CN106912349B - Method for promoting accumulation of luteoloside in honeysuckle flower buds by using fungi - Google Patents
Method for promoting accumulation of luteoloside in honeysuckle flower buds by using fungi Download PDFInfo
- Publication number
- CN106912349B CN106912349B CN201710116171.4A CN201710116171A CN106912349B CN 106912349 B CN106912349 B CN 106912349B CN 201710116171 A CN201710116171 A CN 201710116171A CN 106912349 B CN106912349 B CN 106912349B
- Authority
- CN
- China
- Prior art keywords
- honeysuckle
- cutting
- liquid
- bacterial liquid
- culture medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 241000233866 Fungi Species 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 49
- WJHSRFQBVYHKKL-UHFFFAOYSA-N Oroboside Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=C2C(=O)C(C=3C=C(O)C(O)=CC=3)=COC2=C1 WJHSRFQBVYHKKL-UHFFFAOYSA-N 0.000 title claims abstract description 15
- 238000009825 accumulation Methods 0.000 title claims abstract description 15
- PEFNSGRTCBGNAN-QNDFHXLGSA-N luteolin 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C=C(C=3C=C(O)C(O)=CC=3)OC2=C1 PEFNSGRTCBGNAN-QNDFHXLGSA-N 0.000 title claims abstract description 15
- 230000001737 promoting effect Effects 0.000 title claims abstract description 9
- 241000205585 Aquilegia canadensis Species 0.000 title claims abstract 22
- 238000005520 cutting process Methods 0.000 claims abstract description 74
- 240000006439 Aspergillus oryzae Species 0.000 claims abstract description 32
- 235000002247 Aspergillus oryzae Nutrition 0.000 claims abstract description 32
- 238000011081 inoculation Methods 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims description 82
- 230000001580 bacterial effect Effects 0.000 claims description 53
- 239000001963 growth medium Substances 0.000 claims description 39
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 24
- 238000002360 preparation method Methods 0.000 claims description 23
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 23
- 238000009630 liquid culture Methods 0.000 claims description 21
- 239000000758 substrate Substances 0.000 claims description 21
- 235000001466 Ribes nigrum Nutrition 0.000 claims description 17
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 15
- 244000061456 Solanum tuberosum Species 0.000 claims description 15
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 15
- 239000008103 glucose Substances 0.000 claims description 15
- 229920001817 Agar Polymers 0.000 claims description 13
- 239000008272 agar Substances 0.000 claims description 13
- 238000012258 culturing Methods 0.000 claims description 12
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 12
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 12
- 239000000843 powder Substances 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 11
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 11
- 241000894006 Bacteria Species 0.000 claims description 10
- 240000001890 Ribes hudsonianum Species 0.000 claims description 10
- 235000016954 Ribes hudsonianum Nutrition 0.000 claims description 10
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 10
- IQPNAANSBPBGFQ-UHFFFAOYSA-N luteolin Chemical compound C=1C(O)=CC(O)=C(C(C=2)=O)C=1OC=2C1=CC=C(O)C(O)=C1 IQPNAANSBPBGFQ-UHFFFAOYSA-N 0.000 claims description 10
- LRDGATPGVJTWLJ-UHFFFAOYSA-N luteolin Natural products OC1=CC(O)=CC(C=2OC3=CC(O)=CC(O)=C3C(=O)C=2)=C1 LRDGATPGVJTWLJ-UHFFFAOYSA-N 0.000 claims description 10
- 235000009498 luteolin Nutrition 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 9
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 241001312569 Ribes nigrum Species 0.000 claims description 7
- 229930006000 Sucrose Natural products 0.000 claims description 7
- 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 7
- 238000002156 mixing Methods 0.000 claims description 7
- 229960004793 sucrose Drugs 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 6
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 6
- 238000002791 soaking Methods 0.000 claims description 6
- 238000007598 dipping method Methods 0.000 claims description 5
- 201000010099 disease Diseases 0.000 claims description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 5
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 5
- 241001440127 Phyllodes Species 0.000 claims description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 4
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 claims description 4
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 claims description 4
- 238000010008 shearing Methods 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 239000011782 vitamin Substances 0.000 claims description 4
- 229930003231 vitamin Natural products 0.000 claims description 4
- 229940088594 vitamin Drugs 0.000 claims description 4
- 235000013343 vitamin Nutrition 0.000 claims description 4
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims description 3
- 241000238631 Hexapoda Species 0.000 claims description 3
- 240000007594 Oryza sativa Species 0.000 claims description 3
- 235000007164 Oryza sativa Nutrition 0.000 claims description 3
- 241000607479 Yersinia pestis Species 0.000 claims description 3
- 239000006013 carbendazim Substances 0.000 claims description 3
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims description 3
- 229960002089 ferrous chloride Drugs 0.000 claims description 3
- 239000006260 foam Substances 0.000 claims description 3
- 239000003864 humus Substances 0.000 claims description 3
- 239000002054 inoculum Substances 0.000 claims description 3
- 238000009413 insulation Methods 0.000 claims description 3
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 claims description 3
- 239000012286 potassium permanganate Substances 0.000 claims description 3
- 235000009566 rice Nutrition 0.000 claims description 3
- 239000004576 sand Substances 0.000 claims description 3
- 238000005507 spraying Methods 0.000 claims description 3
- 239000010455 vermiculite Substances 0.000 claims description 3
- 235000019354 vermiculite Nutrition 0.000 claims description 3
- 229910052902 vermiculite Inorganic materials 0.000 claims description 3
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 238000002054 transplantation Methods 0.000 claims description 2
- 239000011159 matrix material Substances 0.000 claims 2
- 241001556138 Phellinus ribis Species 0.000 claims 1
- 241000745988 Phyllostachys Species 0.000 claims 1
- 241000001727 Tropicoporus linteus Species 0.000 claims 1
- 230000003020 moisturizing effect Effects 0.000 claims 1
- 229930000044 secondary metabolite Natural products 0.000 abstract description 10
- 241000123107 Phellinus Species 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 3
- 235000011483 Ribes Nutrition 0.000 abstract description 2
- 241000220483 Ribes Species 0.000 abstract description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 2
