CN112931205A - Dioscorea composita tissue culture breeding method - Google Patents
Dioscorea composita tissue culture breeding method Download PDFInfo
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- CN112931205A CN112931205A CN202110240267.8A CN202110240267A CN112931205A CN 112931205 A CN112931205 A CN 112931205A CN 202110240267 A CN202110240267 A CN 202110240267A CN 112931205 A CN112931205 A CN 112931205A
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- 235000007056 Dioscorea composita Nutrition 0.000 title claims abstract description 29
- 241000156497 Dioscorea composita Species 0.000 title claims abstract description 29
- 238000009395 breeding Methods 0.000 title claims abstract description 21
- 239000001963 growth medium Substances 0.000 claims abstract description 15
- 239000007787 solid Substances 0.000 claims abstract description 13
- 230000001488 breeding effect Effects 0.000 claims abstract description 9
- 238000009630 liquid culture Methods 0.000 claims abstract description 8
- 238000000034 method Methods 0.000 claims abstract description 8
- 230000035755 proliferation Effects 0.000 claims abstract description 8
- 230000001954 sterilising effect Effects 0.000 claims abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 18
- 229930006000 Sucrose Natural products 0.000 claims description 14
- 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 14
- 229960004793 sucrose Drugs 0.000 claims description 14
- 238000002791 soaking Methods 0.000 claims description 13
- 239000002609 medium Substances 0.000 claims description 12
- 229910052799 carbon Inorganic materials 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 229920001817 Agar Polymers 0.000 claims description 7
- 244000017020 Ipomoea batatas Species 0.000 claims description 7
- 235000002678 Ipomoea batatas Nutrition 0.000 claims description 7
- 239000008272 agar Substances 0.000 claims description 7
- 235000020415 coconut juice Nutrition 0.000 claims description 7
- 235000020197 coconut milk Nutrition 0.000 claims description 7
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 239000005720 sucrose Substances 0.000 claims description 7
- 241000196324 Embryophyta Species 0.000 claims description 6
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 6
- 230000004083 survival effect Effects 0.000 claims description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- 241000238631 Hexapoda Species 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 4
- 239000005842 Thiophanate-methyl Substances 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 239000003599 detergent Substances 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 4
- 239000008223 sterile water Substances 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- QGHREAKMXXNCOA-UHFFFAOYSA-N thiophanate-methyl Chemical group COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC QGHREAKMXXNCOA-UHFFFAOYSA-N 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims description 2
- 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 2
- 239000004471 Glycine Substances 0.000 claims description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 2
- 239000004327 boric acid Substances 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 2
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 2
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 2
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims description 2
- 239000011790 ferrous sulphate Substances 0.000 claims description 2
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 2
- 229940064880 inositol 100 mg Drugs 0.000 claims description 2
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 229940099596 manganese sulfate Drugs 0.000 claims description 2
- 239000011702 manganese sulphate Substances 0.000 claims description 2
- 235000007079 manganese sulphate Nutrition 0.000 claims description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 2
- 229960003512 nicotinic acid Drugs 0.000 claims description 2
- 235000001968 nicotinic acid Nutrition 0.000 claims description 2
- 239000011664 nicotinic acid Substances 0.000 claims description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 2
- 239000004323 potassium nitrate Substances 0.000 claims description 2
- 235000010333 potassium nitrate Nutrition 0.000 claims description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 2
- 239000011684 sodium molybdate Substances 0.000 claims description 2
- 235000015393 sodium molybdate Nutrition 0.000 claims description 2
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 2
- 239000008174 sterile solution Substances 0.000 claims description 2
- 239000011691 vitamin B1 Substances 0.000 claims description 2
- 239000011726 vitamin B6 Substances 0.000 claims description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 2
