CN117305217A - Optimized stem tip tissue culture medium, culture method and application - Google Patents
Optimized stem tip tissue culture medium, culture method and application Download PDFInfo
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- 239000003104 tissue culture media Substances 0.000 title claims abstract description 25
- 238000012136 culture method Methods 0.000 title abstract description 6
- 239000001963 growth medium Substances 0.000 claims abstract description 88
- 239000002023 wood Substances 0.000 claims abstract description 57
- 239000000052 vinegar Substances 0.000 claims abstract description 56
- 235000021419 vinegar Nutrition 0.000 claims abstract description 56
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 14
- 238000005286 illumination Methods 0.000 claims description 28
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 27
- 230000001976 improved effect Effects 0.000 claims description 26
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 24
- 244000144730 Amygdalus persica Species 0.000 claims description 23
- 235000006040 Prunus persica var persica Nutrition 0.000 claims description 23
- 238000005406 washing Methods 0.000 claims description 22
- 238000012258 culturing Methods 0.000 claims description 20
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 18
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 18
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 18
- 241000196324 Embryophyta Species 0.000 claims description 17
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 16
- 239000003085 diluting agent Substances 0.000 claims description 16
- 235000016623 Fragaria vesca Nutrition 0.000 claims description 14
- 235000011363 Fragaria x ananassa Nutrition 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
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- 238000002791 soaking Methods 0.000 claims description 12
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- 229920001817 Agar Polymers 0.000 claims description 9
- 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 9
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 9
- 239000004471 Glycine Substances 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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- 239000008272 agar Substances 0.000 claims description 9
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 9
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 9
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 9
- 239000004327 boric acid Substances 0.000 claims description 9
- 229940099596 manganese sulfate Drugs 0.000 claims description 9
- 239000011702 manganese sulphate Substances 0.000 claims description 9
- 235000007079 manganese sulphate Nutrition 0.000 claims description 9
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 9
- 239000002609 medium Substances 0.000 claims description 9
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 9
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- 229960003512 nicotinic acid Drugs 0.000 claims description 9
- 235000001968 nicotinic acid Nutrition 0.000 claims description 9
- 239000011664 nicotinic acid Substances 0.000 claims description 9
- 239000004323 potassium nitrate Substances 0.000 claims description 9
- 235000010333 potassium nitrate Nutrition 0.000 claims description 9
- 239000005720 sucrose Substances 0.000 claims description 9
- JZRWCGZRTZMZEH-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims description 9
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 9
- 229960001763 zinc sulfate Drugs 0.000 claims description 9
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 8
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 8
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 8
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 8
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 7
- 239000008223 sterile water Substances 0.000 claims description 7
- 238000007865 diluting Methods 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- 229930003231 vitamin Natural products 0.000 claims description 3
- 235000013343 vitamin Nutrition 0.000 claims description 3
- 239000011782 vitamin Substances 0.000 claims description 3
- 229940088594 vitamin Drugs 0.000 claims description 3
- 150000003722 vitamin derivatives Chemical class 0.000 claims description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims 1
- 240000009088 Fragaria x ananassa Species 0.000 claims 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims 1
- 229910052742 iron Inorganic materials 0.000 claims 1
- 239000011777 magnesium Substances 0.000 claims 1
- 229910052749 magnesium Inorganic materials 0.000 claims 1
- 230000035755 proliferation Effects 0.000 abstract description 11
- 239000000203 mixture Substances 0.000 abstract description 8
- 230000004069 differentiation Effects 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 6
- 230000006698 induction Effects 0.000 abstract description 4
- 238000012271 agricultural production Methods 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 3
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 abstract description 3
- 238000001784 detoxification Methods 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 17
- 241000220223 Fragaria Species 0.000 description 15
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 12
- 239000005708 Sodium hypochlorite Substances 0.000 description 11
- 230000012010 growth Effects 0.000 description 11
- 238000000197 pyrolysis Methods 0.000 description 11
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 11
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 7
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 7
- 239000002994 raw material Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 6
- 229930003268 Vitamin C Natural products 0.000 description 6
- 239000003610 charcoal Substances 0.000 description 6
- 239000011718 vitamin C Substances 0.000 description 6
- 235000019154 vitamin C Nutrition 0.000 description 6
- 239000003205 fragrance Substances 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 230000001939 inductive effect Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000004161 plant tissue culture Methods 0.000 description 3
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 239000012883 rooting culture medium Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 239000005997 Calcium carbide Substances 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 235000004789 Rosa xanthina Nutrition 0.000 description 1
- 241000220222 Rosaceae Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- -1 and 0.5mg/L Chemical compound 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000021028 berry Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000028446 budding cell bud growth Effects 0.000 description 1
- 238000003763 carbonization Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000001902 propagating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 235000021012 strawberries Nutrition 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000004243 sweat Anatomy 0.000 description 1
- CLZWAWBPWVRRGI-UHFFFAOYSA-N tert-butyl 2-[2-[2-[2-[bis[2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl]amino]-5-bromophenoxy]ethoxy]-4-methyl-n-[2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl]anilino]acetate Chemical compound CC1=CC=C(N(CC(=O)OC(C)(C)C)CC(=O)OC(C)(C)C)C(OCCOC=2C(=CC=C(Br)C=2)N(CC(=O)OC(C)(C)C)CC(=O)OC(C)(C)C)=C1 CLZWAWBPWVRRGI-UHFFFAOYSA-N 0.000 description 1
- 210000002262 tip cell Anatomy 0.000 description 1
- 230000008467 tissue growth Effects 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
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- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an optimized stem tip tissue culture medium, a culture method and application, and belongs to the technical field of agricultural production. The invention provides a general culture medium which can be used for stem tip induction differentiation and proliferation and rooting culture steps, simplifies operation steps, reduces pollution, realizes effective detoxification by using wood vinegar with gradient concentration to replace the disinfection effect of hypochlorous acid, optimizes the composition of the culture medium, and effectively promotes induction differentiation, proliferation and rooting by adding a proper amount of wood vinegar into the culture medium, improves the propagation efficiency of plant tissues, is suitable for large-scale industrialized seedling culture and propagation culture, and has remarkable economic benefit.