- 210000000614 rib Anatomy 0.000 abstract description 2
- 241001570521 Lonicera periclymenum Species 0.000 description 57
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 14
- 241001135342 Phyllobacterium Species 0.000 description 8
- 239000000706 filtrate Substances 0.000 description 8
- 239000002689 soil Substances 0.000 description 8
- 238000001514 detection method Methods 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000000243 solution Substances 0.000 description 6
- 244000167230 Lonicera japonica Species 0.000 description 4
- 241001341042 Phylloporia <moth> Species 0.000 description 4
- 238000009835 boiling Methods 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000002881 soil fertilizer Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000194107 Bacillus megaterium Species 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 239000003337 fertilizer Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 238000004811 liquid chromatography Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 235000017617 Lonicera japonica Nutrition 0.000 description 2
- 240000001396 Malus hupehensis Species 0.000 description 2
- 235000005057 Malus hupehensis Nutrition 0.000 description 2
- 244000239204 Plantago lanceolata Species 0.000 description 2
- 235000010503 Plantago lanceolata Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000012364 cultivation method Methods 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 230000002538 fungal effect Effects 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 238000003973 irrigation Methods 0.000 description 2
- 230000002262 irrigation Effects 0.000 description 2
- -1 luteoloside Natural products 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 241000208828 Caprifoliaceae Species 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 208000002979 Influenza in Birds Diseases 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010068319 Oropharyngeal pain Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 201000007100 Pharyngitis Diseases 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 201000003176 Severe Acute Respiratory Syndrome Diseases 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 206010064097 avian influenza Diseases 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 238000001784 detoxification Methods 0.000 description 1
- 230000005059 dormancy Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000004720 fertilization Effects 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 235000001727 glucose Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000012088 reference solution Substances 0.000 description 1
- 239000009254 shuang-huang-lian Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 125000005504 styryl group Chemical group 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- 238000009333 weeding Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000009916 yin-huang Substances 0.000 description 1
- 239000009792 yinqiao Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G17/00—Cultivation of hops, vines, fruit trees, or like trees
- A01G17/005—Cultivation methods
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention discloses a method for promoting accumulation of luteoloside in honeysuckle buds by using fungi, which adopts a cutting seedling method to cultivate honeysuckle, and performs fungi inoculation on honeysuckle branches in the cutting process to improve the content of the luteoloside in the honeysuckle buds; the fungus is Phellinus Ribes and Aspergillus oryzae. According to the method, the honeysuckle plants are treated by the fungi, so that the growth of the fungi is accompanied in the growth process of the honeysuckle, the fungi grow on the stem base part of the honeysuckle, the growth of the fungi can promote the accumulation of secondary metabolites in honeysuckle buds, and the quality of the honeysuckle is improved. The method has the advantages of wide raw material source, convenient operation and easy implementation, and has important significance for improving the quality of the honeysuckle in production.
Description
Technical Field
The invention relates to a method for promoting accumulation of secondary metabolites in honeysuckle buds, in particular to a method for promoting accumulation of luteoloside in the honeysuckle buds by using fungi.
Background
Along with the rise of the trend of returning to nature, the traditional natural botanical drug is more and more favored by people. Honeysuckle is derived from a dry flower bud or a flower with a primary bloom of Lonicera japonica Thunb of Caprifoliaceae, is cold in nature and sweet in taste, has the effects of clearing heat, removing toxicity, cooling and dispelling wind heat, is a common traditional Chinese medicine, is widely applied to Chinese patent medicines of 1/3 according to statistics, such as Shuanghuanglian, Yinqiao detoxification pills, Yinhuang oral liquid and the like, particularly plays a great role in prevention and treatment of severe epidemic diseases such as SARS and avian influenza and is known as 'penicillin in traditional Chinese medicine'. At present, the demand of the domestic and foreign markets for honeysuckle is increasing day by day, and the requirements on the quality of medicinal materials are also increasing. The secondary metabolites in the bodies of medicinal plants are generally the basis of important substances for the clinical efficacy of traditional Chinese medicinal materials, and the content of luteoloside in the bodies of honeysuckle specified in 'Chinese pharmacopoeia' of 2015 edition is taken as one of important indexes for measuring the quality of honeysuckle medicinal materials, so that effective measures are taken to promote the accumulation of luteoloside in the bodies of honeysuckle, which is more and more important for improving the quality of the honeysuckle medicinal materials.
The Phellinus scriptus Phylloporia ribacter (Schumach.: Fr.) Ryvarden is a fungus from Lonicera japonica plants, which is produced mainly in the Shandong Malus hupehensis and parasitizes the old dry or bare roots of the Lonicera japonica plants, and the fruiting body of the fungus is called the "Yinhua moth". The fungus is a medicinal fungus, has a long history in local, is loaded in the traditional Chinese medicinal material standard of Shandong province in 2002, contains active ingredients such as triterpenes, sterols, polysaccharides and styryl pyrones, and has the effects of clearing away heat and toxic materials, relieving swelling and sore throat, reducing blood sugar, resisting tumors and the like. At present, few researches are carried out on the phyllobacterium ribirum which is mainly prepared into food or medicinal raw materials, and the purposes in other aspects are not reported.
Disclosure of Invention
The invention aims to provide a method for promoting accumulation of luteoloside in honeysuckle flower buds by using fungi.
