CN111587786A - Culture method for efficiently inducing astragalus membranaceus hairy roots - Google Patents
Culture method for efficiently inducing astragalus membranaceus hairy roots Download PDFInfo
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- CN111587786A CN111587786A CN202010383723.XA CN202010383723A CN111587786A CN 111587786 A CN111587786 A CN 111587786A CN 202010383723 A CN202010383723 A CN 202010383723A CN 111587786 A CN111587786 A CN 111587786A
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- mother liquor
- hairy roots
- astragalus
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- 235000006533 astragalus Nutrition 0.000 title claims abstract description 72
- 241000045403 Astragalus propinquus Species 0.000 title claims abstract description 39
- 238000012136 culture method Methods 0.000 title claims abstract description 26
- 230000001939 inductive effect Effects 0.000 title claims abstract description 22
- 239000001963 growth medium Substances 0.000 claims abstract description 82
- 238000012258 culturing Methods 0.000 claims abstract description 38
- 230000006698 induction Effects 0.000 claims abstract description 37
- 241001061264 Astragalus Species 0.000 claims abstract description 34
- 210000004233 talus Anatomy 0.000 claims abstract description 33
- 241000589156 Agrobacterium rhizogenes Species 0.000 claims abstract description 32
- 238000000034 method Methods 0.000 claims abstract description 13
- 239000012452 mother liquor Substances 0.000 claims description 45
- 239000007788 liquid Substances 0.000 claims description 38
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 30
- 229960002727 cefotaxime sodium Drugs 0.000 claims description 24
- AZZMGZXNTDTSME-JUZDKLSSSA-M cefotaxime sodium Chemical compound [Na+].N([C@@H]1C(N2C(=C(COC(C)=O)CS[C@@H]21)C([O-])=O)=O)C(=O)\C(=N/OC)C1=CSC(N)=N1 AZZMGZXNTDTSME-JUZDKLSSSA-M 0.000 claims description 24
- 238000002791 soaking Methods 0.000 claims description 21
- 239000008223 sterile water Substances 0.000 claims description 21
- 238000005406 washing Methods 0.000 claims description 21
- 239000007787 solid Substances 0.000 claims description 20
- 238000009630 liquid culture Methods 0.000 claims description 19
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 9
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- 229960002523 mercuric chloride Drugs 0.000 claims description 7
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 7
- 239000008399 tap water Substances 0.000 claims description 7
- 235000020679 tap water Nutrition 0.000 claims description 7
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 6
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 6
- 229960000367 inositol Drugs 0.000 claims description 6
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 239000002609 medium Substances 0.000 claims description 6
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 6
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 6
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical class [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 6
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- 241000589158 Agrobacterium Species 0.000 claims description 4
- 229910004613 CdTe Chemical class 0.000 claims description 4
- UHYPYGJEEGLRJD-UHFFFAOYSA-N cadmium(2+);selenium(2-) Chemical class [Se-2].[Cd+2] UHYPYGJEEGLRJD-UHFFFAOYSA-N 0.000 claims description 4
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 4
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 4
- 239000003375 plant hormone Substances 0.000 claims description 4
- 239000002096 quantum dot Substances 0.000 claims description 4
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 4
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 4
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- 235000009529 zinc sulphate Nutrition 0.000 claims description 4
- 229920001817 Agar Polymers 0.000 claims description 3
- 229910021580 Cobalt(II) chloride Inorganic materials 0.000 claims description 3
- 229910021592 Copper(II) chloride Inorganic materials 0.000 claims description 3
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 3
- 229910018890 NaMoO4 Inorganic materials 0.000 claims description 3
- 239000001888 Peptone Substances 0.000 claims description 3
- 108010080698 Peptones Proteins 0.000 claims description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 229930003451 Vitamin B1 Natural products 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 3
- ICSSIKVYVJQJND-UHFFFAOYSA-N calcium nitrate tetrahydrate Chemical compound O.O.O.O.[Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ICSSIKVYVJQJND-UHFFFAOYSA-N 0.000 claims description 3
- 239000005018 casein Substances 0.000 claims description 3
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 3
- 235000021240 caseins Nutrition 0.000 claims description 3
- ORTQZVOHEJQUHG-UHFFFAOYSA-L copper(II) chloride Chemical class Cl[Cu]Cl ORTQZVOHEJQUHG-UHFFFAOYSA-L 0.000 claims description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 3
- 235000019800 disodium phosphate Nutrition 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 150000002505 iron Chemical class 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 3
