CN1392252A - Cultivating method for increasing sweet wormwood organ biomass and artemisine content - Google Patents
Cultivating method for increasing sweet wormwood organ biomass and artemisine content Download PDFInfo
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Abstract
The present invention relates to cultivating method of increasing sweet wormwood organ biomass and artemissine content. After improving traditional MS culture medium, single or mixed RE element compound, which may be nitrate, chloride or oxide of La, CE, Lu, Nd or Sm, is added and NAA, 6-BA hormone in the culture medium is reduced or substituted. When the said culture medium is used in solid or liquid culture of sweet wormwood organ, the biomass and artemisine content may be increased greatly, so as to raise the yield of artemisine and reduce the production cost of artemisine.
Description
The invention belongs to the cultural method of plant organ in the biochemical engineering technology, particularly a kind of cultural method that improves sweet wormwood organ biomass and artemislnin content.
Artemisinin is the sesquiterpene lactones that contains peroxy-radical, and molecular formula is C
15H
22O
5It has special efficacy to brain type and anti-chloroquine pernicious malaria.The world market Artemisinin is mainly derived from China and Vietnam, owing to be subjected to multiple factor affecting such as climate, germplasm degeneration, can not satisfy the requirement to the sweet wormwood resource far away.Utilizing plant tissue, organ culture to produce Artemisinin is one of focus of present Artemisinin research, since the eighties, plant tissue, organ culture has been produced Artemisinin carried out more research.Having carried out the Artemisinin synthetic in culture systems such as sweet wormwood callus, suspension cell, bud and hairly root explores.Adopt sweet wormwood organ (hairly root and the bud of growing thickly) large scale culturing will become one of effective way that solves sweet wormwood scarcity of resources in the Artemisinin production, but traditional sweet wormwood organ cultural method, owing to the biomass and the lower Artemisinin production cost that makes of artemislnin content of the sweet wormwood organ of being cultivated are high high, limited the production of Artemisinin.
Studies show that, do not contain or contain extremely low-level Artemisinin in undifferentiated sweet wormwood herb tissue, certain tissue differentiation then can promote the synthetic of Artemisinin.The Artemisinin synthetic is analyzed in to the callus of the callus of sweet wormwood, band bud and the plantlet that produced by the callus differentiation, can draw and not contain Artemisinin in the sweet wormwood callus, when callus follows bud to be differentiated to form, the content that detects Artemisinin is about 0.008% of dry weight, and the content of Artemisinin reaches 0.92% of dry weight in the plant that the differentiation seedling grows up to, and is higher than wild plant (He Xichun etc., Botany Gazette, 1983,25,87-90.); When carrying out sweet wormwood callus suspension culture, in callus, do not detect the existence of Artemisinin, but in suspending nutrient solution, detect the trace Artemisinin (8ug/mL) (Nair MSR, et al, J.Nat.Prod., 1983,49,504-507.).In the callus that has yet confirmed sweet wormwood in the research of Brown (Brown G D, J.Nat.Prod., 1994,57,975～977.), do not contain terpene equally, but in the bud of differentiation, detect the terpene synthetics similar with the parent.When research sweet wormwood suspension cell culture, in culture, do not detect the synthetic of Artemisinin, but in the n-hexane extract of nutrient solution, detect antimalarial activity (Tawfiq N K, et al, Plant cell Rep., 1989,8,425～428.).The content that detects Artemisinin in new inductive sweet wormwood callus is about the 0.1-0.08% of dry weight, this culture is behind three succeeding transfer culture, the content of Artemisinin almost is difficult to detect (Paniego N B in the callus, et al, Plant Cell Tiss.Org.Cult., 1994,36,163～168.).