The lamellar bacteria Phylloporia ribirus (Schumach.: Fr.) Ryvarden is an original wild fungus growing on a honeysuckle plant, and researches show that the lamellar bacteria Phylloporia ribirus growing on the honeysuckle plant in the original wild state has no significant influence on the accumulation of secondary metabolites in honeysuckle buds, which is probably related to the relative substance-ecological balance of the long-term coexistence of the lamellar bacteria Phylloporia ribirrorhii and the honeysuckle flower buds. Through research, the two kinds of bacteria are inoculated together on a honeysuckle plant, and the accumulation of secondary metabolites in honeysuckle buds can be promoted by the joint inoculation of the two kinds of bacteria, so that the content of the secondary metabolites in the honeysuckle buds is greatly improved.
The specific technical scheme of the invention is as follows:
a method for promoting accumulation of luteoloside in honeysuckle buds by using fungi comprises the following steps: cultivating honeysuckle by adopting a cutting seedling method, and performing fungus inoculation on honeysuckle branches in the cutting process so as to improve the content of luteoloside in honeysuckle buds; the fungus is Phellinus Ribes and Aspergillus oryzae.
According to the method, the accumulation of the secondary metabolite luteoloside in the honeysuckle buds is improved in a mode of carrying out fungus inoculation on the honeysuckle branches in the cutting process, so that the content of the luteoloside is improved. The cutting seedling raising method comprises the following steps:
(1) irrigating the substrate and the cutting bed with a carbendazim aqueous solution with the mass concentration of 1/3000 in the first 2 days of cutting until the substrate and the cutting bed are completely wet;
(2) then exposing the substrate and the slotting bed in the sun for 2 days, and placing the substrate in the slotting bed;
(3) irrigating the substrate and the cutting bed with fungus bacterial liquid 2 hours before cutting until the substrate and the cutting bed are completely irrigated, wherein the fungus bacterial liquid is a mixed liquid of black currant leaf layer bacterial liquid and aspergillus oryzae bacterial liquid;
(4) sterilizing and disinfecting a honeysuckle cutting branch, soaking the honeysuckle cutting branch in fungus liquid for 10-12h, taking out the honeysuckle cutting branch, dipping rooting powder, then cutting, and watering thoroughly after cutting;
(5) after cuttage, performing cuttage seedling raising management on the honeysuckle cuttage branches until the cuttage seedlings grow to meet the requirements of transplanting or outplanting, then performing transplantation and seedling stage management (namely field management) on the cuttage seedlings meeting the requirements, and picking buds 15-20 days after the plants bud.
In the method, a honeysuckle plant is inoculated by adopting a fungus bacterial liquid, wherein the fungus bacterial liquid is a mixed liquid of a black currant phyllosphere bacterial liquid and an aspergillus oryzae bacterial liquid. Wherein, the liquid can be obtained by culturing the seed body of Pheyloporium ribirum (Schumach.: Fr.) growing on a wild honeysuckle plant, or by culturing the seed body of Pheyloporium ribirum (Schumach.: Fr.) with the collection number of CGMCC NO 1195; the Aspergillus oryzae bacterial liquid can be obtained by culturing Aspergillus oryzae as strain, and the Aspergillus oryzae can be purchased from the market.
Further, the preparation method of the bacterial liquid of the phyllobacterium ribrum used by the invention comprises the following steps:
A. phenylloporia ribirus ribwort (Schumach.: Fr.) Ryvarden is inoculated on a slant culture medium and cultured at 29 ℃ for 5-7 days; the slant culture medium comprises the following components in percentage by weight: potato 20%, agar 1.5%, glucose 2%, and phosphoric acidPotassium dihydrogen 0.2%, magnesium sulfate 0.05%, and vitamin 1 × 10-3Percent, the balance of water;
B. inoculating the cultured mycelium of Ribes nigrum leaf to liquid culture medium, culturing at 29 deg.C and 150rpm for 5-6 days to obtain Ribes nigrum leaf mycelium liquid, and inoculating 5-8 mycelium masses per bottle per 1000ml liquid culture medium; the liquid culture medium comprises the following components in percentage by weight: 30% of potato, 2% of glucose, 1.4% of agar, 10% of wort (10Bx), 1.5% of sucrose, 0.2% of peptone, 4% of bran, 0.05% of monopotassium phosphate, 0.025% of magnesium sulfate and the balance of water.
Further, the preparation method of the aspergillus oryzae bacterial liquid used by the invention comprises the following steps:
(1) inoculating Aspergillus oryzae to slant culture medium, and culturing at 25-30 deg.C for 3-5 days; the slant culture medium comprises the following components in percentage by weight: 20% of potato, 2% of glucose, 1.6% of agar, 0.2% of peptone, 0.2% of monopotassium phosphate and 0.1% of magnesium sulfate heptahydrate;
(2) inoculating the strain obtained by slant culture into liquid culture medium according to inoculum size of 5-10wt%, and culturing on a shaking table at 25-30 deg.C and 50-150r/min for 3-5 days to obtain Aspergillus oryzae bacterial liquid; the liquid culture medium comprises the following components in percentage by weight: 0.8% of rice bran, 0.5% of cane sugar, 1.5% of glucose, 0.6% of yeast powder, 0.015% of ferrous chloride, 0.10% of magnesium sulfate, 0.15% of dipotassium hydrogen phosphate and the balance of water.
In the fungus bacterial liquid of the invention, the volume ratio of the bacterial liquid of the phyllobacterium ribactericum and the bacterial liquid of the aspergillus oryzae is 2-8:1, and preferably 5: 1. The fungus liquid of the phyllobacterium ribirum and the fungus liquid of aspergillus oryzae are obtained according to the method, and the fungus liquid can be obtained by mixing the fungus liquids according to the specified volume ratio.
In the cuttage method, the substrate used in the step (1) is obtained by mixing river sand, vermiculite and humus according to the mass ratio of 2: 1.