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 3
- 239000010413 mother solution Substances 0.000 claims description 3
- 229960003512 nicotinic acid Drugs 0.000 claims description 3
- 235000001968 nicotinic acid Nutrition 0.000 claims description 3
- 239000011664 nicotinic acid Substances 0.000 claims description 3
- 239000005416 organic matter Substances 0.000 claims description 3
- 235000019319 peptone Nutrition 0.000 claims description 3
- 235000010333 potassium nitrate Nutrition 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 claims description 3
- 150000003839 salts Chemical class 0.000 claims description 3
- 239000011734 sodium Substances 0.000 claims description 3
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 3
- 229910000162 sodium phosphate Inorganic materials 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 229960003495 thiamine Drugs 0.000 claims description 3
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 3
- 239000011573 trace mineral Substances 0.000 claims description 3
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- 239000012528 membrane Substances 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 6
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- 241000589155 Agrobacterium tumefaciens Species 0.000 abstract 1
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- 238000012216 screening Methods 0.000 abstract 1
- 241000196324 Embryophyta Species 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 5
- 229930195732 phytohormone Natural products 0.000 description 4
- 229940107666 astragalus root Drugs 0.000 description 3
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- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
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- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
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- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- 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
- A01H4/001—Culture apparatus for tissue culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The invention provides a culture method for efficiently inducing astragalus hairy roots, which specifically comprises the steps of cultivating astragalus aseptic seedlings, activating agrobacterium rhizogenes, co-culturing astragalus explants and agrobacterium tumefaciens, and performing degerming induction culture and expansion culture. Through screening proper agrobacterium rhizogenes, adjusting culture medium components and a culture process of the hairy roots, the specific agrobacterium rhizogenes capable of efficiently inducing the hairy roots of the astragalus membranaceus is obtained, and the astragalus membranaceus hairy roots which are stable in proliferation and character are screened out.
Description
Technical Field
The invention relates to a method for inducing medicinal plant astragalus to produce hairy roots by utilizing agrobacterium rhizogenes, in particular to a method for efficiently inducing the hairy roots of astragalus, and belongs to the technical field of astragalus biotechnology cultivation.
Background
Astragalus root belongs to perennial herb of leguminosae, and has been used as medicine by root, so far, more than 2000 years of history. The astragalus root has the main pharmacological actions of tonifying qi, strengthening exterior, promoting urination, expelling toxin, discharging pus, promoting granulation and the like, is mainly used for treating short breath, collapse, palpitation, spontaneous perspiration, body weakness, edema, chronic nephritis, dehydration, chronic diarrhea, qi deficiency, deficiency of qi and blood, sinking of middle-jiao, has the effects of resisting bacteria, diminishing inflammation, reducing blood pressure, promoting urination, detoxifying and benefiting gallbladder and the like, and is a common Chinese herbal medicine.
With the increasing enhancement of health care concept of people, the demand of astragalus membranaceus will increase year by year, but at present, the wild resources of astragalus membranaceus decrease year by year, the cultivars decrease, and the astragalus membranaceus is troubled by the problems of pesticide pollution, heavy metal residue and the like. Therefore, the application of modern biotechnology to increase the yield of astragalus, improve the quality of astragalus and explore feasible ways of industrial production has important significance on the production of astragalus.
Infection of plants by Agrobacterium rhizogenes (Agrobacterium rhizogenes) is a natural genetic transformation pattern in nature, which results in the production of hairy roots by integration and expression of a portion (T-DNA) of the root-causing plasmid (Ri) contained in the plant genome. The hairy root has the characteristics of rapid growth, multiple branches, no geotropism and hormone autotrophy, and the physiological, biochemical and genetic properties of the hairy root are stable. The hairy roots also keep certain biochemical functions of normal roots of plants, can efficiently produce secondary metabolites, and is particularly suitable for producing effective components of substituted plant roots.