By sweet wormwood organ growth and the regulation and control of meta-bolites building-up process, utilize transgenic plant all may significantly improve artemislnin content, increase Artemisinin output.Can detect the existence of Artemisinin in the inductive sweet wormwood bud culture, and the influence of and Artemisinin long to the sweet wormwood blastogenesis to nutrition and hormone is studied, find that Plant hormones regulators,gibberellins and caseinhydrolysate etc. have strong hormesis (Woerdenbag H J to Artemisinin in the bud synthetic, et al, PlantCell Tiss.Org.Cult., 1993,32,247～257.).Its artemislnin content is stable in the genetically modified sweet wormwood bud culture, be about 0.02% of dry weight, each metal ion species and the vitamin complex that improve in the substratum are not obvious to the synthetic influence of Artemisinin in the bud, but add Plant hormones regulators,gibberellins and make that the content of Artemisinin has improved 3～4 times of (Paniego N B in the bud, et al, Enzyme Micro.Tech., 1996,18,526～530.).Successfully transform sweet wormwood children stem with Agrobacterium rhizogenes 1601 and obtain the hairly root culture, the feasibility (Qin Mingbo etc., Botany Gazette, 1994,36,165～170.) of carrying out the genetic modification of sweet wormwood with Agrobacterium rhizogenes as genophore is provided.Simultaneously, utilize Agrobacterium rhizogenes 15834 to infect the bud point and the blade of sweet wormwood, obtain sweet wormwood hairly root culture, and the content that detects Artemisinin is about 0.43% of dry weight, and its content is far above content (Weathers P J, the et al of Artemisinin in other sweet wormwood tissue culture, Biotechnol.Lett., 1994,16,1281～1286.).Utilize Agrobacterium rhizogenes 1601 to infect the sweet wormwood blade and set up hairly root cultivation system, and detect Artemisinin in culture, the content of Artemisinin is about 0.2% (Cai Guoqin etc. of dry weight under the condition of adding Plant hormones regulators,gibberellins, the biotechnology journal, 1995,11,315～320.).Utilize agrobacterium tumefaciens infection sweet wormwood blade, artemislnin content is about dry weight 0.17% in the acquisition transfer-gen plant, and the content of the synthetic precursor Artemisinin B of Artemisinin is about 0.22% (Vergauwe A, et al, Plant Cell Rep., 1996,15,929～933.).But the cultural method of above sweet wormwood tissue all exists: the defective that gained sweet wormwood organ biomass and artemislnin content are lower, seriously limited the production of Artemisinin.
Some rare earth element has vegetable cell, tissue and organ growth and the synthetic required different physiological roles of secondary metabolite fast under suitable concentration, as improving photosynthetic efficiency, promote tissue differentiation and absorb etc. nutraceutical;
The effect difference that different rare earth elements is played in plant has only the part element in the light rare earths, as lanthanum, cerium, neodymium, praseodymium, samarium, europium etc., can promote plant-growth; On the contrary, the heavy rare earth element that has, as yttrium etc., even (virtue can tiger, He Youzhao, Zhao Guiwen, the plant physiology effect progress of rare earth element, rare earth, 1998,19 (5): 66-70) to have genetoxic;
The rare earth compound of suitable concentration has the stimulation promoter action to plant-growth, but different rare earth elements is to the difference that influences of plant-growth.The rare earth element of lower concentration can promote seed germination, take root and plant strain growth, and its physiological action mechanism be it is generally acknowledged relevant (Yang Jiapiao, Zhang Shuyuan with enzyme.Rare earth element is to the preliminary study of the wheat resistance of making a draft.China's rare-earth journal, 1986,4 (4): 67).Must the nutritive element this point not obtain conclusive evidence as yet although whether rare earth itself belongs to plant, many researchs have shown that rare earth element truly has the physiologically actives such as absorption, conversion and utilization of promotion plant to nutrient under certain condition; Rare earth element also has obvious facilitation to Nutrition and Metabolism in the plant materials; Some researchs think that rare earth ion can replace Ca
2+Position on cytolemma, this is to keeping cell membrane stability and nutrient absorbing, running all having been played certain promoter action; But the high concentration rare earth element can destroy cell membrane stability, and membrane permeability is increased, and causes K in the tenuigenin
+Run off Deng nutrient ions, the plant nutrition metabolism is obstructed;
Rare earth element has tangible influence to photosynthesis of plants, but also proves that high concentration rare earth has restraining effect to photosynthesis of plant simultaneously; Certain density rare earth can promote the speed that plant PSII protein complexes activity and photoelectron transmit, thereby promotes transform light energy and photochemical reaction process; The restraining effect of high concentration rare earth may be because rare earth ion with Mg