In the cutting method, the preparation method of the honeysuckle cutting branches comprises the following steps: collecting cutting branches at about 9:00 am on cloudy days, shearing 4 mm-thick shoots or hard shoots which are strong and have no diseases and insect pests and grow for two years into cuttings with the length of about 30cm, cutting 6-8 knots on each cutting, cutting 1/2 leaves from each leaf at each knot, flattening the lower end cut of each cutting shoot into an oblique opening, binding 50 cuttings into 1 bundle, spraying moisture on the cuttings by a sprayer, putting the cuttings into a foam heat insulation box after the cuttings are completely wetted, covering a layer of wet towel on the cuttings, and waiting for cutting.
In the cuttage method, the honeysuckle cuttage branches in the step (4) are soaked in 0.06wt% potassium permanganate aqueous solution for 10-12h, and sterilization and disinfection are carried out. And (3) soaking the honeysuckle cutting branches in fungus liquid after sterilization and disinfection so as to inoculate fungi to the cutting branches as much as possible, and dipping rooting powder after soaking so as to perform cutting. The rooting powder is a treating agent for promoting the rooting of the branches, is commonly used in the field, can be prepared by self, and can also be purchased from the market.
In the cuttage method, during cuttage, the bevel end of a cuttage branch is vertically inserted into a seedling bed, the lower end of the cuttage branch is inserted into a substrate of 8-12cm, and the cuttage density is 0.5 multiplied by 0.25 cm. And (5) tightly treading and compacting after cuttage, and completely watering for 1 time. And then carrying out conventional management such as watering, fertilizing and the like in time, generally watering 1 time every 5 days, rooting and sprouting to start growing in 7-15 days, and transplanting and carrying out conventional field management after survival.
In the method, after the cuttage branches meet the transplanting conditions, sandy loam with loose soil, rich and good drainage and a place with convenient irrigation and a water source are selected for transplanting. After land selection, deeply ploughing over 30cm, breaking soil blocks, leveling and harrowing, and applying enough base fertilizer. Then making high ridges or high ridges with the width of 1.3m for planting. The planting land can utilize sporadic land blocks such as barren slopes, land edges, ditch sides, houses and the like, and is generally transplanted before germination in early spring or in dormant period in autumn and winter. During transplanting, digging holes with the width and depth of 30-40cm respectively at the row spacing of 150cm and the plant spacing of 120cm on the prepared planting land, wherein the width and depth are generally per hm2Planting 3750-4200 holes. Applying 5kg of mixed soil fertilizer into each hole, uniformly mixing the mixed soil fertilizer with the bottom soil, planting 1 strong seedling in each hole, filling fine soil, compacting, treading, thoroughly watering for root fixing, watering once every 30 days, performing conventional field management, and picking up 15-20 days after bud emergence. Picking is preferably carried out in the morning of each dayThe fresh flowers are not clamped with impurities of branches and leaves, and should be lightly taken and lightly placed, and then placed in time at a low temperature of 40-50 ℃ for drying and detection.
The method uses the fungus to treat the honeysuckle plants, so that the growth of the fungus is accompanied in the growth process of the honeysuckle, the fungus grows at the stem base part of the honeysuckle, the growth of the fungus can promote the accumulation of a secondary metabolite, namely luteoloside, in honeysuckle buds, and the quality of the honeysuckle is improved. The method has the advantages of wide raw material source, convenient operation and easy implementation, and has important significance for improving the quality of the honeysuckle in production.
Detailed Description
The present invention is further illustrated by the following specific examples, which are intended to be purely exemplary of the invention and are not intended to be limiting thereof.
Example 1
The honeysuckle is cultivated by adopting the following method, and flower buds are adopted:
1. preparation of honeysuckle cutting branches
In the last ten days of 4 months, collecting cutting branches at about 9:00 am on cloudy days, selecting 4mm thick robust tender branches or hard branches which grow robustly, have no diseases, insects, shrink or damage to skins, shearing the robust tender branches or hard branches into cuttings with the length of about 30cm, wherein each cutting has 6-8 knots, 1/2 leaves are respectively sheared from the leaves at each knot, and the cuts at the lower ends of the cuttings are leveled into oblique mouths. Binding every 50 cut branches into 1 bundle, spraying moisture to the branches by using a sprayer, putting the branches into a foam heat insulation box after the branches are completely wetted, covering a layer of wet towel on the box for moisture preservation, and covering the box with a cover for waiting for cuttage.
2. Preparation of fungus liquid
2.1 preparation of liquid fungus of leaf layer of Ribes nigrum
A. Preparation of the culture Medium
Preparation of slant culture medium: purchasing potato on the market, peeling, cutting into 1cm square pieces, adding potato pieces into water, sterilizing at 121 deg.C, boiling for 30min (paying attention to control of fire and adding water), and filtering with gauze to obtain filtrate. Adding agar, glucose, potassium dihydrogen phosphate, magnesium sulfate, and vitamins into the filtrate, and making into tabletStirring, heating to dissolve, adding water, adjusting pH to 6, placing into test tube, sterilizing, cooling, and making into slant culture medium; the formula of the slant culture medium is as follows (wt%): potato 20%, agar 1.5%, glucose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, and vitamin 1 × 10%-3Percent, balance water.
Preparation of liquid medium: purchasing potato on the market, peeling, cutting into 1cm square pieces, adding potato pieces into water, sterilizing at 121 deg.C, boiling for 30min (paying attention to control of fire and adding water), and filtering with gauze to obtain filtrate. Adding glucose, agar, wort (10Bx), sucrose, peptone, bran, potassium dihydrogen phosphate and magnesium sulfate into the filtrate, supplementing water, adjusting pH to 6, and sterilizing to obtain liquid culture medium; the formula of the liquid culture medium is (wt%): 30% of potato, 2% of glucose, 1.4% of agar, 10% of malt extract (10Bx), 1.5% of sucrose, 0.2% of peptone, 4% of bran, 0.05% of monopotassium phosphate, 0.025% of magnesium sulfate and the balance of water.