Since the first successful transformation of higher plants with Agrobacterium rhizogenes in 1997 Ackermann, more than 100 plant hairy root culture systems have been established to date, many of which are medicinal plants. For example, small-scale culture of hairy roots of Nicotiana citrifolia, Datura stramonium, belladonna, hyoscyami, Vinca rosea, Lithospermum erythrorhizon, Horseradish has been successful. The method for producing the effective secondary metabolites of the medicinal plants in a large scale by utilizing the hairy root culture technology has very attractive prospect, and has positive significance for protecting wild resources and ecological environment and realizing sustainable development.
According to literature reports, the induction of astragalus root by infection of agrobacterium rhizogenes has been successful in producing hairy roots, however, the following outstanding problems exist in the induction and culture of hairy roots, for example: the induction effect of the agrobacterium rhizogenes is unstable, the induction rate is low, and the induction rate is possibly determined by inherent characteristics in the interaction process of plant cells and the agrobacterium rhizogenes; and the immature culture process in the liquid culture process leads to the growth of the hairy roots to be alleviated, thereby influencing the harvest yield and the like. Therefore, the invention hopes to further improve the induction culture method of the astragalus mongholicus hairy roots so as to improve the induction efficiency and yield of the hairy roots and effectively solve the problems of the lack of natural resources of the astragalus mongholicus and the shortage of market products.
Disclosure of Invention
The invention aims to overcome the problems of the lack of natural astragalus resources and the shortage of related market products, protects precious soil resources by utilizing biotechnology, provides a culture method for efficiently inducing astragalus hairy roots, and obtains high induction efficiency and short growth cycle of the astragalus hairy roots by selecting different agrobacterium rhizogenes to induce astragalus callus and adjusting components of a culture solution so as to achieve the effect of improving the yield of the astragalus hairy roots.
In order to achieve the aim, the invention provides a culture method for efficiently inducing astragalus mongholicus hairy roots, which comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking radix astragali seed in tap water for 20-24 hr, soaking in 70% ethanol for 10-15min, and washing with sterile water for 3-5 times; soaking in 1% mercuric chloride solution for 8-10 min; washing with sterile water for 3-5 times; inoculating to MS culture medium sterilized in advance, culturing at 15-25 deg.C and humidity of 50-60% to obtain sterile seedling of radix astragali;
step 2, strain activation and culture
Adding agrobacterium rhizogenes into a liquid YEB culture medium, culturing at 26-28 ℃ and 220-250rpm until the OD600 value is 0.5-0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium at the temperature of 26-28 ℃ and the rotation speed of 150-;
step 3 Material Pre-culture
Cutting the leaves, stems, hypocotyls and petioles of the sterile seedlings of astragalus membranaceus in the step 1 into 0.4-0.5cm in size2Inoculating the small blocks as explants to an MS culture medium added with 0.1-0.3mg/L phytohormone, and culturing for 24-48h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 10-15min at the speed of 50-60rpm and the temperature of 25-28 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.2-0.4mg/L of induction factor to culture for 2-4 days; the induction factor is one or more than two of CuCl2, AgNO3, CdSe quantum dots and CdTe quantum dots;
step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef); separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef), subculturing once every other week for three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, carrying out amplification culture at 25-28 ℃ and under the rotation speed of 100-130rpm, and carrying out subculture once every 18-20 days to obtain a large amount of grown astragalus hairy roots.
Further, the astragalus seeds are selected from one of astragalus mongholicus seeds or astragalus membranaceus seeds;
further, the composition per liter of the MS medium is as follows:
macroelements: KNO34-6mM, Ca (NO3) 2.4H 2O 4-6mM, MgSO 4.7H 2O 1.5.5-2.5 mM, KH2PO40.8-1.2mM,
trace elements: h3BO344-48 μ M, MnCl 2.4H2O 8-10 μ M, ZnSO 4.7H2O 0.6-0.8 μ M, CuSO 4.5H2O 0.2-0.4 μ M, Na2MoO 4.2H2O 0.1-0.3 μ M, EDTA-Fe-Na 40-60 μ M;
organic matter: inositol 80-100. mu.M, sucrose 300-450. mu.M, and hydrolyzed casein 300-500. mu.M.
Further, the 1/2MS culture medium is composed by reducing the dosage of macroelements in the MS culture medium by half; the 1/2MS solid medium contains agar 2-4%.