- 229960001763 zinc sulfate Drugs 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims 1
- 239000000203 mixture Substances 0.000 abstract description 5
- 238000011081 inoculation Methods 0.000 abstract description 4
- 238000004904 shortening Methods 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 238000004659 sterilization and disinfection Methods 0.000 abstract 1
- 238000002054 transplantation Methods 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000003203 everyday effect Effects 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000001397 quillaja saponaria molina bark Substances 0.000 description 2
- 229930182490 saponin Natural products 0.000 description 2
- 150000007949 saponins Chemical class 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- BTTWKVFKBPAFDK-UHFFFAOYSA-N (9beta,10alpha)-Androst-4-ene-3,17-dione Natural products OC1CCC2(C)C3CCC(C)(C(CC4)O)C4C3CCC2=C1 BTTWKVFKBPAFDK-UHFFFAOYSA-N 0.000 description 1
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical group C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 1
- 241000735332 Gerbera Species 0.000 description 1
- 229930191978 Gibberellin Natural products 0.000 description 1
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- DWCSNWXARWMZTG-UHFFFAOYSA-N Trigonegenin A Natural products CC1C(C2(CCC3C4(C)CCC(O)C=C4CCC3C2C2)C)C2OC11CCC(C)CO1 DWCSNWXARWMZTG-UHFFFAOYSA-N 0.000 description 1
- AEMFNILZOJDQLW-QAGGRKNESA-N androst-4-ene-3,17-dione Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CCC2=C1 AEMFNILZOJDQLW-QAGGRKNESA-N 0.000 description 1
- AEMFNILZOJDQLW-UHFFFAOYSA-N androstenedione Natural products O=C1CCC2(C)C3CCC(C)(C(CC4)=O)C4C3CCC2=C1 AEMFNILZOJDQLW-UHFFFAOYSA-N 0.000 description 1
- 235000021015 bananas Nutrition 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- WQLVFSAGQJTQCK-VKROHFNGSA-N diosgenin Chemical compound O([C@@H]1[C@@H]([C@]2(CC[C@@H]3[C@@]4(C)CC[C@H](O)CC4=CC[C@H]3[C@@H]2C1)C)[C@@H]1C)[C@]11CC[C@@H](C)CO1 WQLVFSAGQJTQCK-VKROHFNGSA-N 0.000 description 1
- WQLVFSAGQJTQCK-UHFFFAOYSA-N diosgenin Natural products CC1C(C2(CCC3C4(C)CCC(O)CC4=CCC3C2C2)C)C2OC11CCC(C)CO1 WQLVFSAGQJTQCK-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000446 fuel Substances 0.000 description 1
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 1
- 239000003448 gibberellin Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical group N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 1
- 229960001669 kinetin Drugs 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000012015 potatoes Nutrition 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- -1 tetrahydropyran benzyladenine Chemical group 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a dioscorea composita tissue culture breeding method, and belongs to the technical field of crop tissue culture. The method comprises the steps of explant acquisition and sterilization, explant inoculation, proliferation and rooting, test-tube seedling transplantation and field breeding. The invention adopts a culture mode combining early-stage shallow liquid culture and later-stage solid culture, and reduces harmful substances in the process of subculture multiplication of the dioscorea composita seedlings by optimizing the formula composition of the culture medium, thereby obviously improving the subculture multiplication coefficient of the dioscorea composita seedlings, shortening the subculture period and reducing the subculture frequency.
Description
Technical Field
The invention relates to the technical field of crop tissue culture, in particular to a dioscorea composita tissue culture breeding method.
Background
Dioscorea composita is an exotic species from Mexico, and is mainly used for extracting diosgenin from rhizomes and producing fuel ethanol by fermentation. Dioscorea composita has attracted attention because of its high tuber yield, high saponin and starch content, and low production cost of extracting saponin and producing ethanol by fermentation.
Tissue culture and rapid propagation are emerging practical technologies which are most widely applied in the process of seedling propagation in recent years, at present, thousands of plants are successfully cultured in the world through tissue culture, but only dozens of plants, such as bananas, sugarcanes, orchids, gerbera, potatoes and the like, which can be massively applied to production and can generate economic benefits. For dioscorea composita, a tissue culture propagation method which improves the seedling hardening survival rate, is efficient and stable and has low production cost still needs to be explored to realize industrial production.
Disclosure of Invention
The invention aims to provide a dioscorea composita tissue culture breeding method to solve the problems.
