under the condition of 25 ℃, and rooting rate reaches 92%.The root media adopting is: 1/2MS+0.5mg/L IBA+1.0mg/L GGR+2.5mg/L Cys+2.5%% sucrose+0.4% agar+0.05% active carbon, and pH value is 5.6;
(4) acclimatization and transplants: natural lighting lower refining seedling was opened and be placed in to the blake bottle bottle cap that grows to the rooting tube plantlet of 6～8cm after 7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant in the matrix being mixed into by peat soil and river sand (1:1) and be colonizated in large Tanaka, survival rate reaches 95%.
Embodiment tri-
1, explant pretreatment
With running water, rinse the ford dutchmanspipe root or herb band rattan from field acquisition well silt and be placed on 0.05% liquor potassic permanganate and soak 4 hours, then after rinsing 5 times with running water glue plastic bag sealing to be placed on 5 ℃ of refrigerator overnight standby.
2, incubation
(1) evoking adventive bud: ford dutchmanspipe root or herb band joint rattan is after 5 seconds kinds of 80% alcohol disinfecting, with aseptic water washing, be placed on for 4 times in 0.1% mercuric chloride solution and sterilize 13 minutes, then use aseptic water washing 5 times, after aseptic filter paper suck dry moisture, be inoculated on inducing culture, under 25 ℃ of conditions, complete dark cultivation, cultivates and forms indefinite bud after 47 days.The inducing culture adopting is: GS+2.0mg/L KT+0.8mg/L IBA and 0.4mg/L 2, and 4-D+3.5% sucrose+0.4% agar+0.05% active carbon+0.3% acidic hydrolysis casein, pH value is 5.4;
(2) adventitious buds proliferation is cultivated: indefinite bud is inoculated on proliferated culture medium and breed cultivations, inoculate and be placed on illumination every day 13 hours, intensity of illumination is 1200lx, and cultivation temperature is to cultivate under the condition of 27 ℃, within 26 days, transfers once, and growth coefficient is 3～6.The proliferated culture medium adopting is: MS+1.5mg/L 6-BA+0.6mg/L NAA+5.0mg/L vitamin b3+0.5mg/L D-VB5 calcium+6.0mg/L ascorbic acid+4.0mg/L Cys+3.5% sucrose+0.5% agar+0.1% active carbon, and pH value is 5.4;
(3) rooting of vitro seedling: the Multiple Buds that grows to 2～4cm is cut to be inoculated into and carry out root induction on root media, after inoculation, first under 25 ℃ of conditions, entirely secretly cultivate 13 days, then be placed in illumination every day 13 hours, intensity of illumination is 3000lx, cultivation temperature is to cultivate and take root for 20 days under the condition of 25 ℃, and rooting rate reaches 95%.The root media adopting is: 1/2MS+0.2mg/L IBA+0.8mg/L GGR+1.5mg/L Cys+2.5%% sucrose+0.5% agar+0.1% active carbon, and pH value is 5.4;
(4) acclimatization and transplants: natural lighting lower refining seedling was opened and be placed in to the blake bottle bottle cap that grows to the rooting tube plantlet of 6～8cm after 6 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant in the matrix being mixed into by peat soil and river sand (1:1) and be colonizated in large Tanaka, survival rate reaches 94%.
Claims (5)
1. a tissue cultivation rapid breeding method for ford dutchmanspipe root or herb, is characterized in that comprising following processing step:
(1) evoking adventive bud: choosing ford dutchmanspipe root or herb band joint rattan is explant, after 5～15 seconds kinds of 75%～80% alcohol disinfecting, with aseptic water washing, be placed on for 2～5 times in 0.1%～0.3% mercuric chloride solution and sterilize 10～30 minutes, then use aseptic water washing 3～6 times, after aseptic filter paper suck dry moisture, be inoculated on inducing culture, complete dark cultivation under 25～28 ℃ of conditions, until induction forms indefinite bud;
(2) adventitious buds proliferation is cultivated: indefinite bud is inoculated into and on proliferated culture medium, breeds cultivation, inoculation is placed on illumination every day 12～15 hours, intensity of illumination is 1000～1500lx, and cultivation temperature is to cultivate under the condition of 25～28 ℃, and switching in 20～30 days once;
(3) rooting of vitro seedling: the Multiple Buds that grows to 2～4cm is cut to be inoculated into and carry out root induction on root media, after inoculation, first under 25～28 ℃ of conditions, entirely secretly cultivate 7～14 days, then be placed in illumination every day 12～15 hours, intensity of illumination is 2000～3000lx, and cultivation temperature is to be cultured to and to take root under the condition of 25～28 ℃;
(4) acclimatization and transplants: natural lighting lower refining seedling was opened and be placed in to the blake bottle bottle cap that grows to the rooting tube plantlet of 6～8cm after 5～7 days, test-tube plantlet is taken out from blake bottle, wash root medium off, plant in the matrix being mixed into by peat soil and river sand (1:1) and be colonizated in large Tanaka.