B. Preparation of bacterial liquid of phyllobacterium ribis
Inoculation: the inoculation work is carried out on a clean bench. When Phenylloporia ribirus (Schumach.: Fr.) fruiting body of Phenylloporia ribirus growing on wild Lonicera japonica is used as strain, the fruiting body is sterilized with ultraviolet lamp for 40min before inoculation, part of tissue growing at the edge of fruiting body is inoculated into a test tube containing slant culture medium under sterile condition, and cultured at 29 deg.C for 5-7 days. When Phenylloporia ribirus ribwort (Schumach.: Fr.) with the collection number of CGMCC NO 1195 is used as a strain, the strain is directly inoculated into a test tube containing a slant culture medium and cultured for 5-7 days at 29 ℃.
Liquid culture: inoculating the solid cultured Ribes nigrum lamina mycelium in the slant test tube, inoculating into liquid culture medium, removing the slant culture medium containing agar at the bottom, wherein the diameter of the mycelium is about 1-2mm, keeping the size of the mycelium consistent as much as possible, inoculating 8 mycelium into each 1000ml of liquid culture medium, and culturing in a triangular flask at 29 deg.C and 150rpm for 6 days to obtain Ribes nigrum lamina mycelium liquid.
2.2 preparation of Aspergillus oryzae bacterial liquid
A. Preparation of the culture Medium
Preparation of slant culture medium: purchasing potato on the market, peeling, cutting into 1cm square pieces, adding potato pieces into water, sterilizing at 121 deg.C, boiling for 30min (paying attention to control of fire and adding water), and filtering with gauze to obtain filtrate. Adding glucose, agar, peptone, potassium dihydrogen phosphate, and magnesium sulfate heptahydrate into the filtrate, stirring with glass rod, heating to dissolve, adding water, adjusting pH to 6, placing into test tube, sterilizing, cooling, and making into slant culture medium; the formula of the slant culture medium is as follows (wt%): 20% of potato, 2% of glucose, 1.6% of agar, 0.2% of peptone, 0.2% of monopotassium phosphate and 0.1% of magnesium sulfate heptahydrate.
Preparation of liquid medium: the formula of the liquid culture medium is (wt%): 0.8% of rice bran, 0.5% of cane sugar, 1.5% of glucose, 0.6% of yeast powder, 0.015% of ferrous chloride, 0.10% of magnesium sulfate, 0.15% of dipotassium hydrogen phosphate and the balance of water. Mixing the above components, sterilizing at 121 deg.C, boiling for 30min (with attention paid to control fire power and water supplementing), adjusting pH to 6, and sterilizing to obtain liquid culture medium.
B. Preparation of Aspergillus oryzae bacterial liquid
Inoculation: the inoculation work is carried out on a clean bench. Aspergillus oryzae is used as strain, and the strain is directly inoculated into a test tube containing a slant culture medium and cultured for 3-5 days at 30 ℃.
Liquid culture: inoculating the strain obtained by slant culture into liquid culture medium according to the inoculum size of 8 wt%, and culturing on a shaking table at 30 deg.C and 50-150r/min for 5 days to obtain Aspergillus oryzae bacterial liquid.
2.3 preparation of fungal solutions
And (3) uniformly mixing the obtained bacterial liquid of the phyllobacterium ribirum and the obtained bacterial liquid of aspergillus oryzae according to the volume ratio of 5:1 to obtain the fungal liquid.
3. Preparation of honeysuckle cutting seedling raising bed
The honeysuckle cutting and transplanting bed is a plastic container with water leakage holes at the bottom and the length of 2m, the width of 1m and the height of 0.25m, and culture mediums filled in the plastic container are river sand, vermiculite and humus which are mixed according to the mass ratio of 2: 1. And irrigating the substrate and the cutting bed with an aqueous solution of carbendazim with the mass concentration of 1/3000 in the first 2 days of cutting until the substrate and the cutting bed are completely wetted. The substrate and the bed were then exposed to the sun for 2 days, and the substrate was placed in the bed. Irrigating with fungus liquid 2 hours before cuttage until the substrate and the cutting bed are completely irrigated.
4. Cuttage of honeysuckle branches
Putting the honeysuckle stem cutting branches into 0.06wt% potassium permanganate aqueous solution, soaking for 10-12h, and sterilizing. Then soaking the mixture in fungus liquid (the volume ratio of the liquid of the fungus of the black currant phyllodes to the liquid of the fungus of aspergillus oryzae is 5:1) for 12h, taking out the mixture, dipping the mixture in rooting powder, quickly dipping the mixture in the rooting powder for 20S, and then carrying out cuttage. When in cuttage, the oblique mouth end of a cuttage branch is vertically inserted into a seedling bed, the lower end of the cuttage branch is inserted into a substrate by 8-12cm, and the cuttage density is generally 0.5 multiplied by 0.25 cm. And (5) tightly treading and compacting after cuttage, and completely watering for 1 time. And then carrying out conventional management such as watering, fertilizing and the like in time, generally watering 1 time every 5 days, rooting and sprouting to start growing in 7-15 days, and transplanting after survival.
5. Transplanting process
After the cuttage branches meet the transplanting conditions, sandy loam with loose soil, rich fertilizer and good drainage and a place with convenient irrigation and a water source are selected for transplanting. After land selection, deeply ploughing over 30cm, breaking soil blocks, leveling and harrowing, and applying enough base fertilizer. Then making high ridges or high ridges with the width of 1.3m for planting. The planting land can be transplanted before germination in early spring or in dormancy stage in autumn and winter by using barren slopes, land sides, ditch sides and sporadic land blocks in front of houses and behind houses. During transplanting, digging holes with the width and depth of 30-40cm respectively at the row spacing of 150cm and the plant spacing of 120cm on the prepared planting land, wherein the width and depth are generally per hm2Planting 3750-4200 holes. Applying 5kg of mixed soil fertilizer into each hole, uniformly mixing the mixed soil fertilizer with the bottom soil, planting 1 strong seedling in each hole, filling fine soil, compacting, treading, thoroughly watering for root fixing, watering once every 30 days, and performing conventional field management. The plants can be picked up 15-20 days after the plants grow to bud. Picking in morning every day, not sandwiching impurities of branches and leaves, and lightly picking, and drying the picked fresh flowers at 40-50 deg.C in time.