According to one aspect of the invention, a dioscorea composita tissue culture breeding method is provided, which comprises the following steps:
(1) obtaining and sterilizing explants: selecting a stem section of dioscorea composita with strong growth, no disease and insect harm, 3-4 leaves and a stem tip as an explant, cutting off the leaves, leaving the stem section with axillary buds, putting the stem section with the axillary buds into a detergent solution, stirring, washing with flowing water, soaking the washed stem section with alcohol, then soaking with NaClO, and then rinsing with sterile water to obtain a sterile explant;
(2) inoculation of explants: inoculating the dioscorea composita aseptic explant on a liquid culture medium to obtain a test-tube plantlet with complete roots and leaves;
(3) and (3) proliferation and rooting: transferring the test-tube plantlet obtained in the step (2) on a solid culture medium for proliferation and rooting;
(4) transplanting test-tube seedlings: dividing seedlings with complete roots and leaves into individual plants before the test-tube seedlings are transplanted out of bottles, soaking the seedlings out of the bottles in a sterile solution, then transplanting the seedlings into a transplanting container with top glass, growing under the conditions that the temperature is 20-25 ℃ and the humidity is 75-85%, and indicating that the seedlings are transplanted to survive when the height of the seedlings is 2-3 cm;
(5) and (3) field breeding: hardening the transplanted survival seedlings for 3-5 days under natural conditions, and then transplanting the seedlings to a field for breeding.
In the present invention, PBA represents tetrahydropyran benzyladenine, NAA represents naphthylacetic acid, and GA3It is gibberellin, KT is kinetin, BA is 6-benzylaminopurine, and AD is androst-4-ene-3, 17-dione.
In some embodiments, the liquid medium comprises the following components: 2/3MS + PBA0.1-0.8mg/L + NAA0.2-0.8mg/L + GA30.2-0.5mg/L, 0.2-0.4mg/L of KT, 3 percent of cane sugar, 0.1 percent of active carbon, 15-20g/L of sweet potato juice and 30-40g/L of coconut juice, and the pH value is 5.8-6.2.
In some embodiments, the solid medium comprises the following components: MS + BA0.5-1.0mg/L + AD5-10mg/L + KT0.2-0.4mg/L + 3% sucrose + 0.75% agar + 0.2-0.3% active carbon +20-40g/L coconut milk, and the pH value is 6.0-6.5.
In some embodiments, the MS comprises the following components: 1650mg/L of ammonium nitrate, 1900mg/L of potassium nitrate, 440mg/L of calcium chloride, 370mg/L of magnesium sulfate, 170mg/L of monopotassium phosphate, 22.3mg/L of manganese sulfate, 8.6mg/L of zinc sulfate, 0.025mg/L of cobalt chloride, 0.025mg/L of copper sulfate, 6.2mg/L of boric acid, 0.25mg/L of sodium molybdate, 0.83mg/L of potassium iodide, 27.8mg/L of ferrous sulfate, 37.2mg/L of disodium ethylenediaminetetraacetate, 0.5mg/L of nicotinic acid, and 0.5mg/L of vitamin B60.5mg/L, vitamin B10.1mg/L, inositol 100mg/L and glycine 2 mg/L.
In some embodiments, the liquid medium further comprises PVP 100 mg/L. Thereby, the browning of the explant can be suppressed.
In some embodiments, the culture temperature in step (2) and step (3) is 24 + -2 deg.C, and the culture is performed in a low light environment of 1000-2000 lx by using red light or white light and 16 hours per day.
In some embodiments, in step (4), the sterilizing solution is 700-fold and 1000-fold thiophanate methyl.
Preferably, the dioscorea composita tissue culture breeding method of the present invention comprises the following steps:
(1) obtaining and sterilizing explants: selecting a stem section of dioscorea composita with strong growth, no disease and insect harm, 3-4 leaves and a stem tip as an explant, cutting off the leaves, leaving the stem section with axillary buds, putting the stem section with the axillary buds into a detergent solution, stirring for 5-10min, washing for 10-20min with flowing water, soaking the cleaned stem section in 75% alcohol for 30-40s, soaking in 5-20% NaClO for 5-15min, and rinsing for 4-6 times with sterile water to obtain the sterile explant;
(2) inoculation of explants: inoculating the dioscorea composita aseptic explant to a liquid culture medium to obtain a test-tube plantlet with complete roots and leaves; the liquid culture medium comprises the following components: 2/3MS + PBA0.1-0.8mg/L + NAA0.2-0.8mg/L + GA30.2-0.5mg/L + KT0.2-0.4mg/L + 3% sucrose + 0.1% active carbon +15-20g/L sweet potato juice +30-40g/L coconut milk, and the pH value is 5.8-6.2;
(3) and (3) proliferation and rooting: transferring the test-tube plantlet obtained in the step (2) on a solid culture medium for proliferation and rooting; the solid culture medium comprises the following components: MS + BA0.5-1.0mg/L + AD5-10mg/L + KT0.2-0.4mg/L + 3% sucrose + 0.75% agar + 0.2-0.3% active carbon +20-40g/L coconut milk, pH value is 6.0-6.5;
(4) transplanting test-tube seedlings: dividing seedlings with complete roots and leaves into single plants before transplanting the test-tube seedlings out of the bottles, soaking the seedlings out of the bottles for 10-15 minutes by using 700-times thiophanate methyl for 1000 times, then transplanting the seedlings into a transplanting container with top glass, growing under the conditions that the temperature is 20-25 ℃ and the humidity is 75-85%, and indicating that the seedlings are transplanted to survive when the height of the seedlings is 2-3 cm;
(5) and (3) field breeding: and (4) hardening the transplanted survival seedlings under natural conditions for 3-5 days, and transplanting the seedlings to a field for breeding.