2+Competitive adsorption takes place and replace Mg
2+, so that influence chlorophyllous synthetic; Rare earth element also has certain influence to the resistance of plant, can improve water conservation and the heat-resisting ability of plant, can also strengthen the resistance against diseases of plant;
High concentration rare earth can impel plant stomata to close, and suppresses transpiration, thereby reduces the Metabolic activity of plant or make its imbalance.High concentration rare earth can form complex compound with ATP, thereby suppresses the catalyzed reaction of hexokinase and glycolysis-is obstructed, and causes that soluble sugar increases in the body, and the plasma membrane permeability increases, and ionogen and tenuigenin exosmose, thereby reduce and destroy the degeneration-resistant resistance against diseases of plant;
The physiological action of rare earth element in plant-growth caused plant cell engineering personnel's attention gradually, also carried out certain research, as the preceding exponential phase in the Vinca cell suspension culture add lanthanum nitrate no matter the accumulation of the growth of pair cell and secondary metabolite promoter action is all arranged (first English advances, Hu Zongding, rare earth element is to the influence of periwinkle cell cultures, rare earth, 1993,14 (3): 38-40); La
3+In the growth course of Chinese cymbidium root stock organoid be by film on acceptor produce and regulate its growth (Luo Hong, Chen Rumin, the influence that La3+ grows some organoid of Chinese cymbidium root stock, tropical and subtropical zone Botany Gazette, 1996,4 (3): 56-59) after second information; Produce in the taxol in the yew cell suspension culture, the adding of lanthanum, cerium can obviously change the cell growth pattern, and help the synthetic of taxol and discharge that (first English advances, Hu Guowu, Wang Chuangui, etc., lanthanum, cerium synthesize and the influence that discharges yew cell growth and taxol, China's rare-earth journal, 1998,16 (1): 56-60); Add lanthanum and not only can promote its growth in hair weeds cells is cultivated, (lanthanum is to the influence of hair weeds cells cultivation and aminoacid component, Chinese rare-earth journal, 1999,17 (1): 60-64) for Liu Shiming, Liang Shizhong but also changed aminoacids content;
But rare earth element yet there are no report in the application aspect plant organ (particularly sweet wormwood organ) cultivation, and along with vegetable cell large scale culturing continuous advancement in technology, rare earth element will show more and more great potential in plant tissue, organ culture.
The objective of the invention is to overcome the defective that exists in above-mentioned many sweet wormwood organ cultural methods, can improve the biomass of sweet wormwood organ and the cultural method of artemislnin content significantly and provide a kind of.
Technical scheme of the present invention is as follows:
The cultural method of raising sweet wormwood organ biomass provided by the invention and artemislnin content is characterized in that step is as follows:
(1) obtaining liq or solid medium:
A. liquid nutrient medium preparation: in the MS liquid nutrient medium, add the sucrose of 20-30g/L, 0 or the 6-BA of 0.5mg/L, 0 or the NAA of 0.05mg/L; Adding contains single or the mixed rare-earth elements compound solution again, and the horizontal high voltage steam sterilizing of going forward side by side is handled; The content of single or mixed rare-earth elements in substratum that is added is 0.0001-0.5mmol/L;
B. solid medium preparation: in the aforesaid liquid substratum, add 6-10g/L agar;
(2) sweet wormwood organ is inserted in aforesaid liquid or the solid medium and to cultivate 20-30 days; Its culture temperature is 25-30 ℃, and illumination is 2000-5000lux;
Described compound single or mixed rare-earth elements comprises nitrate, muriate, the oxide compound of lanthanum, cerium, praseodymium, neodymium or samarium; Described sweet wormwood organ is sweet wormwood bud or sweet wormwood hairly root.
Use method provided by the invention that sweet wormwood organ is cultivated, because increasing substantially of gained culture biomass and artemislnin content, not only can solve the difficult problem of sweet wormwood scarcity of resources in the Artemisinin production, and can improve the Artemisinin turnout, and reduce the Artemisinin production cost; Owing to added cheap rare-earth compound in the substratum that the present invention uses, reduce even save the consumption of NAA, 6-BA hormone, further reduced the Artemisinin production cost, under the dual function that sweet wormwood organ biomass increases and artemislnin content increases, the output of Artemisinin production can significantly improve, and cost reduces.