Example 2
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the volume ratio of the bacterial liquid of the phyllobacterium ribirum to the bacterial liquid of aspergillus oryzae is 2: 1.
Example 3
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the volume ratio of the bacterial liquid of the phyllobacterium ribirum to the bacterial liquid of aspergillus oryzae is 8: 1.
Comparative example 1
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the fungus bacterial liquid is single bacterial liquid of black currant leaf-shaped layer bacteria.
Comparative example 2
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the fungus bacterial liquid is single aspergillus oryzae bacterial liquid.
Comparative example 3
Honeysuckle was cultivated according to the method of example 1, with the exception that the flower buds were collected: the fungus bacterial liquid is a mixed liquid of a black currant leaf-shaped lamina bacterial liquid and a bacillus megaterium bacterial liquid in a volume ratio of 5: 1.
The preparation method of the bacillus megaterium liquid comprises the following steps:
inoculating Bacillus megaterium into liquid culture medium according to the inoculation amount of 1 wt%, adjusting the temperature to 35-38 deg.C, controlling the aseptic air quantity at 90-100m3/h, and fermenting for 2 days. The formula of the liquid culture medium is as follows: 1.2% of corn flour, 0.3% of yeast powder, 0.4% of bean cake powder, 0.05% of sodium chloride, 0.06% of magnesium sulfate, 0.2% of dipotassium hydrogen phosphate, 0.1% of calcium carbonate and the balance of water, sterilizing at 121 ℃, and keeping the pH value at 7.0-7.2.
Comparative example 4
Honeysuckle plants were cultivated according to the cutting seedling method of example 1, with flower buds collected, except that: the substrate, the cutting bed and the honeysuckle cutting branches are not treated by fungus liquid.
In order to verify the influence of the cultivation method on the content of the secondary metabolite of the flower bud, a cultivation experiment is carried out on the Shandong Malus hupehensis. The experimental method comprises the following steps: selecting proper transplanting land, and dividing into multiple small blocksArea of 66.7m per cell2Honeysuckle was cultivated in a random block arrangement in each cell according to one of examples 1-3 and comparative examples 1-4, each of which was repeated 4 times. Besides different treatments of fungus liquid, other seedling management is kept consistent, such as fertilization, pesticide application, watering and weeding.
In the experimental process, the growth condition of the honeysuckle plant is recorded, the luteolin in the flower buds is detected after the flower buds are collected, and the influence of various methods on the content of the luteolin is evaluated.
The method for detecting the content of the luteoloside comprises the following steps:
1. preparation of control solutions: taking appropriate amount of luteolin control, precisely weighing, placing into brown measuring flask, adding 70% ethanol to obtain solution containing 40 μ g per 1ml (preserving at below 10 deg.C).
2. Preparation of sample solution: the bud sample is collected, placed at the low temperature of 40-50 ℃ for drying, ground, about 2g of fine powder (passing through a No. 4 sieve) of the sample to be measured is taken, precisely weighed, placed in a conical flask with a plug, 50ml of 70% ethanol is precisely added, the weight is weighed, ultrasonic treatment (power 250W, frequency 35kHz) is carried out for 1h, the mixture is cooled, the weight is weighed again, the lost weight is complemented with 70% ethanol, the mixture is shaken evenly and filtered. Precisely measuring 10mL of filtrate, recovering the solvent until the filtrate is dry, dissolving the residue with 70% ethanol, transferring the solution to a 5mL volumetric flask, and adding 70% ethanol until the volume is scaled to obtain the product. The solution was filtered through an organic membrane (pore size 0.2 μm) into a liquid phase vial for use.
3. And (3) content detection: detecting by liquid chromatography, with column C18(4.60mm × 250mm, 1.7 μm), acetonitrile as mobile phase A, and 0.5% glacial acetic acid solution as mobile phase B, and performing gradient elution according to the specification in the following table; the detection wavelength was 350 nm. The number of theoretical plates should not be less than 20000 calculated according to luteolin peak. The detection wavelength is 327nm, the sample injection amount is 20uL, and the column temperature is 40 ℃.
| Time (minutes) | Mobile phase A (%) | Mobile phase B (%) |
| 0-15 | 10→20 | 90→80 |
| 15-30 | 20 | 80 |
| 30-40 | 20→30 | 80→70 |
The specific detection method comprises the following steps: preparing the reference solution into different concentrations, and detecting by liquid chromatography to obtain a standard curve with the concentration and peak area as horizontal and vertical coordinates. Performing liquid chromatography detection on each sample solution by the same method, and calculating the content of luteolin in each sample according to peak area.
The experimental process shows that when the methods of examples 1-3 and comparative examples 2 and 3 are used for honeysuckle cultivation, the growth speed of honeysuckle plants is higher than that of comparative examples 1 and 4, the buds of the plants are earlier than that of comparative examples 1 and 4, and the average height of the plants is about 1.6 cm. And the disease and pest incidence rate of the honeysuckle plants of the examples 1-3 and the comparative examples 2 and 3 is low. The results of the luteolin detection of the collected flower buds are shown in the following table 1, and it can be seen from the table that the content of luteolin in the flower buds obtained by the method of the embodiment 1-3 is obviously higher than that of the flower buds obtained by the method of each proportion, but the effect of using the bacterial liquid of the lamellar bacterium of scripus triqueter or the bacterial liquid of aspergillus oryzae alone or using other bacteria and the bacterial liquid of the lamellar bacterium of scripus triqueter in combination is not obvious, and the bacterial liquid of the invention has a good promotion effect on the accumulation of the secondary metabolites of the honeysuckle buds.
