CN101015275A - Method for inducing and culturing sweet wormwood polyploid - Google Patents
Method for inducing and culturing sweet wormwood polyploid Download PDFInfo
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- CN101015275A CN101015275A CN 200710034499 CN200710034499A CN101015275A CN 101015275 A CN101015275 A CN 101015275A CN 200710034499 CN200710034499 CN 200710034499 CN 200710034499 A CN200710034499 A CN 200710034499A CN 101015275 A CN101015275 A CN 101015275A
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Abstract
It relates to a new method for inducing sweet worm wood polyploidy. The main processes comprises: taking the tender leaf of sweet worm wood with high content of artemisinin as explants, adsorbing surface water with aseptic paper after sterilization, inducing the callus of explants on culture medium, then inducing polyploidy with colchicine for induced callus, carrying out differentiation culture, identifying polyploid, and rooting culturing for polyploid. The invention is characterized in that it overcomes various limits, such as low artemisinin concentration and instability under natural conditions, sensitivity to collection time, collection site, temperature and fertilization in geographical environment,, and difficulty for quality control; it can cultivate a new stable variety with high artemisinin content and accelerated life cycle.
Description
Technical field the present invention relates to a kind of cultivation of plants technology, specifically, relates to a kind of cultivation method of sweet wormwood.
Medicinal sweet wormwood is feverfew artemisia annua (ArtemisiaannuaL.) in the background technology, has heat-clearing preventing malaria, the antipruritic effect of wind dispelling, is used to hinder heat, malaria, hectic fever, child convulsion, heat and rushes down, dislikes sore mange etc." qinghaosu " is isolated antimalarial effective monomer from the Chinese medicine sweet wormwood, be and the diverse new compound of known antimalarial, be called unique in the world effective malaria treatment medicine by the World Health Organization, it is not only best antimalarial specific drug behind chloroquine, pyrimethamine, uncle's quinoline and sulfanilamide (SN), and is the medicine that onset is the fastest, effect is best, toxicity is minimum in the used anti-medicine.
Present medicinal qinghaosu is mainly from plant extracts, seminal propagation.Field cultivation is the main path that obtains sweet wormwood herb at present, but in natural plants, artemislnin content is generally on the low side, and unstable, again because of being subjected to geographical environment to gather the influence of period, factors such as collection position, temperature and fertilising, quality is wayward, the processing of qinghaosu and extraction link are more, waste time and energy, a large amount of simultaneously natural resourcess of gathering, will inevitably destroy the environment and the ecological balance, cause resource exhaustion.For a long time, countries in the world are all in the developmental research that steps up to carry out qinghaosu and derivative thereof.
Producing at present provides the main path of sweet wormwood raw material to have: field production under the natural environment, utilize methods such as tissue culture, cell culture to cultivate sweet wormwoods, wherein the seminal propagation field cultivation is the main path that obtains sweet wormwood herb.Be but the general content of these method qinghaosus is low, and unstable.Cultivate content height and stable new varieties and become the research topic that the various countries scientist faces.
Summary of the invention the object of the present invention is to provide sweet wormwood polyploid to induce and enlarges cultivation method, it adopts the polyploid of callus induction to carry out the expanding propagation cultivation, the method can be cultivated the high and stable new varieties of content, can improve artemislnin content and accelerates life cycle.
For achieving the goal, embodiment of the present invention are: a kind of sweet wormwood polyploid is induced and cultivation method, it is characterized in that may further comprise the steps:
(1) get the sweet wormwood tender leaf, with the wine cleaning/disinfecting of 0.1% mercuric chloride and 75%, be inoculated in the MS inducing culture, condition of culture is: temperature is 20～30 ℃, illumination 8～20 hours, and humidity is 75%～95%;
(2) callus be impregnated in the colchicine solution of filtration sterilization, liquor strength is 2500～10000mg/L, processing time is 12～50h, handle back aseptic water washing 3 times, transfer on the fresh MS inducing culture that does not contain colchicin, secretly cultivated 6 days, the low light level was cultivated 6 days, transfer to then and carry out illumination cultivation on the MS+6-BAlmg/L+IAA0.1mg/L differential medium, cultivate and carry out the polyploid evaluation after 50 days, determine that it is the tetraploid sweet wormwood;
(3) the sweet wormwood seedling stem apex that will have a tetraploid feature changes over to and differentiates the tetraploid aseptic seedling in the proliferated culture medium;
When (4) treating seedling length to 4～6cm, be divided into individual plant, be inoculated on the root media; Begin hardening when treating root length to 1～2cm, open bottle cap, placed under the room temperature 2～4 days, take out little bacterium, wash the root medium, plant in red worker and the humus soil (2: 1), water permeable, earlier with plastics membrane cover 3-6 days;
(5) will be at 3-6 days sweet wormwood of plastics membrane cover, transplantation of seedlings is regularly watered after the transplanting to the land for growing field crops, manages.