Further, the agrobacterium strain is selected from one of ATCC15834, ATCC11325, ACCC10060, LBA9402, R1000, R1601, a 4.
Further, the composition of the liquid YEB medium in step 2 is as follows: dissolving yeast powder 1g, beef extract 5g, peptone 5g, sucrose 5g, MgSO4 & 7H2O 0.5g in distilled water 1L, adjusting pH to 7.4 with sodium hydroxide, sterilizing at 121 deg.C under high pressure for 20 min;
further, the plant hormone in the step 3 is one of IBA and NAA.
Further, the composition and preparation method of the 6,7-V liquid culture medium in the step 6 are as follows:
A. preparation of component mother liquor
(1) Macroelement mother liquor: the mother liquor contains 16g KNO3, 2g (NH4)2SO4, 5g MgSO4 & 7H2O, 3g NaH2PO4, 4g KCl and 0.4g Na2HPO4 per liter;
(2) CaCl2 mother liquor: the mother liquor contains 4g of CaCl2 & 2H2O per liter
(3) And (3) a microelement mother solution: the mother liquor contains 4g of MnSO4 & H2O, 1.5g of ZnSO4 & 7H2O, 5g H3BO3, 0.05g of KI, 0.25g of NaMoO4 & 2H2O, 0.25g of CuSO4 & 5H2O and 0.25g of CoCl2 & 6H2O per liter
(4) Mother liquor of iron salt: the mother liquor contains 1.86g of EDTA-Na2 and 1.39g of FeSO 4.7H 2O per liter
(5) Vitamin mother liquor: the mother liquor contains nicotinic acid 0.125g, vitamin B1 0.05g, vitamin B6 0.05g, and inositol 10g per liter
B. The preparation method of the 6,7-V liquid culture medium comprises the following steps: taking 50mL of macroelement mother liquor, 50mL of CaCl2 mother liquor, 1mL of microelement mother liquor, 10mL of ferric salt mother liquor, 10mL of vitamin mother liquor and 30g of cane sugar; dissolving in 1L distilled water to adjust pH to 5.8.
Further, the step-wise reduction of the concentration of cefotaxime sodium (cef) in the step 4 is performed according to the manners of 400mg/L, 300mg/L, 200mg/L and 100 mg/L.
The invention has the following beneficial effects:
(1) according to the method, the proper agrobacterium rhizogenes is screened, the components of the culture medium are adjusted, and the culture process of the hairy roots is adopted, so that the specific agrobacterium rhizogenes capable of efficiently inducing the hairy roots of the astragalus membranaceus is obtained, and the astragalus membranaceus hairy roots which are stable in proliferation and character are screened out, so that the method has the advantages of short production period, uniform production quality and adaptability to normal-temperature growth;
(2) when the explant prepared from the astragalus aseptic seedlings and agrobacterium rhizogenes are co-cultured, a specific induction factor is applied, so that the induction development of the hairy roots can be stimulated, and the induction rate of the hairy roots is greatly improved;
(3) according to the invention, the method for culturing the astragalus membranaceus hairy roots with efficient induction and large-scale rapid growth is obtained by adjusting the culture process of the astragalus membranaceus hairy roots, specifically controlling the composition change of a culture medium, the culture temperature, the culture application and the like, and the problems of the lack of natural resources of astragalus membranaceus and the shortage of market products are effectively solved.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
1. The following raw materials were prepared
(1) Preparing an MS culture medium, wherein the composition of each liter of MS culture medium is as follows:
macroelements: KNO 36 mM, Ca (NO3) 2.4H 2O 6mM, MgSO 4.7H 2O2.5mM, KH2PO41.2mM,
trace elements: h3BO 348 mu M, MnCl2 & 4H2O 10 mu M, ZnSO4 & 7H2O 0.6.6 mu M, CuSO4 & 5H2O0.2 mu M, Na2MoO4 & 2H2O 0.3 mu M, EDTA-Fe-Na 60 mu M;
organic matter: inositol 100. mu.M, sucrose 450. mu.M, and hydrolyzed casein 500. mu.M.
