CN102524075A - Method for culturing chrysanthemum morifolium ramat with high flavones content - Google Patents
Method for culturing chrysanthemum morifolium ramat with high flavones content Download PDFInfo
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- CN102524075A CN102524075A CN2012100283016A CN201210028301A CN102524075A CN 102524075 A CN102524075 A CN 102524075A CN 2012100283016 A CN2012100283016 A CN 2012100283016A CN 201210028301 A CN201210028301 A CN 201210028301A CN 102524075 A CN102524075 A CN 102524075A
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Abstract
The invention discloses a method for culturing chrysanthemum morifolium ramat with high flavones content, which comprises the following steps: inducing calluses by using the scotyledons of chrysanthemum morifolium ramat as explants, subculturing the calluses till a multiplication period, pre-culturing the calluses under a dark condition, performing agrobacterium tumefaciens coculture transformation, transferring the calluses to a screening culture medium, performing three rounds of screening, inducing resistant calluses to differentiate into seedlings, performing gene marking histochemical staining and gene screening molecular detection on resistant plants, identifying differentiated seedlings having the insertion sequence of a target gene, namely chalcone synthases (CHS), detecting the total flavonoids content in petals of the screened transgenic plants, selecting transgenic plants with total flavonoids content of above 10 percent, continuing to evaluate the expression stability of the total flavonoids content, and selecting high-quality plants with stable total flavonoids content of above 10 percent. The chrysanthemum morifolium ramat with high flavones content cultured by the invention can be developed into novel health-care food additive and nutrition reinforcing agent.
Description
Technical field
The present invention relates to a kind of breeding method of FLOS CHRYSANTHEMI ALBA from Haizhou of China, especially improve the breeding method of the FLOS CHRYSANTHEMI ALBA from Haizhou of China of flavones content.
Background technology
FLOS CHRYSANTHEMI ALBA from Haizhou of China is the dry capitulum of feverfew chrysanthemum, and cultivation history is long, according to the record of relevant historical data, at least surplus in the of existing 360 year, is one of " Zhejiang eight flavors " authentic medicinal herbs so far.FLOS CHRYSANTHEMI ALBA from Haizhou of China contains cyanidenon-7-β-D-glucoside, apiolin-7-β-D-glucoside, acacetin-7-β-multiple flavonoids effective constituents such as D-glucoside, and antioxidation is good, has the effect of " clearing away heat to dispel wind, flat liver make eye bright ".Discover in early days to show that effects such as that FLOS CHRYSANTHEMI ALBA from Haizhou of China has is analgesic, antibacterial, resisiting influenza virus, anti-cardiovascular, anti-inflammatory have research to show that FLOS CHRYSANTHEMI ALBA from Haizhou of China has effects such as anti-oxidant, antitumor, anti-AIDS in recent years both at home and abroad.At present, the processing corporate boss will extract the separation chrysanthemum total flavone, and therefore, the FLOS CHRYSANTHEMI ALBA from Haizhou of China new varieties of seed selection high flavones content become the main direction of breeding.
Summary of the invention
Under above-mentioned background, the objective of the invention is to propose a kind of breeding method of high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China just,, carry out the exploitation of flavonoids, be used for food and medicine industry to obtain the high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China.
The breeding method of high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China of the present invention may further comprise the steps:
1) cotyledon of getting FLOS CHRYSANTHEMI ALBA from Haizhou of China is an explant, is seeded to inducing culture, under 25 ± 1 ℃, light intensity 3000 lux optical condition, induces the formation callus;
2) will induce the callus that formed for 3 weeks, and be transferred to subculture medium, successive transfer culture to callus gets into breeds the phase at double;
3) callus that will breed at double carries out the preparatory cultivation 2 days of acceptor callus under the dark condition of 25 ± 1 ℃ of lucifuges;
4) will pass through pre-incubated callus, in agrobacterium suspension, soak 15 minutes, discard bacterium liquid afterwards, blot, be inoculated on the common medium, under the dark condition of 25 ± 1 ℃ of lucifuges, cultivate altogether 2 days with aseptic filter paper;
5) with above-mentioned cultured calli altogether, after cleaning, drying, transfer to screening culture medium; Under the dark condition of 25 ± 1 ℃ of lucifuges, cultivated for 6 weeks, collect the resistant calli that newly grows, be transferred on the new screening culture medium; Carry out continuous 2 again and take turns the subculture screening, whenever continue 1 time at a distance from 3 turnovers;
6) get 3 resistant callis of taking turns after the screening; Be transferred to differential medium; Under 25 ± 1 ℃, light intensity 3000 lux optical condition, induce seedling differentiation; The antagonism plant carries out histochemical stain and Molecular Detection; Select and remain contain the genes of interest insetion sequence transfer-gen plant, said genes of interest insetion sequence is meant the insetion sequence of genes of interest chalcone synthase gene C HS, marker gene glucuronidase gene GUS and the anti-hygromycin gene HPT of screening-gene, genes of interest chalcone synthase gene C HS sheet segment length 960bp;
7) be taken into the transfer-gen plant of choosing, field planting is measured general flavone content in the petal, and the general flavone content of selecting and remain is higher than 3% transfer-gen plant;
8) continue the transgenic line that the plantation step 7) is screened, estimate the expression stability of flavones content, stable 3% the elite plant strain that is higher than of breeding general flavone content is the high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China of cultivating.