Of the present invention facts have proved: in sweet wormwood organ (hairly root and the bud of growing thickly) growth and Artemisinin building-up process, the proper amount of rare-earth element can significantly improve the output of sweet wormwood and the content of Artemisinin; This promoter action of rare earth element and its influence to the sweet wormwood organ cell membrane permeability promote nutrition to absorb metabolism, and it is relevant to regulate the inside and outside water balance of cell; The sweet wormwood organ growing environment is relevant with the intravital active oxygen of sweet wormwood with removing; With the phytokinin effect that rare earth element has, growth is relevant fast to promote sweet wormwood organ; Be proportionate because sweet wormwood organ differentiation degree and speed and Artemisinin are synthetic, so the adding of rare earth element has improved the artemislnin content in the sweet wormwood organ simultaneously again significantly; Under the dual function that sweet wormwood organ biomass increases and artemislnin content increases, the Artemisinin output that sweet wormwood organ is cultivated can significantly improve.
The growth of sweet wormwood organ (hairly root and the bud of growing thickly) and the synthetic dependency that exists of Artemisinin, the differentiation of organ is one of Artemisinin synthetic essential condition.In substratum, add rare earth nitrate, muriate, vitriol and be equal to substratum and sterilize together, perhaps earlier earth solution is transferred to certain pH value, in culturing process, add in the substratum by sterilizing filter.The add-on of rare earth element is generally at 0.0001-0.5mmol/L, and the concentration range that single rare earth that each is different or mixed rare-earth elements suit is difference to some extent, and identical rare earth element is also different at the hairly root or the bud of growing thickly.
Further describe the present invention below in conjunction with embodiment:
Embodiment 1: carry out the sweet wormwood bud with method of the present invention and cultivate, its step is as follows:
1. prepare following four kinds of solid mediums:
Preparation solid medium 1: in the MS liquid nutrient medium, add the sucrose of 20g/L, 0.5mg/L 6-BA, 0.05mg/LNAA; The salpeter solution and the 8g/L agar that add lanthanum sesquioxide again, the concentration 0.1mmol/L of lanthanum in this solid medium;
Preparation solid medium 2: in the MS liquid nutrient medium, add the sucrose of 20g/L, 0.5mg/L 6-BA, 0.05mg/L NAA; The oxide compound salpeter solution and the 8g/L agar that add cerium again, the concentration 0.5mmol/L of cerium in this solid medium;
Preparation solid medium 3: in the MS liquid nutrient medium, add the sucrose of 20g/L, 0.5mg/L 6-BA, 0.05mg/L NAA; Add the tri-chlorination two neodymium aqueous solution and 8g/L agar again, the concentration 0.05mmol/L of neodymium in this solid medium;
Preparation solid medium 4: in the MS liquid nutrient medium, add the sucrose of 20g/L, 0.5mg/L 6-BA, 0.05mg/L NAA; Add mishmetal Re oxide compound salpeter solution solution and 8g/L agar again, the concentration 0.04mmol/L of Re in this solid medium; The composition of the Re oxide compound here and content (W/W): La
2O
371.5%+CeO
225.9%+Pr
6O
112.44%+Sm
2O
30.3%;
2. the sweet wormwood bud is inserted respectively in above-mentioned four kinds of solid mediums, at 25 ℃, under the illumination 3000lux condition, cultivated 30 days, its result is as follows:
In solid medium 1, cultivate, gained culture biomass than control group (under same culture conditions, carry out the sweet wormwood bud with conventional MS solid medium and cultivate abbreviation " control group ") increase by 80%, artemislnin content increases by 124% than control group, and unit volume Artemisinin output has improved 305% than control group;
Cultivate in solid medium 2, the biomass of gained culture increases by 1.3 times than control group, and artemislnin content increases nearly 1.5 times than control group, and unit volume Artemisinin output contrast has improved 4.7 times;
Cultivate in solid medium 3, the biomass of gained culture increases by 70.9% than control group, and artemislnin content increases by 160% than control group, and unit volume Artemisinin output has improved 270.9% than control group;
Cultivate in solid medium 4, the biomass of gained culture has increased by 155.5% than control group, and artemislnin content increases by 87.8% than control group, and unit volume Artemisinin output has improved 260.4% than control group.