Description
Technical Field
The invention belongs to the technical field of agricultural production, and particularly relates to an optimized stem tip tissue culture medium, a culture method and application.
Background
Strawberry belongs to Rosaceae, fragaria, perennial herb. In the world production of small berries, strawberry is the first place. Strawberry is a fruit tree with the shortest result, earliest maturity, smallest plant body, shortest period, convenient management, less diseases and insects, easy processing and convenient regulation. It can be harvested after cultivation for several months, and is supplied on the market in early spring for 5 months, thus filling the light season market of fruits. It can also be used for forcing culture or production for several times in one year, and supplied all year round. The strawberry propagation mainly provides seedlings by dividing the stems of the grape into the branches, the efficiency is low, the popularization of good varieties is not facilitated, in the long-term asexual propagation, the plants often accumulate various viruses, the seedlings are degraded, and the yield and the quality are reduced, so that the strawberry propagation has become a main problem for obstructing the strawberry production. By applying the in vitro culture technology, a large amount of high-quality strawberry seedlings can be quickly propagated, the variety purification and updating are facilitated, the method can be used for removing viruses and propagating virus-free seedlings, and the popularization of new varieties is accelerated.
However, the current strawberry stem tip tissue culture seedling raising technology generally adopts 3 culture mediums for inducing differentiation, proliferation and rooting, the culture mediums need to be frequently prepared and inoculated in production, the procedures are complicated, and the factors such as cutting damage and inoculation pollution affecting the production benefit are more. For example, a Chinese patent application with the application number of 201310351128.8 discloses a special culture medium for tissue culture of strawberry stem tips, which belongs to the technical field of tissue culture seedlings. Including primary culture medium, secondary culture medium and rooting culture medium. The primary culture medium is prepared by adding 6-benzyl amino purine with the concentration of 0.2-0.5mg/L and naphthylacetic acid with the concentration of 0.1-0.2mg/L into an MS culture medium; the secondary culture medium is prepared by adding 6-benzyl amino purine with the concentration of 0.2-0.5mg/L into an MS culture medium; the rooting culture medium is 1/2MS culture medium.
Meanwhile, in the plant propagation and growth process, pollution can be caused by factors such as non-strict sterilization of inoculation devices and culture mediums, irregular operation and the like. In addition, in the conventional plant tissue culture process, bacteria carried by plants themselves are difficult to remove, and thus, the bacteria are contaminated in the medium. These factors make the traditional tissue culture plants slow in growth and low in survival rate, and inhibit the wide application and popularization of the plant tissue culture in actual agricultural production.
Therefore, how to develop a comprehensive culture medium can be used for inducing differentiation of strawberry stem tips, proliferation and rooting culture, and solves the pollution problem of the prior culture medium.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an optimized stem tip tissue culture medium and a culture method, and the components of the culture medium are optimized to realize efficient culture of tissues.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
an optimized stem tip tissue culture medium is a modified MS culture medium, wherein each liter of modified MS culture medium comprises an MS basic culture medium, and is supplemented with 15-20g/L of sucrose, 1-5g/L of agar, 1700-1800mg/L of potassium nitrate, 1000-1200mg/L of ammonium sulfate, 400-500mg/L of magnesium sulfate, 80mg/L of monopotassium phosphate, 450mg/L of calcium chloride, 37mg/L of disodium edetate, 22mg/L of manganese sulfate, 12mg/L of zinc sulfate, 4.5mg/L of boric acid, 1.0mg/L of potassium iodide, 8mg/L of ferric ethylenediamine o-dihydroxyacetate, 3mg/L of glycine, 0.5mg/L, IAA0.2-0.4mg/L, NAA 0.1.1-0.2 mg/L of nicotinic acid, and 0.5mg/L, IBA of BA0.5mg/L0.5mg/L, vitamin C1mg/L, vitamin B 1 1mg/L, vitamin B 6 1mg/L, wood vinegar 5-10ml/L.