(2) Preparation of 1/2MS medium: reducing the dosage of macroelements in the MS culture medium by half to prepare 1/2MS culture medium;
adding 3% agar into 1/2MS culture medium, and cooling to obtain 1/2MS solid culture medium;
(3) the method of formulating liquid YEB medium is as follows: dissolving yeast powder 1g, beef extract 5g, peptone 5g, sucrose 5g, MgSO4 & 7H2O 0.5g in distilled water 1L, adjusting pH to 7.4 with sodium hydroxide, sterilizing at 121 deg.C under high pressure for 20 min;
(4) preparing a 4.6,7-V liquid culture medium:
A. preparation of component mother liquor
(1) Macroelement mother liquor: the mother liquor contains 16g KNO3, 2g (NH4)2SO4, 5g MgSO4 & 7H2O, 3g NaH2PO4, 4g KCl and 0.4g Na2HPO4 per liter;
(2) CaCl2 mother liquor: the mother liquor contains 4g of CaCl2 & 2H2O per liter
(3) And (3) a microelement mother solution: the mother liquor contains 4g of MnSO4 & H2O, 1.5g of ZnSO4 & 7H2O, 5g H3BO3, 0.05g of KI, 0.25g of NaMoO4 & 2H2O, 0.25g of CuSO4 & 5H2O and 0.25g of CoCl2 & 6H2O per liter
(4) Mother liquor of iron salt: the mother liquor contains 1.86g of EDTA-Na2 and 1.39g of FeSO 4.7H 2O per liter
(5) Vitamin mother liquor: the mother liquor contains nicotinic acid 0.125g, vitamin B1 0.05g, vitamin B6 0.05g, and inositol 10g per liter
The preparation method of the B.6,7-V liquid culture medium comprises the following steps: taking 50mL of macroelement mother liquor, 50mL of CaCl2 mother liquor, 1mL of microelement mother liquor, 10mL of ferric salt mother liquor, 10mL of vitamin mother liquor and 30g of cane sugar; dissolving in 1L distilled water to adjust pH to 5.8.
2. Testing the induction rate of different agrobacterium rhizogenes on astragalus seedling explants
Step 1. preparation of Astragalus aseptic seedling
Soaking Mongolian radix astragali seed in tap water for 24 hr, soaking in 70% ethanol for 13min, and washing with sterile water for 5 times; soaking in 1% mercuric chloride solution for 10 min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, culturing at 25 deg.C and humidity of 60% to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding different kinds of agrobacterium into a liquid YEB culture medium, and culturing at 26 ℃ and 250rpm until the OD600 value is 0.5; then, culturing the bacterial liquid in 1/2MS liquid culture medium, and culturing for 30min at 26 ℃ and 150 rpm;
step 3, cutting the leaves, stems, hypocotyls and petioles of the astragalus membranaceus aseptic seedlings in the step 1 into 0.5cm in size2Inoculating the small blocks as explants to an MS culture medium added with 0.3mg/L phytohormone, and culturing for 48h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium for culturing for 2-4 days;
step 5, degerming induction culture of hairy roots
And (3) washing the explant obtained in the step (4) with sterile water, inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef), culturing at 25 ℃ in a dark place, counting the number of the induced hairy roots after 15 days, and calculating the induction rate. The relevant experimental data are shown in table 1:
TABLE 1
| Agrobacterium rhizogenes | Stem of a tree | Leaf of Chinese character | Hypocotyl | Leaf stalk |
| ATCC15834 | 68％ | 60％ | 55％ | 46％ |
| ATCC11325 | 70％ | 75％ | 66％ | 59％ |
| ACCC10060 | 56％ | 60％ | 46％ | 55％ |
| LBA9402 | 72％ | 80％ | 69％ | 65％ |
| R1601 | 60％ | 49％ | 52％ | 46％ |
| A4 | 55％ | 46％ | 40％ | 50％ |
According to the experimental data in table 1, ATCC11325 and LBA9402 are the best agrobacterium rhizogenes species for the induction of astragalus hairy roots, and the induction efficiency is different in different parts of astragalus, and the induction rate of leaves and stems is higher than that of hypocotyls and petioles. Considering the comprehensive induction rate, the following experiment selects LBA9402 as the specific species of Agrobacterium rhizogenes, and uses the leaves of the seedling of Astragalus membranaceus to prepare explants for inducing the growth of hairy roots.