TABLE 1 content of luteolin in honeysuckle buds obtained by the example and comparative example cultivation methods
| Luteolin content (%) | |
| Example 1 | 0.081 |
| Example 2 | 0.076 |
| Example 3 | 0.075 |
| Comparative example 1 | 0.062 |
| Comparative example 2 | 0.059 |
| Comparative example 3 | 0.065 |
| Comparative example 4 | 0.058 |
Claims (6)
1. A method for promoting accumulation of luteoloside in honeysuckle flower buds by using fungi adopts a cutting seedling method to cultivate honeysuckle, and is characterized in that: performing fungus inoculation on the honeysuckle branches in the cutting process to improve the content of luteolin in honeysuckle buds; the fungus is Phellinus ribis and Aspergillus oryzae; the method specifically comprises the following steps:
(1) irrigating the substrate and the cutting bed with aqueous solution of carbendazim with the mass concentration of 1/3000 2 days before cutting until the substrate and the cutting bed are completely wetted;
(2) then exposing the substrate and the slotting bed in the sun for 2 days, and placing the substrate in the slotting bed;
(3) irrigating the substrate and the cutting bed with fungus bacterial liquid 2 hours before cutting until the substrate and the cutting bed are completely irrigated, wherein the fungus bacterial liquid is a mixed liquid of black currant leaf layer bacterial liquid and aspergillus oryzae bacterial liquid;
(4) sterilizing and disinfecting a honeysuckle cutting branch, soaking the honeysuckle cutting branch in fungus liquid for 10-12h, taking out the honeysuckle cutting branch, dipping rooting powder, then cutting, and watering thoroughly after cutting;
(5) after cuttage, performing cuttage seedling raising management on the honeysuckle cuttage branches until the cuttage seedlings grow to meet the requirements of transplanting or outplanting, then performing transplantation and seedling stage management on the cuttage seedlings meeting the requirements, and picking flower buds 15-20 days after the plants bud;
the fungus bacterial liquid is a mixed liquid of a scripus triqueter lamina bacterial liquid and an aspergillus oryzae bacterial liquid, and the volume ratio of the scripus triqueter lamina bacterial liquid to the aspergillus oryzae bacterial liquid is 2-8: 1;
the preparation method of the bacterial liquid of the phyllostachys ribis comprises the following steps:
A. inoculating Phellinus Linteus to slant culture medium, and culturing at 29 deg.C for 5-7 days; the slant culture medium comprises the following components in percentage by weight: potato 20%, agar 1.5%, glucose 2%, potassium dihydrogen phosphate 0.2%, magnesium sulfate 0.05%, and vitamin 1 × 10%-3Percent, the balance of water;
B. inoculating the cultured mycelium of Ribes nigrum leaf to liquid culture medium, culturing at 29 deg.C and 150rpm for 5-6 days to obtain Ribes nigrum leaf mycelium liquid, and inoculating 7-8 mycelium masses per 1000ml liquid culture medium; the liquid culture medium comprises the following components in percentage by weight: 30% of potato, 2% of glucose, 1.4% of agar, 10% of 10Bx malt extract, 1.5% of sucrose, 0.2% of peptone, 4% of bran, 0.05% of monopotassium phosphate, 0.025% of magnesium sulfate and the balance of water;
the preparation method of the aspergillus oryzae bacterial liquid comprises the following steps:
(1) inoculating Aspergillus oryzae to slant culture medium, and culturing at constant temperature of 25-30 deg.C for 2-3 days; the slant culture medium comprises the following components in percentage by weight: 20% of potato, 2% of glucose, 1.6% of agar, 0.2% of peptone, 0.2% of monopotassium phosphate and 0.1% of magnesium sulfate heptahydrate;
(2) inoculating the strain obtained by slant culture into liquid culture medium according to the inoculum size of 5-10wt%, and culturing on a shaker at 25-30 deg.C and 200r/min for 6-7 days to obtain Aspergillus oryzae bacterial liquid; the liquid culture medium comprises the following components in percentage by weight: 0.8% of rice bran, 0.5% of cane sugar, 1.5% of glucose, 0.6% of yeast powder, 0.015% of ferrous chloride, 0.10% of magnesium sulfate, 0.15% of dipotassium hydrogen phosphate and the balance of water.
2. The method of claim 1, further comprising: the bacterial liquid of the black currant phyllodes takes the fruiting body of the black currant phyllodes growing on a honeysuckle plant in a wild state as a strain, or takes the black currant phyllodes with the preservation number of CGMCC NO 1195 as the strain; the Aspergillus oryzae bacterial liquid takes Aspergillus oryzae as a strain.
3. The method of claim 1, further comprising: in the fungus bacterial liquid, the volume ratio of the bacterial liquid of the black currant phylliform bacteria to the bacterial liquid of the aspergillus oryzae is 5: 1.
4. The method of claim 1, further comprising: in the step (1), the matrix is obtained by mixing river sand, vermiculite and humus according to the mass ratio of 2: 1; in the step (4), the honeysuckle cutting branches are soaked in 0.06wt% potassium permanganate aqueous solution for 10-12h, and then are sterilized and disinfected.
5. The method of claim 1, further comprising: the preparation method of the honeysuckle cutting branch comprises the following steps: collecting cutting branches at about 9:00 am on cloudy days, shearing 4 mm-thick shoots or hard shoots which are strong and have no diseases and insect pests and grow for two years, shearing the shoots into cuttings with the length of about 30cm, cutting 6-8 knots on each cutting, respectively cutting 1/2 leaves from the leaves at each knot, flattening the cuts at the lower ends of the cutting sticks into inclined openings, binding 50 sticks into 1 bundle, spraying moisture on the branches by using a sprayer, putting the branches into a foam heat insulation box after the branches are completely wetted, covering a layer of wet towel on the branches for moisturizing, and covering a cover for waiting for cutting.