Preferably, the culture medium is modified MS culture medium, wherein each liter of modified MS culture medium comprises MS basic culture medium, and is supplemented with 20g/L of sucrose, 5g/L of agar, 1800mg/L of potassium nitrate, 1200mg/L of ammonium sulfate, 500mg/L of magnesium sulfate, 80mg/L of monopotassium phosphate, 450mg/L of calcium chloride, 37mg/L of disodium ethylenediamine tetraacetate, 22mg/L of manganese sulfate, 12mg/L of zinc sulfate, 4.5mg/L of boric acid, 1.0mg/L of potassium iodide, 8mg/L of ethylenediamine o-dihydroxyacetic acid, 3mg/L of glycine, 0.5mg/L of nicotinic acid, 0.4mg/L of IAA, 0.2mg/L of NAA, 0.5mg/L of BA0.5mg/L, 0.5mg/L of IBA, 1mg/L of vitamin C and 1mg/L of vitamin B 1 1mg/L, vitamin B 6 1mg/L, wood vinegar 10ml/L.
More preferably, the pH of the medium is adjusted to 5.5-6.0.
More preferably, the medium is sterilized with high pressure steam at 120℃and a pressure of 0.1-0.15MPa for 20-30min before use.
More preferably, the wood vinegar is peach shell wood vinegar with pH of 2.8-2.9.
The MS minimal medium is designed by Murashige and Skoog, has high inorganic salt content and ion concentration, has good balance among elements, meets the nutrition and physiology required by plant growth, and can be used as a minimal medium for rapid propagation of most plant tissue culture. Can be prepared according to a formula list or can be a commercially available finished product culture medium.
The peach shell pyroligneous liquor is purchased from Shijia Hongsen charcoal limited company, and is obtained by condensing and separating a steam gas mixture which is obtained by taking peach shells as raw materials and conducting dry distillation in carbonization equipment, and the obtained liquid is light yellow and semitransparent, has fumigating fragrance and has a pH value of 2.8.
The optimized stem tip tissue culture medium is mainly applied to tissue culture of strawberry stem tips.
A method of culturing using optimized shoot tip tissue culture medium comprising the steps of:
(1) Preparing an improved MS culture medium according to a formula, and carrying out ultraviolet sterilization on all the tools used in the culture for 30-60min;
(2) Pretreatment of stem tips: washing the stem tip in flowing water for 20-30min, washing with 70% ethanol for one time, washing with sterile water for 3-4 times, sequentially soaking the stem tip in 600 times of diluent, 300 times of diluent and 100 times of diluent of wood vinegar, respectively soaking for 30min, and taking out;
(3) Inoculating the stem tip into a modified MS culture medium for primary culture under the following culture conditions: culturing at 22-26deg.C for 14h/d under 1600-2000lx for 3-4 days;
(4) Transferring the cluster buds obtained by primary culture nutrient enrichment into an improved MS culture medium for culture for 25-30 days, wherein the illumination intensity is 2000-3000lx, the illumination time is 14h/d, the temperature is 22-26 ℃, and the secondary culture algebra is controlled to be 5-7 generations;
(5) Transferring the plant seedling to the improved MS culture medium to promote rooting, rooting culture for 25-40 days at 22-26 deg.c and illumination intensity of 2500-3000lx, illumination period of 14 hr/d and relative air humidity of not higher than 50%.
Further, 600 times, 300 times and 100 times of the diluted solution of the wood vinegar in the step (2) are obtained by diluting the peach shell wood vinegar by using sterile deionized water for 600 times, 300 times and 100 times. The peach shell pyroligneous liquor used in the invention is purchased from Shijia Hongsen charcoal limited company.
The wood vinegar is also called plant acid, is an organic mixture obtained by condensing gas derived from dry distillation of biomass such as wood in dry distillation equipment, and contains acetic acid as main component, and also contains less than 200 organic matters such as phenols, acids, alcohols and microelements. The wood vinegar not only contains various nutrient elements, but also has the effects of sterilization and bacteriostasis. In the prior art, sodium hypochlorite is often used for disinfecting tissues to be cultivated, and the following disadvantages are caused by using the sodium hypochlorite for disinfection:
1. workers who frequently touch sodium hypochlorite with hands sweats a lot of the palm, thin nails and shed hair.