3. Testing the induction rate of different induction factors on astragalus seedling explants
Agrobacterium rhizogenes LBA9402 and the use of the leaves of the seedlings of Astragalus for experiments, the specific steps are as follows:
step 1. preparation of Astragalus aseptic seedling
Soaking Mongolian radix astragali seed in tap water for 24 hr, soaking in 70% ethanol for 13min, and washing with sterile water for 5 times; soaking in 1% mercuric chloride solution for 10 min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, culturing at 25 deg.C and humidity of 60% to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding different kinds of agrobacterium into a liquid YEB culture medium, and culturing at 26 ℃ and 250rpm until the OD600 value is 0.5; then, culturing the bacterial liquid in 1/2MS liquid culture medium, and culturing for 30min at 26 ℃ and 150 rpm;
step 3, cutting the leaves, stems, hypocotyls and petioles of the astragalus membranaceus aseptic seedlings in the step 1 into 0.5cm in size2Inoculating the small blocks as explants to an MS culture medium added with 0.3mg/L phytohormone, and culturing for 48h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, inoculating the bacteria liquid into an MS culture medium containing 0.4mg/L of induction factors, and culturing for 2 days;
step 5, degerming induction culture of hairy roots
And (3) washing the explant obtained in the step (4) with sterile water, inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef), culturing at 25 ℃ in a dark place, counting the number of the induced hairy roots after 15 days, and calculating the induction rate. The relevant experimental data are shown in table 2:
TABLE 2
According to the experimental data in table 2, when the explant of the astragalus seedling and agrobacterium rhizogenes are co-cultured, different induction factors are applied to the culture medium to promote the induction rate to be increased, and the induction rate can be greatly increased by adding CdSe and CdTe quantum dots.
Example 1
A culture method for efficiently inducing astragalus mongholicus hairy roots comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking Mongolian radix astragali seed in tap water for 24 hr, soaking in 70% ethanol for 15min, and washing with sterile water for 5 times; soaking in 1% mercuric chloride solution for 10 min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, culturing at 15 deg.C and humidity of 60% to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding agrobacterium rhizogenes LBA9402 into liquid YEB culture medium, culturing at 28 ℃ and 220rpm until OD600 value is 0.5; then, culturing the bacterial liquid in 1/2MS liquid culture medium, culturing for 20min at 28 ℃ and 180 rpm;
step 3 Material Pre-culture
Cutting leaves of the sterile seedlings of astragalus membranaceus in the step 1 into 0.4cm in size2Inoculating the small blocks as explants to an MS culture medium added with 0.3mg/L phytohormone IBA, and culturing for 48h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.4mg/L CdSe quantum dots to culture for 4 days; step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef); separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef), subculturing once every other week for three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, carrying out amplification culture at 28 ℃ and 130rpm, and subculturing once every 18 days to obtain a large amount of grown astragalus hairy roots.
Example 2
A culture method for efficiently inducing astragalus mongholicus hairy roots comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking Astragalus membranaceus seed in tap water for 20 hr, soaking in 70% ethanol for 10min, and washing with sterile water for 5 times; soaking in 1% mercuric chloride solution for 8 min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, culturing at 25 deg.C and humidity of 60% to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding agrobacterium rhizogenes LBA9402 into liquid YEB culture medium, culturing at 26 ℃ and 220rpm until OD600 value is 0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium, culturing for 20min at 28 ℃ and 180 rpm;
step 3 Material Pre-culture
Cutting leaves of the sterile seedlings of astragalus membranaceus in the step 1 into 0.4cm in size2Inoculating the small blocks as explants to an MS culture medium added with 0.2mg/L plant hormone NAA, and culturing for 24h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.3mg/L CdTe quantum dots for culture for 4 days; step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef); separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef), subculturing once every other week for three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, carrying out amplification culture at 26 ℃ and 120rpm, and carrying out subculture once every 20 days to obtain a large amount of grown astragalus hairy roots.