Among the present invention, said inducing culture adds IAA0.3mg, 6-BA12mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
Among the present invention, said subculture medium adds IAA0.3mg, 6-BA12mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
Among the present invention, said medium altogether adds IAA0.3mg, 6-BA12mg, acetosyringone 1mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
Among the present invention, said screening culture medium is added IAA0.3mg, 6-BA12mg, hygromycin 25mg, carboxylic Bian 200 mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
Among the present invention, said differential medium adds IAA0.3mg, 6-BA4mg/L, carboxylic Bian 200mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
Above-mentioned MS minimal medium is (NH
4)
2NO
31650mg, KNO
31900mg, CaCl
22H
2O 440mg, MgSO
47H
2O 370mg, KH
2P0
4170mg, MnSO
44H
2O 22.3mg, ZnSO
47H
2O 8.6mg, Na
2MoO
42H2O 0.25 mg, CuSO
45H
2O 0.025mg, CoCl
26H
2O 0.025mg, Na
2-EDTA2H2O 37.3mg, FeSO
47H
2O 27.8mg, H
3BO
36.2mg, KI 0.83mg, inositol 100mg, nicotinic acid 0.5mg, vitamin B6 0.5mg, vitamin B1 0.5mg and glycine 2mg.
Among the present invention; The bacterial strain of said Agrobacterium is EHA105; Include plasmid pCAM1305; The genes of interest insetion sequence is carried in the T-DNA zone of pCAM1305, comprises the insetion sequence of genes of interest chalcone synthase gene C HS, marker gene glucuronidase gene GUS and the anti-hygromycin gene HPT of screening-gene.Genes of interest chalcone synthase gene C HS sheet segment length 960bp constitutes the expression framework jointly with 35S and no terminator.Chalcone synthase gene C HS is first key enzyme in the flavonoids biosynthesis, and the activity of CHS is directly connected to the synthetic of flavonoids, can regulate and control to improve the content of flavones.Bacterial strain is that EHA10 conveniently buys or give acquisition, in general plant genetic laboratory preservation is arranged all, referring to document Wu Guan front yard etc., and Scientia Agricultura Sinica, 2005,38 (12): 2395-2402.
The histochemical stain method of above-mentioned marker gene glucuronidase gene GUS is referring to Rueb and Hensgens. Rice Genetics Newsletters; 1989; (6): 168-169, the molecular detecting method of the anti-hygromycin gene HPT of screening-gene is referring to document Wu Guan front yard etc., Scientia Agricultura Sinica; 2005,38 (12): 2395-2402.
Among the present invention, said flavones content detection method is that the colorimetric method of standard specimen is measured at wavelength 510nm place with the rutin; Referring to document Yang Mei China; Lu fills big, Kuang Yanwei, etc. content of total flavone in column chromatography-determined by ultraviolet spectrophotometry Desmodium styracifolium. Chinese herbal medicine; 2004,35 (6): 688 ~ 690.
Above-mentioned stability be meant flavones content in the selected FLOS CHRYSANTHEMI ALBA from Haizhou of China between different year between (more than 2 years), season the content of (more than 2 seasons) and intersite (2 more than the place) not remarkable.
The FLOS CHRYSANTHEMI ALBA from Haizhou of China of the high flavones content that the present invention cultivates has the following advantages:
The present invention at first utilizes chalcone synthase gene C HS; Activity through synthetic first key enzyme of regulation and control flavonoids; Significantly improve the content of flavones, cultivate the high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China, carry out high-performance bio and extract; Development novel healthy food additive and nutrition fortifier are used for food, field of medicaments.