2. Sodium hypochlorite has sensitization effect.
3. The chlorine evolved by the sodium hypochlorite reaction or decomposition may cause poisoning.
Sodium hypochlorite can only treat bacteria, fungi and part of spores in water.
5. The sodium hypochlorite reacts with water to generate trihalogen compounds, which causes secondary pollution to the water body. The trihalogen compound is a chemical substance which is extremely serious to human body, and sodium hypochlorite has no deeper positive effect on plant tissues, and also has only the effect of killing viruses.
Therefore, on one hand, the invention uses wood vinegar with gradient concentration to replace hypochlorous acid stem tip for treatment, the wood vinegar treatment from high concentration to low concentration has good sterilization and bacteriostasis effects on the stem tip, and simultaneously, the wood vinegar from high concentration to low concentration repeatedly stimulates the stem tip tissue and has good growth promoting effect on plant tissue, and in the subsequent primary culture, secondary culture and rooting culture, a small amount of wood vinegar is added into the culture medium, so that on the one hand, various nutrient elements can be provided for the tissue, and the induction differentiation, proliferation and rooting can be promoted, and the culture efficiency can be improved.
In conclusion, the technical scheme of the invention has the beneficial effects that:
the invention provides a general culture medium which can be used for inducing differentiation of plant stem tips and also can be used for proliferation and rooting culture steps, simplifies operation steps, reduces pollution, realizes effective detoxification by using wood vinegar with gradient concentration to replace the disinfection effect of hypochlorous acid, optimizes the composition of the culture medium, and effectively promotes inducing differentiation, proliferation and rooting by adding a proper amount of wood vinegar into the culture medium, improves the propagation efficiency of plant tissues, is suitable for large-scale industrialized seedling culture and propagation culture, and has remarkable economic benefit.
Detailed Description
The technical scheme of the present invention is further described below with reference to specific examples, but is not limited thereto.
Example 1
An optimized tissue culture medium of stem tip, which is a modified MS culture medium, wherein each liter of modified MS culture medium comprises MS basic culture medium and is supplemented with 15g/L of sucrose, 1g/L of agar, 1700mg/L of potassium nitrate, 1000mg/L of ammonium sulfate, 400mg/L of magnesium sulfate, 80mg/L of monopotassium phosphate and chlorine450mg/L of calcium carbide, 37mg/L of disodium ethylenediamine tetraacetate, 22mg/L of manganese sulfate, 12mg/L of zinc sulfate, 4.5mg/L of boric acid, 1.0mg/L of potassium iodide, 8mg/L of ferric ethylenediamine ortho-dihydroxyacetate, 3mg/L of glycine, 0.5mg/L of nicotinic acid, 0.2mg/L of IAA, 0.1mg/L of NAA, 0.5mg/L of BA, 0.5mg/L of IBA, 1mg/L of vitamin C and 1mg/L of vitamin B 1 1mg/L, vitamin B 6 1mg/L, wood vinegar 5ml/L.
The pH of the culture medium is adjusted to 5.5-6.0.
The medium is sterilized with high pressure steam at 120deg.C under 0.1Mpa for 20min before use.
The wood vinegar is peach shell wood vinegar with pH of 2.8-2.9.
The peach hull pyroligneous liquor of the embodiment is purchased from Shijia Hongsen charcoal limited company, and is obtained by condensing and separating a steam gas mixture obtained by dry distillation in dry distillation equipment by taking peach hulls as raw materials, and has light yellow semitransparent color, fumigating fragrance and pH value of 2.8.
A method of culturing using optimized shoot tip tissue culture medium comprising the steps of:
(1) Preparing an improved MS culture medium according to a formula, and carrying out ultraviolet sterilization on all the tools used in the culture for 30min;
(2) Pretreatment of stem tips: washing the stem tip in flowing water for 20min, washing with 70% ethanol for one time, washing with sterile water for 3-4 times, sequentially soaking the stem tip in 600 times of diluent, 300 times of diluent and 100 times of diluent of wood vinegar, respectively soaking for 30min, and taking out;
(3) Inoculating the stem tip into a modified MS culture medium for primary culture under the following culture conditions: culturing at 22-26deg.C for 14h/d under 1600-2000lx for 3 days;
(4) Transferring the cluster buds obtained by primary culture nutrient enrichment into an improved MS culture medium for culture for 25 days, wherein the illumination intensity is 2000-3000lx, the illumination time is 14h/d, the temperature is 22-26 ℃, and the subculture algebra is controlled to be 5-7 generations;
(5) Transferring the plant seedlings subjected to the subculture into the improved MS culture medium to promote rooting, culturing for 25 days at 22-26 ℃ under the illumination intensity of 2500-3000lx and the illumination duration of 14h/d, wherein the relative air humidity is not higher than 50%.