2. the tissue cultivation rapid breeding method of a kind of ford dutchmanspipe root or herb according to claim 1, it is characterized in that the described ford dutchmanspipe root or herb band rattan explant of step (1) needed to carry out pretreatment before carrying out adventitious bud inducing, the method of processing is: with running water, rinse the ford dutchmanspipe root or herb band rattan from field acquisition well silt and be placed on 0.01%～0.05% liquor potassic permanganate and soak 4～8 hours, then after rinsing 3～5 times with running water glue plastic bag sealing to be placed on 3～5 ℃ of refrigerator overnight standby.
3. the tissue cultivation rapid breeding method of a kind of ford dutchmanspipe root or herb according to claim 1, it is characterized in that the described inducing culture of step (1) is: take GS as minimal medium, additional 2.0%～3.5% sucrose, 0.35%～0.5% agar, 0.05%～0.1% active carbon, 0.05%～0.5% acidic hydrolysis casein and 0.5～2.0mg/L KT, 0.1～2.0mg/L IBA and 0.1～1.0mg/L 2,4-D, pH value is 5.4～5.8.
4. the tissue cultivation rapid breeding method of a kind of ford dutchmanspipe root or herb according to claim 1, it is characterized in that the described proliferated culture medium of step (2) is: take MS as minimal medium, additional 0.2～3.0mg/L 6-BA, 0.1～1.5mg/L NAA, 1.0～6.0mg/L vitamin b3,0.1～0.5mg/L D-VB5 calcium, 1.0～6.0mg/L ascorbic acid, 1.0～5.0mg/L Cys and 2.5%～3.5% sucrose, 0.35%～0.5% agar, 0.05%～0.1% active carbon, pH value is 5.4～5.8.
5. the tissue cultivation rapid breeding method of a kind of ford dutchmanspipe root or herb according to claim 1, it is characterized in that the described root media of step (3) is: take 1/2MS as minimal medium, additional 0.1～0.8mg/L IBA, 0.1～1.0mg/L GGR(note: two gill-GGR, purchased from Beijing Ai Bidi research and development centre), 0.5～2.5mg/L Cys and 2.0%～2.5% sucrose, 0.4%～0.6% agar, 0.05%～0.1% active carbon, pH value is 5.4～5.8.
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Cited By (9)
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| CN104770296A (en) * | 2015-04-03 | 2015-07-15 | 广西壮族自治区药用植物园 | Method for rapidly propagating through adoption of aristolochia fordiana leaves |
| CN104798684A (en) * | 2015-04-25 | 2015-07-29 | 玉林师范学院 | Tissue culture rapid propagation method for plukenetia volubilis L. |
| CN107027634A (en) * | 2017-06-22 | 2017-08-11 | 玉林市林业科学研究所 | A kind of tissue cultivation rapid breeding method of high-quality shatian pomelo |
| CN107494282A (en) * | 2017-10-17 | 2017-12-22 | 李正美 | A kind of Yellowmouth Dutchmanspipe Root method for tissue culture |
| CN109329063A (en) * | 2018-11-27 | 2019-02-15 | 广西玉林市华睿茶业有限公司 | A kind of tissue cultivation rapid breeding method of moellendorf spikemoss herb |
| CN109380118A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of tissue cultivation rapid breeding method of radish seed |
| CN109380084A (en) * | 2018-11-23 | 2019-02-26 | 张世燊 | A kind of tissue cultivation rapid breeding method of the seed of feather cockscomb |
| CN109496844A (en) * | 2018-11-23 | 2019-03-22 | 韦宇 | A kind of tissue cultivation rapid breeding method of Changshan |
| CN116458429A (en) * | 2023-04-28 | 2023-07-21 | 广西壮族自治区南宁良凤江国家森林公园 | Application and method for tissue culture propagation of sargentgloryvine stem seeds and embryo |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104770296A (en) * | 2015-04-03 | 2015-07-15 | 广西壮族自治区药用植物园 | Method for rapidly propagating through adoption of aristolochia fordiana leaves |
| CN104798684A (en) * | 2015-04-25 | 2015-07-29 | 玉林师范学院 | Tissue culture rapid propagation method for plukenetia volubilis L. |
| CN107027634A (en) * | 2017-06-22 | 2017-08-11 | 玉林市林业科学研究所 | A kind of tissue cultivation rapid breeding method of high-quality shatian pomelo |
| CN107494282A (en) * | 2017-10-17 | 2017-12-22 | 李正美 | A kind of Yellowmouth Dutchmanspipe Root method for tissue culture |
| CN109380084A (en) * | 2018-11-23 | 2019-02-26 | 张世燊 | A kind of tissue cultivation rapid breeding method of the seed of feather cockscomb |
| CN109496844A (en) * | 2018-11-23 | 2019-03-22 | 韦宇 | A kind of tissue cultivation rapid breeding method of Changshan |
| CN109380118A (en) * | 2018-11-25 | 2019-02-26 | 钟天路 | A kind of tissue cultivation rapid breeding method of radish seed |
| CN109329063A (en) * | 2018-11-27 | 2019-02-15 | 广西玉林市华睿茶业有限公司 | A kind of tissue cultivation rapid breeding method of moellendorf spikemoss herb |
| CN116458429A (en) * | 2023-04-28 | 2023-07-21 | 广西壮族自治区南宁良凤江国家森林公园 | Application and method for tissue culture propagation of sargentgloryvine stem seeds and embryo |
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| CN104082138B (en) | 2016-08-24 |
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