Embodiment 2: carry out the sweet wormwood bud with method of the present invention and cultivate, its step is as follows:
1. prepare following four kinds of liquid nutrient mediums:
The preparation of liquid nutrient medium 1: do not contain agar, the concentration of lanthanum in this liquid nutrient medium is 0.008mmol/L, and other component and content are identical with solid medium 1 among the embodiment;
The preparation of liquid nutrient medium 2: do not contain agar, the concentration of cerium in this liquid nutrient medium is 0.002mmol/L, and other component and content are identical with solid medium 2 among the embodiment;
The preparation of liquid nutrient medium 3: do not contain agar, the concentration of neodymium in this liquid nutrient medium is 0.002mmol/L, and other component and content are identical with solid medium 3 among the embodiment;
The preparation of liquid nutrient medium 4: do not contain agar, Re (La
2O
371.5%+CeO
225.9%+Pr
6O
112.44%+Sm
2O
30.3%.) concentration in this liquid nutrient medium is 0.008mmol/L, other component and content are identical with solid medium 3 among the embodiment;
2. the sweet wormwood bud is inserted respectively in above-mentioned four kinds of liquid nutrient mediums, at 30 ℃, under the condition of illumination 5000lux, cultivated 20 days, its result is as follows:
Cultivate in the liquid medium within 1, the biomass of gained culture improves 89% than control group; Artemislnin content improves 43% than control group; Artemisinin unit volume output is improving 154.5% than control group;
Cultivate in the liquid medium within 2, the biomass of gained culture improves 73% than control group, and artemislnin content improves 5% than control group, and Artemisinin unit volume output improves 82% than control group;
Cultivate in the liquid medium within 3, the biomass of gained culture improves 67.6% than control group, and artemislnin content improves 19.6% than control group, and Artemisinin unit volume output improves 104.3% than control group;
Cultivate in the liquid medium within 4, the biomass of gained culture improves 74% than control group, and artemislnin content improves 55.4% than control group, and Artemisinin unit volume output improves 169.46% than control group.
Embodiment 3: in the substratum that present embodiment is prepared, each rare earth element concentration sees Table 1 respectively to table 4, and all the other components,, were cultivated 25 days under the illumination 4000lux 28 ℃ of culture temperature with embodiment 2, its result as table 1 to shown in the table 4:
Experimental result when table 1 is lanthanum for rare ten elements
| Condition | ?La 3+Concentration (mmol/L) | Fresh weight Fw (g/L) | Dry weight Wd (g/L) | Artemislnin content (mg/g) | Artemisinin output (mg/L) | Artemisinin output increases (%) |
| Control group | ????0 | ??184.80 | ????6.54 | ????10.62 | ????69.46 | ????0 |
| No NAA and 6BA | ???0.01 | ??161.42 | ????11.48 | ????6.94 | ????79.67 | ????14.70 |
| No NAA has 6BA | ???0.5 | ??197.50 | ????7.22 | ????9.81 | ????70.83 | ????1.97 |
| There is NAA not have 6BA | ???0.1 | ??179.40 | ????11.66 | ????13.55 | ????157.99 | ????127.46 |
Experimental result when table 2 is cerium for rare earth element
| Condition | Ce 3+Concentration (mmol/L) | Fresh weight Fw (g/L) | Dry weight Wd (g/L) | Artemislnin content (mg/g) | Artemisinin output (mg/L) | Artemisinin output increases (%) |
| Contrast | ????0 | ??184.80 | ???6.54 | ????10.62 | ????69.46 | ????0 |
| No NAA and 6BA | ???0.001 | ??237.28 | ???12.02 | ????6.94 | ????83.42 | ????20.10 |
| No NAA has 6BA | ???0.01 | ??215.74 | ???8.18 | ????15.92 | ????130.23 | ????87.49 |
| There is NAA not have 6BA | ???0.1 | ??197.20 | ???12.42 | ????15.19 | ????188.66 | ????171.61 |
Experimental result when table 3 is neodymium for rare earth element
| Condition | ??Nd 3+Concentration (mmol/L) | Fresh weight Fw (g/L) | Dry weight Wd (g/L) | Artemislnin content (mg/g) | Artemisinin output (mg/L) | Artemisinin output increases (%) |
| Contrast | ????0 | ??184.80 | ???6.54 | ????10.62 | ????69.46 | ????0 |
| No NAA and 6BA | ????0.3 | ??155.24 | ???9.18 | ????7.27 | ????66.74 | ????-3.92 |
| No NAA has 6BA | ????0.1 | ??228.44 | ???10.06 | ????10.38 | ????104.42 | ????50.33 |