The 600 times, 300 times and 100 times of the diluted solution of the step (2) wood vinegar is obtained by diluting the peach shell wood vinegar by using sterile deionized water 600 times, 300 times and 100 times.
Example 2
An optimized stem tip tissue culture medium, which is a modified MS culture medium, wherein each liter of modified MS culture medium comprises MS basic culture medium, and is supplemented with 18g/L of sucrose, 3g/L of agar, 1700mg/L of potassium nitrate, 1100mg/L of ammonium sulfate, 450mg/L of magnesium sulfate, 80mg/L of monopotassium phosphate, 450mg/L of calcium chloride, 37mg/L of disodium ethylenediamine tetraacetate, 22mg/L of manganese sulfate, 12mg/L of zinc sulfate, 4.5mg/L of boric acid, 1.0mg/L of potassium iodide, 8mg/L of ethylenediamine o-dihydroxyacetic acid, 3mg/L of glycine, 0.5mg/L of nicotinic acid, 0.3mg/L of IAA, 0.2mg/L of NAA, 0.5mg/L of BA0.5mg/L, 0.5mg/L of IBA, 1mg/L of vitamin C and 1mg/L of vitamin B 1 1mg/L, vitamin B 6 1mg/L, wood vinegar 7ml/L.
The pH of the culture medium is adjusted to 5.5-6.0.
The culture medium is sterilized at 120deg.C with high pressure steam of 0.1-0.15Mpa for 25min.
The wood vinegar is peach shell wood vinegar with pH of 2.8-2.9.
The peach hull pyroligneous liquor of the embodiment is purchased from Shijia Hongsen charcoal limited company, and is obtained by condensing and separating a steam gas mixture obtained by dry distillation in dry distillation equipment by taking peach hulls as raw materials, and has light yellow semitransparent color, fumigating fragrance and pH value of 2.8.
A method of culturing using optimized shoot tip tissue culture medium comprising the steps of:
(1) Preparing an improved MS culture medium according to a formula, and carrying out ultraviolet sterilization on all the tools used in the culture for 40min;
(2) Pretreatment of stem tips: washing the stem tip in flowing water for 25min, washing with 70% ethanol for one time, washing with sterile water for 3-4 times, sequentially soaking the stem tip in 600 times of diluent, 300 times of diluent and 100 times of diluent of wood vinegar, respectively soaking for 30min, and taking out;
(3) Inoculating the stem tip into a modified MS culture medium for primary culture under the following culture conditions: culturing at 22-26deg.C for 14h/d under 1600-2000lx for 3 days;
(4) Transferring the cluster buds obtained by primary culture nutrient enrichment into an improved MS culture medium for culturing for 28 days, wherein the illumination intensity is 2000-3000lx, the illumination time is 14h/d, the temperature is 22-26 ℃, and the subculture algebra is controlled to be 5-7 generations;
(5) Transferring the plant seedlings subjected to the subculture into the improved MS culture medium to promote rooting, culturing for 30 days at 22-26 ℃ under the illumination intensity of 2500-3000lx and the illumination duration of 14h/d, wherein the relative air humidity is not higher than 50%.
The 600 times, 300 times and 100 times of the diluted solution of the step (2) wood vinegar is obtained by diluting the peach shell wood vinegar by using sterile deionized water 600 times, 300 times and 100 times.
Example 3
An optimized stem tip tissue culture medium, which is a modified MS culture medium, wherein each liter of modified MS culture medium comprises MS basic culture medium, and is supplemented with 20g/L of sucrose, 5g/L of agar, 1800mg/L of potassium nitrate, 1200mg/L of ammonium sulfate, 500mg/L of magnesium sulfate, 80mg/L of monopotassium phosphate, 450mg/L of calcium chloride, 37mg/L of disodium ethylenediamine tetraacetate, 22mg/L of manganese sulfate, 12mg/L of zinc sulfate, 4.5mg/L of boric acid, 1.0mg/L of potassium iodide, 8mg/L of ethylenediamine o-dihydroxyacetic acid, 3mg/L of glycine, 0.5mg/L of nicotinic acid, 0.4mg/L of IAA, 0.2mg/L of NAA, 0.5mg/L of BA0.5mg/L, 0.5mg/L of IBA, 1mg/L of vitamin C and 1mg/L of vitamin B 1 1mg/L, vitamin B 6 1mg/L, wood vinegar 10ml/L.
The pH of the culture medium is adjusted to 5.5-6.0.
The culture medium is sterilized with high pressure steam at 120 deg.C under 0.1-0.15Mpa for 30min.
The wood vinegar is peach shell wood vinegar with pH of 2.8-2.9.
The peach hull pyroligneous liquor of the embodiment is purchased from Shijia Hongsen charcoal limited company, and is obtained by condensing and separating a steam gas mixture obtained by dry distillation in dry distillation equipment by taking peach hulls as raw materials, and has light yellow semitransparent color, fumigating fragrance and pH value of 2.8.