In step (1), (2), described MS inducing culture is MS+NAA2mg/L+KT0.1mg/L.
In the step (2), the method that described polyploid is identified is: gather the variant stem apex during 9-10 the morning, 0.002mol/L oxine solution preliminary treatment 3h, with fixing 2～24h under 4 ℃ of conditions of the fixing liquefaction in Kano, the 12min that dissociates under the 60 ℃ of conditions of hydrochloric acid with 1mol steams and stays water to clean 5～6 times again, with compressing tablet behind the carbolfuchsin dyeing 15min, with the cell division phase and to chromosome counting, chromosome number is 2n=4x=36, determines that it is tetraploid plant.
In the step (3), described proliferated culture medium is: MS+6-BA2～4mg/L+NAA0.1～0.5mg/L.
Step (4), described root media is: MS+2,4-D1～2mg/L+KT0.1～0.2mg/L.
The present invention is as explant with sweet wormwood children tender leaf, after conventional sterilization processing, blot surface water with aseptic filter paper, with medium the explant callus is induced then, the callus that induces is carried out carrying out with colchicin the mutagenesis of polyploid, carry out differentiation culture after the mutagenesis, carry out the evaluation of polyploid, carry out culture of rootage when polyploid is planted.It is low that the present invention has overcome under the nature the general content of qinghaosu, and it is unstable, be subjected to geographical environment to gather period, gather the influence of factors such as position, temperature and fertilising, the restriction of factors such as the wayward border of quality, can cultivate the high and stable new varieties of content, can improve artemislnin content and accelerate life cycle.
Specific implementation method
(1) callus induction: in the sweet wormwood tender leaf of fine mining height artemislnin content, mercuric chloride sterilization 10min with 0.1% uses 75% alcohol disinfecting 30s, aseptic water washing 7 times then, shred blade, be inoculated on the MS+NAA2mg/L+KT0.1mgg/ medium and induce.
(2) multiploid induction: the callus that induces is immersed in the colchicine solution of filtration sterilization, liquor strength is 2500mg/L～10000mg/L, processing time 20～50h, handle back aseptic water washing 3 times, be transferred on the fresh culture that does not contain colchicin, secretly cultivated 6 days, the low light level was cultivated 6 days again, transfer to then and carry out illumination cultivation on the MS+6-BA1mg/L+IAA0.1mg/L differential medium, carry out polyploid after 50 days and identify.
(3) evaluation of polyploid: gathered the variant stem apex at 10 o'clock in the morning, 0.002mol/Lr8-oxyquinoline solution preliminary treatment 3h, again with fixing 2～24h under 4 ℃ of conditions of the fixing liquefaction in Kano, 12min dissociates under 60 ℃ of conditions of hydrochloric acid with 1mol, steaming stays water to clean 5～6 times, with compressing tablet behind the carbolfuchsin dyeing 15min, with the cell division phase and to chromosome counting, chromosome number is 2n=4x=36, determines that it is tetraploid plant.
(4) cultivation of polyploid seedling: will have tetraploid feature seedling stem apex and change in the proliferated culture medium, and all can differentiate the limbs aseptic seedling, proliferated culture medium is: MS+6-BA2～4mg/L+NAA0.1～0.5mg/L.
(5) culture of rootage and hardening: carry out culture of rootage and hardening when treating that seedling grows to 4～6cm, be divided into individual plant, remove the callus of base portion, be inoculated in MS+2,4-D1～2mg/L+KT0.1～0.2mg/L carries out culture of rootage, begins hardening when treating root length to 1～2cm, open bottle cap, placed under the room temperature 2～4 days, and took out little bacterium, wash the root medium, plant in red worker and the humus soil (2: 1), water permeable, earlier with plastics membrane cover 5 days
(6) transplant the land for growing field crops: 5 days seedling of growth in the plastic foil is moved to the land for growing field crops, when regularly watering after the transplanting in the pipe of row field.