| There is NAA not have 6BA | ????0.01 | ??155.62 | ???13.40 | ????11.86 | ????158.92 | ????128.79 |
The experimental result that table 4 for rare earth element for mishmetal Re is
| Condition | Re concentration (mmol/L) | Fresh weight Fw (g/L) | Dry weight Wd (g/L) | Artemislnin content (mg/g) | Artemisinin output (mg/L) | Artemisinin output increases (%) |
| Contrast | ???0 | ??184.80 | ????6.54 | ????10.62 | ????69.46 | ????0 |
| No NAA and 6BA | ???0.005 | ??184.10 | ????7.40 | ????11.68 | ????86.43 | ????24.43 |
| No NAA has 6BA | ???0.04 | ??198.16 | ????7.30 | ????17.78 | ????127.79 | ????86.86 |
| There is NAA not have 6BA | ???0.4 | ??183.48 | ????11.62 | ????8.77 | ????101.91 | ????46.72 |
Embodiment 4: present embodiment is joined in four kinds of substratum, the content of sucrose is 3%, rare earth element concentration is respectively 0.04mmol/L lanthanum (in the substratum 1), 0.005mmol/L cerium (in the substratum 2), 0.01mmol/L neodymium (in the substratum 3), 0.04mmol/L mishmetal Re (in the substratum 4), other component and content are with embodiment 2 in the substratum, at 28 ℃, under the condition of illumination 4000lux, cultivated the sweet wormwood bud 25 days in 2.5 liters of reactors, its result is as follows:
Cultivate in substratum 1, can make the biomass of culture improve 15% than control group, artemislnin content improves 10.8% than control group, and unit volume Artemisinin output improves 24.4% than control group;
Cultivate in substratum 2, can make the biomass of culture improve 14.3% than control group, artemislnin content improves 2.5% than control group, and unit volume Artemisinin output improves 17.03% than control group;
Cultivate in substratum 3, can make the biomass of culture improve 15.9% than control group, artemislnin content improves 0.10% than control group; Unit volume Artemisinin output improves 16.03% than control group;
Cultivate in substratum 4, can make the biomass of culture improve 16.8% than control group, artemislnin content improves 3.61% than control group, and unit volume Artemisinin output improves 17.78% than control group.
Embodiment 5: four kinds of liquid nutrient mediums of present embodiment preparation are (to contain 3% sucrose, 0.4g/L NH at the MS liquid nutrient medium
4NO
3, 2.02g/L KNO
3, 0.204g/L KH
2PO
4, 0.37g/L MgSO
4.7H
2O, 0.33g/LCaCl
2) in add rare earth element respectively, the rare earth element concentration that is added: lanthanum is 0.04mmol/L in the liquid nutrient medium 1; Cerium is 0.01mmol/L in the liquid medium within 2; Neodymium is 0.01mmol/L in the liquid medium within 3; Mishmetal Re (La in the liquid medium within 4
2O
371.5%+CeO
225.9%+Pr
6O
112.44%+Sm
2O
30.3%.) be 0.04mmol/L; At 28 ℃, the 3000lux illumination condition, shake-flask culture sweet wormwood hairly root, incubation time are 25 days, its result is as follows:
When cultivating in the liquid medium within 1, culture unit volume Artemisinin output improves 110.2% than control group;
When cultivating in the liquid medium within 2, culture unit volume Artemisinin output increases by 118.73% than control group;
When cultivating in the liquid medium within 3, culture unit volume Artemisinin output improves 79.2% than control group;
When cultivating in the liquid medium within 4, culture unit volume Artemisinin output improves 56.72% than control group.
Embodiment 6: in four kinds of substratum of present embodiment preparation, sucrose concentration is 5%, rare earth element concentration is respectively 0.1mmol/L lanthanum (in the substratum 1), 0.05mmol/L cerium (in the substratum 2), 0.03mmol/L neodymium (in the substratum 3), 0.02mmol/L mishmetal Re (in the substratum 4), all the other components and content are with embodiment 5,28 ℃ of culture temperature, under the illumination 4000lux, cultivated the sweet wormwood hairly root 25 days in 2.5 liters of reactors, its result is as follows:
When cultivating in substratum 1, culture unit volume Artemisinin output improves 42.2% than control group;
When cultivating in substratum 2, culture unit volume Artemisinin output improves 48.32% than control group;
When cultivating in substratum 3, culture unit volume Artemisinin output improves 45.77% than control group;
When cultivating in substratum 4, culture unit volume Artemisinin output improves 28.1% than control group.