A method of culturing using optimized shoot tip tissue culture medium comprising the steps of:
(1) Preparing an improved MS culture medium according to a formula, and carrying out ultraviolet sterilization on all the tools used in the culture for 60min;
(2) Pretreatment of stem tips: washing the stem tip in flowing water for 30min, washing with 70% ethanol for one time, washing with sterile water for 3-4 times, sequentially soaking the stem tip in 600 times of diluted solution, 300 times of diluted solution and 100 times of diluted solution of wood vinegar, respectively soaking for 30min, and taking out;
(3) Inoculating the stem tip into a modified MS culture medium for primary culture under the following culture conditions: culturing at 22-26deg.C for 14h/d under 1600-2000lx for 4 days;
(4) Transferring the cluster buds obtained by primary culture nutrient enrichment into an improved MS culture medium for culture for 30 days, wherein the illumination intensity is 2000-3000lx, the illumination time is 14h/d, the temperature is 22-26 ℃, and the subculture algebra is controlled to be 5-7 generations;
(5) Transferring the plant seedlings subjected to the subculture into the improved MS culture medium to promote rooting, culturing for 40 days at 22-26 ℃ under the illumination intensity of 2500-3000lx and the illumination duration of 14h/d, wherein the relative air humidity is not higher than 50%.
The 600 times, 300 times and 100 times of the diluted solution of the step (2) wood vinegar is obtained by diluting the peach shell wood vinegar by using sterile deionized water 600 times, 300 times and 100 times.
Comparative example 1
A tissue culture medium of stem tip is an improved MS culture medium, wherein each liter of the improved MS culture medium comprises MS basic culture medium, and is supplemented with 20g/L of sucrose, 5g/L of agar, 1800mg/L of potassium nitrate, 1200mg/L of ammonium sulfate, 500mg/L of magnesium sulfate, 80mg/L of monopotassium phosphate, 450mg/L of calcium chloride, 37mg/L of disodium ethylenediamine tetraacetate, 22mg/L of manganese sulfate, 12mg/L of zinc sulfate, 4.5mg/L of boric acid, 1.0mg/L of potassium iodide, 8mg/L of ethylenediamine o-dihydroxyacetic acid, 3mg/L of glycine, 0.5mg/L, IAA 0.2.2-0.4 mg/L of nicotinic acid, 0.2mg/L of NAA, 0.5mg/L of BA0.5mg/L, 0.5mg/L of IBA, 1mg/L of vitamin C and B 1 1mg/L, vitamin B 6 1mg/L。
The comparative example was conducted in the same manner as in example 3, except that wood vinegar was not added to the medium.
Comparative example 2
A stem tip tissue culture medium is a modified MS culture medium, and each liter of modified MS culture medium comprises MS basic culture medium, and is supplemented with 20g/L of sucrose, 5g/L of agar, 1800mg/L of potassium nitrate, 1200mg/L of ammonium sulfate, 500mg/L of magnesium sulfate, 80mg/L of monopotassium phosphate, 450mg/L of calcium chloride, 37mg/L of disodium ethylenediamine tetraacetate, 22mg/L of manganese sulfate, 12mg/L of zinc sulfate, 4.5mg/L of boric acid, 1.0mg/L of potassium iodide, 8mg/L of ethylenediamine o-dihydroxyacetic acid, 3mg/L of glycine, 0.5mg/L of nicotinic acid, 0.4mg/L of IAA, 0.2mg/L of NAA, 0.5mg/L of BA0.5mg/L, 0.5mg/L of IBA, 1mg/L of vitamin C and 1mg/L of vitamin B 1 1mg/L, vitamin B 6 1mg/L, wood vinegar 10ml/L.
The pH of the culture medium is adjusted to 5.5-6.0.
The culture medium is sterilized with high pressure steam at 120 deg.C under 0.1-0.15Mpa for 30min.
The wood vinegar is peach shell wood vinegar with pH of 2.8-2.9.
The comparative peach shell pyroligneous liquor is purchased from Shijia Hongsen charcoal limited company, and is obtained by condensing and separating a steam gas mixture obtained by dry distillation in dry distillation equipment by taking peach shells as raw materials to obtain a liquid which is light yellow and semitransparent, has fumigating fragrance and has a pH value of 2.8.