6. The method of claim 1 or 5, wherein: when in cuttage, the oblique mouth end of a cuttage branch is vertically inserted into a seedling bed, the lower end of the cuttage branch is inserted into a matrix of 8-12cm, and the cuttage density is 0.5cm multiplied by 0.25 cm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710116171.4A CN106912349B (en) | 2017-03-01 | 2017-03-01 | Method for promoting accumulation of luteoloside in honeysuckle flower buds by using fungi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710116171.4A CN106912349B (en) | 2017-03-01 | 2017-03-01 | Method for promoting accumulation of luteoloside in honeysuckle flower buds by using fungi |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106912349A CN106912349A (en) | 2017-07-04 |
| CN106912349B true CN106912349B (en) | 2020-03-13 |
Family
ID=59454400
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710116171.4A Expired - Fee Related CN106912349B (en) | 2017-03-01 | 2017-03-01 | Method for promoting accumulation of luteoloside in honeysuckle flower buds by using fungi |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106912349B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108207476A (en) * | 2018-03-02 | 2018-06-29 | 芜湖欧标农业发展有限公司 | A kind of cuttage implantation methods of carpinus turczaninowii |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103749126A (en) * | 2014-01-09 | 2014-04-30 | 浙江省林业科学研究院 | Sustainable three-nerved spicebush cultivation method |
| JP5552649B2 (en) * | 2009-03-16 | 2014-07-16 | 独立行政法人農業生物資源研究所 | Method for preventing or suppressing microbial infection of plant and microbial infection resistant plant |
| CN105660098A (en) * | 2016-01-08 | 2016-06-15 | 华南师范大学 | Method for reducing honeysuckle cadmium accumulation and increasing yield of honeysuckle by means of glomus intraradices |
| CN106358821A (en) * | 2016-08-26 | 2017-02-01 | 滁州菲扬农业科技有限公司 | Cuttage treatment method in cuttage seedling culturing process of honeysuckles |
-
2017
- 2017-03-01 CN CN201710116171.4A patent/CN106912349B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5552649B2 (en) * | 2009-03-16 | 2014-07-16 | 独立行政法人農業生物資源研究所 | Method for preventing or suppressing microbial infection of plant and microbial infection resistant plant |
| CN103749126A (en) * | 2014-01-09 | 2014-04-30 | 浙江省林业科学研究院 | Sustainable three-nerved spicebush cultivation method |
| CN105660098A (en) * | 2016-01-08 | 2016-06-15 | 华南师范大学 | Method for reducing honeysuckle cadmium accumulation and increasing yield of honeysuckle by means of glomus intraradices |
| CN106358821A (en) * | 2016-08-26 | 2017-02-01 | 滁州菲扬农业科技有限公司 | Cuttage treatment method in cuttage seedling culturing process of honeysuckles |
Non-Patent Citations (2)
| Title |
|---|
| 《丛枝菌根真菌对西南岩溶地区干旱及干湿交替下金银花根系生长的影响》;刘锦春 马晔 陶建平 高凯敏 梁千慧;《北京林业大学学报》;20151031;第37卷(第10期);第110-115页 * |
| 《内生真菌与药用植物宿主间的交流》;程玉鹏，李天聪，林进华，陈琦，马爱萍，李弘琨;《中医药信息》;20161130;第33卷(第6期);第120-122页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106912349A (en) | 2017-07-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104770179B (en) | Cultivation method for Dendrobium officinale | |
| CN105340554A (en) | Planting method of Chinese wolfberries | |
| CN103814723A (en) | Efficient cultivation method for high-altitude area radix dipsaci | |
| CN102960226B (en) | Dendrobium officinale Kimura et Migo tissue culture seedling acclimatization method | |
| CN104396641A (en) | Pest control method of pollution-free grape | |
| CN105075586A (en) | Planting method of muskmelon | |
| CN106986691A (en) | A kind of cultural method of spun gold emperor chrysanthemum | |
| CN106359087B (en) | A kind of lucid asparagus tissue-culturing rapid propagation method for culturing seedlings | |
| CN105746168A (en) | Imitated wild cultivation method for roxburgh anoectochilus terminal buds | |
| CN109006101A (en) | A kind of implantation methods of fructus lycii | |
| CN113207536A (en) | Woody honeysuckle cultivation method | |
| CN107896756A (en) | A kind of method for integrated control of vine pest and disease damage | |
| CN107439187A (en) | A kind of green high yield and high quality chrysanthemum implantation methods | |
| CN106912349B (en) | Method for promoting accumulation of luteoloside in honeysuckle flower buds by using fungi | |
| CN107360842A (en) | A kind of organic Momordica grosvenori high-yield planting method | |
| CN107455123A (en) | A kind of organic hot pepper implantation methods | |
| CN106508572A (en) | Technique for planting figs on balcony | |
| CN107637417B (en) | Cultivation method of radix ophiopogonis | |
| CN106550731A (en) | Southern lean ground planted Rheum technology | |
| CN106134670A (en) | A kind of implantation methods of Fructus Fragariae Ananssae | |
| CN108739403A (en) | A kind of tissue culture and rapid propagation method of rose wood | |
| CN108575599A (en) | A kind of method for cultivating tomato | |
| CN106922349B (en) | Method for promoting accumulation of chlorogenic acid in honeysuckle flower buds by using fungi | |
| CN107926468A (en) | A kind of raspberry implantation methods | |
| CN114097560A (en) | Seedling raising method for medicinal and edible plant rhizoma smilacis glabrae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20211130 Address after: 273300 room 607 in the courtyard of e-commerce Park, south of Chengnan, South Outer Ring North Road, east of Mengyang Road, Pingyi County, Linyi City, Shandong Province Patentee after: Shandong Zhongping Pharmaceutical Co.,Ltd. Address before: 250022 No. 336, South Xin Zhuang West Road, Shizhong District, Ji'nan, Shandong Patentee before: University of Jinan |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20200313 |