Example 3
A culture method for efficiently inducing astragalus mongholicus hairy roots comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking Astragalus membranaceus seed in tap water for 20 hr, soaking in 70% ethanol for 10min, and washing with sterile water for 5 times; soaking in 1% mercuric chloride solution for 8 min; washing with sterile water for 5 times; inoculating to MS culture medium sterilized in advance, culturing at 25 deg.C and humidity of 60% to obtain radix astragali aseptic seedling;
step 2, strain activation and culture
Adding agrobacterium rhizogenes LBA9402 into liquid YEB culture medium, culturing at 26 ℃ and 220rpm until OD600 value is 0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium, and culturing for 20min at 26 ℃ and 160 rpm;
step 3 Material Pre-culture
Cutting leaves of the sterile seedlings of astragalus membranaceus in the step 1 into 0.4cm in size2Inoculating the small blocks as explants to an MS culture medium added with 0.2mg/L plant hormone NAA, and culturing for 24h in a dark place;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 15min at the conditions of 60rpm and 25 ℃; sucking off the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.3mg/L CuCl2 to culture for 3 days; step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef); separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef), subculturing once every other week for three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, carrying out amplification culture at 26 ℃ and 120rpm, and carrying out subculture once every 20 days to obtain a large amount of grown astragalus hairy roots.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (9)
1. A culture method for efficiently inducing astragalus mongholicus hairy roots is characterized by comprising the following steps: the method comprises the following steps:
step 1. preparation of Astragalus aseptic seedling
Soaking radix astragali seed in tap water for 20-24 hr, soaking in 70% ethanol for 10-15min, and washing with sterile water for 3-5 times; soaking in 1% mercuric chloride solution for 8-10 min; washing with sterile water for 3-5 times; inoculating to MS culture medium sterilized in advance, culturing at 15-25 deg.C and humidity of 50-60% to obtain sterile seedling of radix astragali;
step 2, strain activation and culture
Adding agrobacterium rhizogenes into a liquid YEB culture medium, culturing at 26-28 ℃ and 220-250rpm until the OD600 value is 0.5-0.6; then, culturing the bacterial liquid in 1/2MS liquid culture medium at the temperature of 26-28 ℃ and the rotation speed of 150-;
step 3 Material Pre-culture
Cutting the leaves, stems, hypocotyls and petioles of the sterile seedlings of astragalus membranaceus in the step 1 into 0.4-0.5cm in size2The pieces are used as explants to be inoculated with 0.1-0.3mg/L phytohormoneCulturing in MS culture medium in dark for 24-48 hr;
step 4. Co-culture of explant and Agrobacterium rhizogenes
Fully contacting the explant in the step 3 with the bacterial liquid in the step 2 for 10-15min at the speed of 50-60rpm and the temperature of 25-28 ℃; sucking the redundant bacteria liquid on the surface by using sterile filter paper, and inoculating the bacteria liquid into an MS culture medium added with 0.2-0.4mg/L of induction factor to culture for 2-4 days; the induction factor is one or more than two of CuCl2, AgNO3, CdSe quantum dots and CdTe quantum dots;
step 5, degerming induction culture of hairy roots
Washing the explant obtained in the step 4 with sterile water, and then inoculating the washed explant to 1/2MS solid culture medium containing 500mg/L cefotaxime sodium (cef) to be cultured in the dark at 25 ℃; replacing the culture medium with new one week at intervals, and gradually reducing the concentration of cefotaxime sodium (cef); separating the roots after the roots reach 3cm, placing the roots on 1/2MS solid culture medium containing 50mg/L cefotaxime sodium (cef), subculturing once every other week for three times, and then placing the roots on 1/2MS solid culture medium without cefotaxime sodium (cef) for culture;
step 6, enlarged cultivation of hairy root
Transferring the hairy roots in the step 5 into a 6,7-V liquid culture medium, carrying out amplification culture at 25-28 ℃ and under the rotation speed of 100-130rpm, and carrying out subculture once every 18-20 days to obtain a large amount of grown astragalus hairy roots.
2. The culture method for efficiently inducing the hairy roots of astragalus membranaceus as claimed in claim 1, wherein the culture method comprises the following steps: the radix astragali seed is selected from one of Mongolian radix astragali seed or membrane pod radix astragali seed.