A method for culturing using shoot tip tissue culture medium comprising the steps of:
(1) Preparing an improved MS culture medium according to a formula, and carrying out ultraviolet sterilization on all the tools used in the culture for 60min;
(2) Pretreatment of stem tips: washing the stem tip in flowing water for 30min, washing with 70% ethanol for one time, washing with sterile water for 3-4 times, soaking in 600 times diluted wood vinegar for 90min, and taking out;
(3) Inoculating the stem tip into a modified MS culture medium for primary culture under the following culture conditions: culturing at 22-26deg.C for 14h/d under 1600-2000lx for 4 days;
(4) Transferring the cluster buds obtained by primary culture nutrient enrichment into an improved MS culture medium for culture for 30 days, wherein the illumination intensity is 2000-3000lx, the illumination time is 14h/d, the temperature is 22-26 ℃, and the subculture algebra is controlled to be 5-7 generations;
(5) Transferring the plant seedlings subjected to the subculture into the improved MS culture medium to promote rooting, culturing for 40 days at 22-26 ℃ under the illumination intensity of 2500-3000lx and the illumination duration of 14h/d, wherein the relative air humidity is not higher than 50%.
The 600-time diluent of the wood vinegar in the step (2) is obtained by diluting the peach shell wood vinegar by 600 times with sterile deionized water.
This comparative example was conducted in the same manner as in example 3, except that in the pretreatment of the stem tip, the stem tip was treated with only 600-fold diluted wood vinegar, i.e., the treatment of gradient concentration was not conducted.
Comparative example 3
As in comparative example 2, the raw materials and the cultivation steps were the same as in example 3 except that in the pretreatment of the stem tip, the stem tip was treated with only 300-fold dilution of pyroligneous liquor, i.e. no treatment of pyroligneous liquor of gradient concentration was performed.
Comparative example 4
As in comparative example 2, the raw materials and the cultivation steps were the same as in example 3 except that in the pretreatment of the stem tip, the stem tip was treated with only 100-fold dilution of pyroligneous liquor, i.e. no treatment of pyroligneous liquor of gradient concentration was performed.
Comparative example 5
As in comparative example 2, the same procedure as in example 3 was followed except that in pretreatment of the stem tip, the stem tip was sterilized with sodium hypochlorite at a mass concentration of 5%. The method comprises the following steps:
a method for culturing using shoot tip tissue culture medium comprising the steps of:
(1) Preparing an improved MS culture medium according to a formula, and carrying out ultraviolet sterilization on all the tools used in the culture for 60min;
(2) Pretreatment of stem tips: washing the stem tip in flowing water for 30min, washing with 70% alcohol for one time, washing with sterile water for 3-4 times, soaking with 5% sodium hypochlorite for 10 min, and washing with sterile deionized water for 4-5 times;
(3) Inoculating the stem tip into a modified MS culture medium for primary culture under the following culture conditions: culturing at 22-26deg.C for 14h/d under 1600-2000lx for 4 days;
(4) Transferring the cluster buds obtained by primary culture nutrient enrichment into an improved MS culture medium for culture for 30 days, wherein the illumination intensity is 2000-3000lx, the illumination time is 14h/d, the temperature is 22-26 ℃, and the subculture algebra is controlled to be 5-7 generations;
(5) Transferring the plant seedlings subjected to the subculture into the improved MS culture medium to promote rooting, culturing for 40 days at 22-26 ℃ under the illumination intensity of 2500-3000lx and the illumination duration of 14h/d, wherein the relative air humidity is not higher than 50%.
Experimental test
The test variety is red, the stolons of the strawberries planted in the greenhouse are collected in 5-6 months, and terminal buds with full and young stolons of 2-4cm long are selected from the robust red strawberry parent strain.
According to the present invention, the culture media were prepared and cultured according to the culture methods of examples 1 to 3 and comparative examples 1 to 5. Eight treatment groups S1-S8 were divided into 10 flasks. Counting the pollution rate, browning rate and proliferation bud number after 30d inoculation, and calculating proliferation coefficient and rooting rate. Other indicators were recorded every 30d 1 investigation.
The stem tip induced bud growth state comprises the induced bud emergence speed, leaf color and growth vigor. The growth state of the cluster bud proliferation buds comprises the growth speed, the leaf color and the growth vigor of the buds. The growth state of the tissue culture seedling comprises rooting growth vigor, root state and the like of the tissue culture seedling.
Stem tip induction rate (%) =number of induced bud stem tips/number of inoculated stem tips×100%
Cluster bud proliferation coefficient = number of proliferating buds/number of inoculated buds
Rooting rate (%) = rooting tissue culture seedling number/inoculation tissue culture seedling number×100%
The specific experimental results are shown in table 1:
table 1 experimental test results
As can be seen from the data in the table, the culture medium of the embodiment of the invention provides rich nutrient substances for the stem tip of the strawberry, and is suitable for culture at each stage, but the culture conditions of the culture medium lack of comparative example 1 of wood vinegar, comparative example 2 treated only with 600 times of diluent of wood vinegar, comparative example 3 treated only with 300 times of diluent of wood vinegar, comparative example 4 treated only with 100 times of diluent of wood vinegar and comparative example 5 treated with sodium hypochlorite are different from those of the embodiment. The wood vinegar with gradient concentration not only realizes the purpose of effective sterilization, but also has the function of stimulating and promoting growth of stem tip cells, and in the subsequent culture, the growth is more vigorous, and wood vinegar components are added into the culture medium, so that various nutrients and trace elements can be provided, the tissue growth is promoted, and a certain antibacterial effect can be achieved.