Description of drawings
Fig. 1 is the vector construction sketch map of the used chalcone synthase gene C HS insetion sequence of the present invention.
Embodiment
Below further specify the present invention through instantiation.
Embodiment 1:
Cotyledon to get FLOS CHRYSANTHEMI ALBA from Haizhou of China kind " late small ocean chrysanthemum " is an explant, is seeded to inducing culture, under 25 ± 1 ℃, light intensity 3000 lux optical condition, induces the formation callus;
Get and induce the callus that formed for 3 weeks, be transferred to subculture medium, successive transfer culture to callus gets into breeds the phase at double;
Get at double the callus of propagation, under 25 ± 1 ℃, the dark condition of lucifuge, carry out the acceptor callus
Preparatory cultivation 2 days;
The pre-incubated callus (about 400 culture dishes contain the callus more than 2500) of learning from else's experience soaked 15 minutes in agrobacterium suspension; Discard bacterium liquid afterwards; Blot with aseptic filter paper, be inoculated on the common medium, under 25 ± 1 ℃, the dark condition of lucifuge, cultivated altogether 2 days;
Get above-mentioned cultured calli altogether; After cleaning, drying, transfer to screening culture medium, under 25 ± 1 ℃, the dark condition of lucifuge, cultivated for 6 weeks; Collect the resistant calli (about 420 callus) that newly grows; Be transferred on the new screening culture medium, carry out continuous 2 again and take turns the subculture screening, whenever continue 1 time at a distance from 3 turnovers;
Get 3 resistant callis (about 60 callus) of taking turns after the screening; Be transferred to differential medium; Under 25 ± 1 ℃, light intensity 3000 lux optical condition, induce seedling differentiation 75 strains; The antagonism plant carries out marker gene GUS histochemical stain and screening-gene HPT Molecular Detection, selects and remain to contain transfer-gen plant 21 strains (vector construction of genes of interest insetion sequence is as shown in Figure 1) of genes of interest chalcone synthase gene C HS insetion sequence.
Be taken into the transfer-gen plant of choosing, gather in the crops seed behind the field planting, carry out flavones content detection in the blade, the general flavone content of selecting and remain is higher than 10% transfer-gen plant 7 strains, continues the plantation transgenic line;
Continuous 3 years of 2008-2010; Estimate the expression stability of flavones content respectively in Zhejiang Hangzhou, Linan, three seasons of three locations, Fuyang; Stable 10% the elite plant strain that is higher than of breeding general flavone content; Final 2 of the high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China of cultivating: T-HBJ-1 and T-HBJ-2, general flavone content is respectively 11.9%, 10.3% in its petal, and the general flavone content of former FLOS CHRYSANTHEMI ALBA from Haizhou of China is merely 6.93%.
Claims (7)
1. the breeding method of a high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China may further comprise the steps:
1) cotyledon of getting FLOS CHRYSANTHEMI ALBA from Haizhou of China is an explant, is seeded to inducing culture, under 25 ± 1 ℃, light intensity 3000 lux optical condition, induces the formation callus;
2) will induce the callus that formed for 3 weeks, and be transferred to subculture medium, successive transfer culture to callus gets into breeds the phase at double;
3) callus that will breed at double carries out the preparatory cultivation 2 days of acceptor callus under the dark condition of 25 ± 1 ℃ of lucifuges;
4) will pass through pre-incubated callus, in agrobacterium suspension, soak 15 minutes, discard bacterium liquid afterwards, blot, be inoculated on the common medium, under the dark condition of 25 ± 1 ℃ of lucifuges, cultivate altogether 2 days with aseptic filter paper;
5) with above-mentioned cultured calli altogether, after cleaning, drying, transfer to screening culture medium; Under the dark condition of 25 ± 1 ℃ of lucifuges, cultivated for 6 weeks, collect the resistant calli that newly grows, be transferred on the new screening culture medium; Carry out continuous 2 again and take turns the subculture screening, whenever continue 1 time at a distance from 3 turnovers;
6) get 3 resistant callis of taking turns after the screening; Be transferred to differential medium; Under 25 ± 1 ℃, light intensity 3000 lux optical condition, induce seedling differentiation; The antagonism plant carries out histochemical stain and Molecular Detection; Select and remain contain the genes of interest insetion sequence transfer-gen plant, said genes of interest insetion sequence is meant the insetion sequence of genes of interest chalcone synthase gene C HS, marker gene glucuronidase gene GUS and the anti-hygromycin gene HPT of screening-gene, genes of interest chalcone synthase gene C HS sheet segment length 960bp;
7) be taken into the transfer-gen plant of choosing, field planting is measured general flavone content in the petal, and the general flavone content of selecting and remain is higher than 3% transfer-gen plant;
8) continue the transgenic line that the plantation step 7) is screened, estimate the expression stability of flavones content, stable 3% the elite plant strain that is higher than of breeding general flavone content is the high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China of cultivating.