3. The culture method for efficiently inducing the hairy roots of astragalus membranaceus according to any one of claims 1-2, wherein the culture method comprises the following steps: the composition per liter of the MS medium was as follows:
macroelements: KNO34-6mM, Ca (NO3) 2.4H 2O 4-6mM, MgSO 4.7H 2O 1.5.5-2.5 mM, KH2PO40.8-1.2mM,
trace elements: h3BO344-48 μ M, MnCl 2.4H2O 8-10 μ M, ZnSO 4.7H2O 0.6-0.8 μ M, CuSO 4.5H2O 0.2-0.4 μ M, Na2MoO 4.2H2O 0.1-0.3 μ M, EDTA-Fe-Na 40-60 μ M;
organic matter: inositol 80-100. mu.M, sucrose 300-450. mu.M, and hydrolyzed casein 300-500. mu.M.
4. The culture method for efficiently inducing the hairy roots of astragalus membranaceus according to any one of claims 1 to 3, wherein the culture method comprises the following steps: the 1/2MS culture medium is composed by halving the dosage of macroelements in the MS culture medium; the 1/2MS solid medium contains agar 2-4%.
5. The culture method for efficiently inducing the hairy roots of astragalus membranaceus according to any one of claims 1 to 4, wherein the culture method comprises the following steps: the agrobacterium strain is selected from one of ATCC15834, ATCC11325, ACCC10060, LBA9402, R1000, R1601 and A4.
6. The culture method for efficiently inducing the hairy roots of astragalus membranaceus according to any one of claims 1 to 5, wherein the culture method comprises the following steps: the composition of the liquid YEB medium in step 2 is as follows: dissolving yeast powder 1g, beef extract 5g, peptone 5g, sucrose 5g, MgSO4 & 7H2O 0.5g in 1L distilled water, adjusting pH to 7.4 with sodium hydroxide, sterilizing at 121 deg.C under high pressure for 20 min.
7. The culture method for efficiently inducing the hairy roots of astragalus membranaceus according to any one of claims 1 to 5, wherein the culture method comprises the following steps: the plant hormone in the step 3 is one of IBA and NAA.
8. The culture method for efficiently inducing the hairy roots of astragalus membranaceus according to any one of claims 1 to 5, wherein the culture method comprises the following steps: the composition and preparation method of the 6,7-V liquid culture medium in the step 6 are as follows:
A. preparation of component mother liquor
(1) Macroelement mother liquor: the mother liquor contains 16g KNO3, 2g (NH4)2SO4, 5g MgSO4 & 7H2O, 3g NaH2PO4, 4g KCl and 0.4g Na2HPO4 per liter;
(2) CaCl2 mother liquor: the mother liquor contains 4g of CaCl2 & 2H2O per liter
(3) And (3) a microelement mother solution: the mother liquor contains 4g of MnSO4 & H2O, 1.5g of ZnSO4 & 7H2O, 5g H3BO3, 0.05gKI, 0.25g of NaMoO4 & 2H2O, 0.25g of CuSO4 & 5H2O and 0.25g of CoCl2 & 6H2O per liter
(4) Mother liquor of iron salt: the mother liquor contains 1.86g of EDTA-Na2 and 1.39g of FeSO 4.7H 2O per liter
(5) Vitamin mother liquor: the mother liquor contains nicotinic acid 0.125g, vitamin B1 0.05g, vitamin B6 0.05g, and inositol 10g per liter
B. The preparation method of the 6,7-V liquid culture medium comprises the following steps: taking 50mL of macroelement mother liquor, 50mL of CaCl2 mother liquor, 1mL of microelement mother liquor, 10mL of ferric salt mother liquor, 10mL of vitamin mother liquor and 30g of cane sugar; dissolving in 1L distilled water to adjust pH to 5.8.
9. The culture method for efficiently inducing the hairy roots of astragalus membranaceus according to any one of claims 1 to 5, wherein the culture method comprises the following steps: the step-wise reduction of the concentration of cefotaxime sodium (cef) in the step 4 is performed according to the modes of 400mg/L, 300mg/L, 200mg/L and 100 mg/L.
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| CN102776223A (en) * | 2012-06-05 | 2012-11-14 | 上海师范大学 | Method for improving tanshinone content of hairy roots of salvia miltiorrhiza through quantum dots |
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| KR20140122533A (en) * | 2013-04-10 | 2014-10-20 | (주)아모레퍼시픽 | Mass propagation method for adventitious root of Astragali Radix containing the increased amount of Astragaloside IV |
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