It should be noted that the above-mentioned embodiments are merely some, but not all embodiments of the preferred mode of carrying out the invention. It is evident that all other embodiments obtained by a person skilled in the art without making any inventive effort, based on the above-described embodiments of the invention, shall fall within the scope of protection of the invention.
Claims (8)
1. An optimized stem tip tissue culture medium is characterized in that the culture medium is an improved MS culture medium, wherein each liter of the improved MS culture medium comprises an MS basic culture medium, and is supplemented with 15-20g/L of sucrose, 1-5g/L of agar, 1700-1800mg/L of potassium nitrate, 1000-1200mg/L of ammonium sulfate and 400-500mg/L of magnesium sulfatemg/L, potassium dihydrogen phosphate 80mg/L, calcium chloride 450mg/L, disodium ethylenediamine tetraacetate 37mg/L, manganese sulfate 22mg/L, zinc sulfate 12mg/L, boric acid 4.5mg/L, potassium iodide 1.0mg/L, ethylenediamine o-dihydroxyacetic acid iron 8mg/L, glycine 3mg/L, nicotinic acid 0.5mg/L, IAA0.2-0.4mg/L, NAA0.1-0.2mg/L, BA0.5mg/L, IBA0.5mg/L, vitamin C1mg/L, vitamin B 1 1mg/L, vitamin B 6 1mg/L, wood vinegar 5-10ml/L.
2. The optimized tissue culture medium of stem tip according to claim 1, wherein the culture medium is modified MS culture medium, each liter of modified MS culture medium comprises MS basic culture medium, and is supplemented with sucrose 20g/L, agar 5g/L, potassium nitrate 1800mg/L, ammonium sulfate 1200mg/L, magnesium sulfate 500mg/L, potassium dihydrogen phosphate 80mg/L, calcium chloride 450mg/L, disodium edetate 37mg/L, manganese sulfate 22mg/L, zinc sulfate 12mg/L, boric acid 4.5mg/L, potassium iodide 1.0mg/L, ethylenediamine o-dihydroxyiron acetate 8mg/L, glycine 3mg/L, nicotinic acid 0.5mg/L, IAA0.4mg/L, NAA0.2mg/L, BA0.5mg/L, IBA0.5mg/L, vitamin C1mg/L, vitamin B 1 1mg/L, vitamin B 6 1mg/L, wood vinegar 10ml/L.
3. The optimized tissue culture medium of stem tips of claim 1 or 2, wherein the medium is pH adjusted to 5.5-6.0.
4. The optimized tissue culture medium of stem tip according to claim 1 or 2, characterized in that the medium is sterilized with high pressure steam at 120 ℃ for 20-30min at a pressure of 0.1-0.15Mpa before use.
5. The optimized stem tip tissue culture medium according to claim 1 or 2, wherein the wood vinegar is peach shell wood vinegar,
the pH is 2.8-2.9.
6. Use of the optimized stem tip tissue culture medium according to any one of claims 1-5 for strawberry stem tip tissue culture.
7. A method of culturing using the optimized stem tip tissue culture medium of any one of claims 1-5, comprising the steps of:
(1) Preparing an improved MS culture medium according to a formula, and carrying out ultraviolet sterilization on all the tools used in the culture for 30-60min;
(2) Pretreatment of stem tips: washing the stem tip in flowing water for 20-30min, washing with 70% ethanol for one time, washing with sterile water for 3-4 times, sequentially soaking the stem tip in 600 times of diluent, 300 times of diluent and 100 times of diluent of wood vinegar, respectively soaking for 30min, and taking out;
(3) Inoculating the stem tip into a modified MS culture medium for primary culture under the following culture conditions: culturing at 22-26deg.C for 14h/d under 1600-2000lx for 3-4 days;
(4) Transferring the cluster buds obtained by primary culture nutrient enrichment into an improved MS culture medium for culture for 25-30 days, wherein the illumination intensity is 2000-3000lx, the illumination time is 14h/d, the temperature is 22-26 ℃, and the secondary culture algebra is controlled to be 5-7 generations;
(5) Transferring the plant seedling to the improved MS culture medium to promote rooting, rooting culture for 25-40 days at 22-26 deg.c and illumination intensity of 2500-3000lx, illumination period of 14 hr/d and relative air humidity of not higher than 50%.
8. The method of claim 6, wherein 600-fold, 300-fold and 100-fold dilutions of the wood vinegar in step (2) are obtained by diluting the peach shell wood vinegar with sterile deionized water 600-fold, 300-fold and 100-fold dilutions.
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