2. the breeding method of high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China according to claim 1 is characterized in that said inducing culture adds IAA0.3mg, 6-BA12mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
3. the breeding method of high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China according to claim 1 is characterized in that said subculture medium adds IAA0.3mg, 6-BA12mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
4. the breeding method of high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China according to claim 1 is characterized in that said medium altogether adds IAA0.3mg, 6-BA12mg, acetosyringone 1mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
5. the breeding method of high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China according to claim 1; It is characterized in that said screening culture medium is MS minimal medium interpolation IAA0.3mg, 6-BA12mg, hygromycin 25mg, carboxylic Bian 200 mg, sucrose 30g and agar powder 6.8g, pH sterilization preceding 5.7.
6. the breeding method of high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China according to claim 1 is characterized in that said differential medium adds IAA0.3mg, 6-BA4mg/L, carboxylic Bian 200mg, sucrose 30g and agar powder 6.8g for the MS minimal medium, pH sterilization preceding 5.7.
7. according to the breeding method of the described high flavones content FLOS CHRYSANTHEMI ALBA from Haizhou of China of claim 2-6, it is characterized in that said MS minimal medium is KNO
31900mg, (NH
4)
2NO
31650mg, CaCl
22H
2O 440mg, MgSO
47H
2O 370mg, KH
2P0
4170mg, MnSO
44H
2O 22.3mg, ZnSO
47H
2O 8.6mg, FeSO
47H
2O 27.8mg, Na
2MoO
42H2O 0.25 mg, CuSO
45H
2O 0.025mg, CoCl
26H
2O 0.025mg, Na
2-EDTA2H2O 37.3mg, H
3BO
36.2mg, KI 0.83mg, inositol 100mg, nicotinic acid 0.5mg, vitamin B6 0.5mg, vitamin B1 0.5mg and glycine 2mg.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004585A (en) * | 2012-12-12 | 2013-04-03 | 南京农业大学 | Method for quickly screening transgene chrysanthemum plant by kanamycin |
| CN103430840A (en) * | 2013-08-01 | 2013-12-11 | 南京农业大学 | Breeding method for disease-resistant variety of dendranthema morifolium |
| CN103975851A (en) * | 2014-03-31 | 2014-08-13 | 中国计量学院 | Chrysanthemum morifolium tissue culturing and breeding method |
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| CN1888070A (en) * | 2005-06-28 | 2007-01-03 | 北京林业大学 | Agrobacterium tumefaciens mediated ground cover chrysanthemum genetically modifying method |
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2012
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1888070A (en) * | 2005-06-28 | 2007-01-03 | 北京林业大学 | Agrobacterium tumefaciens mediated ground cover chrysanthemum genetically modifying method |
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| 余琪等: "杭白菊提取物中活性成分及总黄酮的含量测定", 《中国现代应用药学》, vol. 28, no. 6, 30 June 2011 (2011-06-30) * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103004585A (en) * | 2012-12-12 | 2013-04-03 | 南京农业大学 | Method for quickly screening transgene chrysanthemum plant by kanamycin |
| CN103004585B (en) * | 2012-12-12 | 2015-03-18 | 南京农业大学 | Method for quickly screening transgene chrysanthemum plant by kanamycin |
| CN103430840A (en) * | 2013-08-01 | 2013-12-11 | 南京农业大学 | Breeding method for disease-resistant variety of dendranthema morifolium |
| CN103975851A (en) * | 2014-03-31 | 2014-08-13 | 中国计量学院 | Chrysanthemum morifolium tissue culturing and breeding method |
| CN103975851B (en) * | 2014-03-31 | 2016-01-27 | 中国计量学院 | A kind of method of FLOS CHRYSANTHEMI ALBA from Haizhou of China tissue cultures and breeding |
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Application publication date: 20120704 |