Claims (3)
1. cultural method that improves sweet wormwood organ biomass and artemislnin content is characterized in that step is as follows:
(1) obtaining liq or solid medium:
A. liquid nutrient medium preparation: in the MS liquid nutrient medium, add the sucrose of 20-50g/L, 0 or 0.5mg/L
6-BA, 0 or the NAA of 0.05mg/L; Adding contains single or the mixed rare-earth elements compound solution again,
The horizontal high voltage steam sterilizing of going forward side by side is handled; In the single or mixed rare-earth elements liquid medium within that is added
Content is 0.0001-0.5mmol/L;
B. solid medium preparation: in the aforesaid liquid substratum, add 6-10g/L agar;
(2) sweet wormwood organ being inserted in aforesaid liquid or the solid medium, is 25-30 ℃ in temperature, and illumination is under the condition of 2000-5000lux, cultivates 20-30 days.
2. by the cultural method of described raising sweet wormwood organ biomass of claim 1 and artemislnin content, it is characterized in that: described compound single or mixed rare-earth elements comprises nitrate, muriate, the oxide compound of lanthanum, cerium, praseodymium, neodymium or samarium;
3. by the cultural method of described raising sweet wormwood organ biomass of claim 1 and artemislnin content, it is characterized in that: described sweet wormwood organ is sweet wormwood bud or sweet wormwood hairly root.
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| CN102165917A (en) * | 2011-01-11 | 2011-08-31 | 福建农林大学 | Method for raising deguelin content with rare earth element |
| CN101182544B (en) * | 2007-11-15 | 2011-09-21 | 上海柏泰来生物技术有限公司 | Method for enhancing arteannuin content in southernwood by transforming ads gene |
| CN104186318A (en) * | 2014-08-18 | 2014-12-10 | 雷桅 | Method for increasing flavone compound content of scutellaria viscidula |
| CN104472358A (en) * | 2014-11-26 | 2015-04-01 | 浙江理工大学 | Induction method capable of promoting accumulation of tanshinone in hairy roots of salvia miltiorrhiza |
| CN106518888A (en) * | 2016-10-29 | 2017-03-22 | 常州亚环环保科技有限公司 | Bio-extraction method of high-purity artemisinin |
-
2001
- 2001-06-15 CN CNB011159804A patent/CN1137992C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006051987A1 (en) * | 2004-11-15 | 2006-05-18 | Periodock, Co., Ltd. | Novel drug for inhibiting estrogen-dependent diseases, prostaglandin d (pgd)-dependent gynecological diseases, immunological diseases, cancer and angiogenesis |
| JPWO2006051987A1 (en) * | 2004-11-15 | 2008-05-29 | 株式会社ピリオドック | Estrogen-dependent diseases, prostaglandin D (PGD) -dependent gynecological diseases, immune diseases, cancer, and novel pharmaceuticals for inhibiting angiogenesis |
| CN101182544B (en) * | 2007-11-15 | 2011-09-21 | 上海柏泰来生物技术有限公司 | Method for enhancing arteannuin content in southernwood by transforming ads gene |
| CN102165917A (en) * | 2011-01-11 | 2011-08-31 | 福建农林大学 | Method for raising deguelin content with rare earth element |
| CN104186318A (en) * | 2014-08-18 | 2014-12-10 | 雷桅 | Method for increasing flavone compound content of scutellaria viscidula |
| CN104472358A (en) * | 2014-11-26 | 2015-04-01 | 浙江理工大学 | Induction method capable of promoting accumulation of tanshinone in hairy roots of salvia miltiorrhiza |
| CN104472358B (en) * | 2014-11-26 | 2016-08-24 | 浙江理工大学 | The abductive approach of tanshinone accumulation in Hairy Root Cultures of Salvia miltiorrhiza can be promoted |
| CN106518888A (en) * | 2016-10-29 | 2017-03-22 | 常州亚环环保科技有限公司 | Bio-extraction method of high-purity artemisinin |
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