Claims (5)
1, a kind of sweet wormwood polyploid is induced and cultivation method, it is characterized in that, may further comprise the steps:
(1) get the sweet wormwood tender leaf, with the wine cleaning/disinfecting of 0.1% mercuric chloride and 75%, be inoculated in the MS inducing culture, condition of culture is: temperature is 20～30 ℃, illumination 8～20 hours, and humidity is 75%～95%;
(2) callus be impregnated in the colchicine solution of filtration sterilization, liquor strength is 2500～10000mg/L, processing time is 12～50h, handle back aseptic water washing 3 times, transfer on the fresh MS inducing culture that does not contain colchicin, secretly cultivated 6 days, the low light level was cultivated 6 days, transfer to then and carry out illumination cultivation on the MS+6-BA1mg/L+IAA0.1mg/L differential medium, cultivate and carry out the polyploid evaluation after 50 days, determine that it is the tetraploid sweet wormwood;
(3) the sweet wormwood seedling stem apex that will have a tetraploid feature changes over to and differentiates the tetraploid aseptic seedling in the proliferated culture medium;
When (4) treating seedling length to 4～6cm, be divided into individual plant, be inoculated on the root media; Begin hardening when treating root length to 1～2cm, open bottle cap, placed under the room temperature 2～4 days, take out little bacterium, wash the root medium, plant in red worker and the humus soil (2: 1), water permeable, earlier with plastics membrane cover 3-6 days;
(5) will be at 3-6 days sweet wormwood of plastics membrane cover, transplantation of seedlings is regularly watered after the transplanting to the land for growing field crops, manages.
2, sweet wormwood polyploid according to claim 1 is induced and cultivation method, it is characterized in that, in step (1), (2), described MS inducing culture is MS+NAA2mg/L+KT0.1mg/L.
3, sweet wormwood polyploid according to claim 1 is induced and cultivation method, it is characterized in that, in the step (3), described proliferated culture medium is: MS+6-BA2～4mg/L+NAA0.1～0.5mg/L.
4, sweet wormwood polyploid according to claim 1 is induced and cultivation method, it is characterized in that, and step (4), described root media is: MS+2,4-D1～2mg/L+KT0.1～0.2mg/L.
5, sweet wormwood polyploid according to claim 1 is induced and cultivation method, it is characterized in that, in the step (2), the method that described polyploid is identified is: gathered the variant stem apex at 10 o'clock in the morning, 0.002mol/Lr oxine solution preliminary treatment 3h, again with fixing 2～24h under 4 ℃ of conditions of the fixing liquefaction in Kano, 12min dissociates under 60 ℃ of conditions of hydrochloric acid with 1mol, steaming stays water to clean 5～6 times, with compressing tablet behind the carbolfuchsin dyeing 15min, with the cell division phase and to chromosome counting, chromosome number is 2n=4x=36, determines that it is tetraploid plant.
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| CN2007100344998A CN101015275B (en) | 2007-03-07 | 2007-03-07 | Method for inducing and culturing sweet wormwood polyploid |
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| CN2007100344998A CN101015275B (en) | 2007-03-07 | 2007-03-07 | Method for inducing and culturing sweet wormwood polyploid |
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| CN101015275A true CN101015275A (en) | 2007-08-15 |
| CN101015275B CN101015275B (en) | 2011-04-20 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102893860A (en) * | 2011-10-19 | 2013-01-30 | 崔广荣 | Stevia rebaudiana bertoni polyploid in vitro induction technology |
| CN105638478A (en) * | 2016-03-02 | 2016-06-08 | 广西壮族自治区农业科学院生物技术研究所 | Method for inducing rhizoma bletillae tetraploid through calluses |
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2007
- 2007-03-07 CN CN2007100344998A patent/CN101015275B/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102893860A (en) * | 2011-10-19 | 2013-01-30 | 崔广荣 | Stevia rebaudiana bertoni polyploid in vitro induction technology |
| CN105638478A (en) * | 2016-03-02 | 2016-06-08 | 广西壮族自治区农业科学院生物技术研究所 | Method for inducing rhizoma bletillae tetraploid through calluses |
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| CN101015275B (en) | 2011-04-20 |
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