The invention adopts a culture mode combining early-stage shallow liquid culture and later-stage solid culture, and reduces harmful substances in the process of subculture multiplication of the dioscorea composita seedlings by optimizing the formula composition of the culture medium, thereby obviously improving the subculture multiplication coefficient of the dioscorea composita seedlings, shortening the subculture period and reducing the subculture frequency.
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1
The dioscorea composita tissue culture breeding method comprises the following steps:
(1) obtaining and sterilizing explants:
selecting a stem section of dioscorea composita with strong growth, no disease and insect harm, 3-4 leaves and a stem tip as an explant, cutting off the leaves to leave the stem section with axillary buds, putting the stem section with the axillary buds into a detergent solution, stirring for 5min, washing for 20min with flowing water, soaking the washed stem section in 75% alcohol for 30s, then soaking in 20% NaClO for 10min, and then rinsing for 5 times with sterile water to obtain a sterile explant;
(2) inoculation of explants:
shearing the dioscorea composita aseptic explant, and inoculating 9 sections of budded segments onto a liquid culture medium, wherein the liquid culture medium comprises the following components: 2/3MS + PBA 0.5mg/L + NAA0.6mg/L + GA30.5mg/L + KT0.4mg/L + 3% sucrose + 0.1% active carbon +20g/L sweet potato juice +30g/L coconut juice, the pH value is 5.9, the culture temperature is 24 +/-2 ℃, white light is adopted in 1100lx low-light environment, the illumination time is 16 hours every day, the culture is carried out for 35 days, aseptic detection is carried out periodically during the period to ensure the sterility of a mother bottle and the reliability of seedlings, and test-tube seedlings with complete roots and leaves are obtained;
(3) and (3) proliferation and rooting:
transferring the test-tube plantlet obtained in the step (2) on a solid culture medium for proliferation and rooting, and soaking the test-tube plantlet in MS nutrient solution for 4min when the culture medium is changed, wherein the solid culture medium comprises the following components: MS + BA1.0mg/L + AD8mg/L + KT0.3mg/L + 3% cane sugar + 0.75% agar + 0.3% active carbon +40g/L coconut juice, the pH value is 6.5, the culture temperature is 24 +/-2 ℃, and in a 1700lx weak light environment, white light is adopted, the illumination time is 16 hours every day, and the culture period is 45 days;
(4) transplanting test-tube seedlings:
dividing seedlings with complete roots and leaves into single plants before the test-tube seedlings are taken out of bottles and transplanted, soaking the taken-out seedlings in 800 times of thiophanate methyl for 12 minutes, then transplanting the seedlings into a transplanting container with top glass, growing under the conditions that the temperature is 24 ℃ and the humidity is 80 percent, and indicating that the seedlings are transplanted to survive when the height of the seedlings is 2-3 cm;
(5) and (3) field breeding:
and (4) hardening the transplanted survival seedlings under natural conditions for 4 days, and transplanting the seedlings to a field for breeding.
Example 2
This example differs from example 1 in that, in step (2), the liquid medium had the following composition: 2/3MS + PBA0.8mg/L + NAA0.5mg/L + GA30.3mg/L, KT0.4mg/L, 3% of cane sugar, 0.1% of activated carbon, 15g/L of sweet potato juice and 35g/L of coconut juice, and the pH value is 6.1.
In the step (3), the solid medium comprises the following components: MS + BA0.8mg/L + AD10mg/L + KT0.4mg/L + 3% sucrose + 0.75% agar + 0.3% activated carbon +40g/L coconut milk, pH 6.2.
Example 3
This example differs from example 1 in that, in step (2), the liquid medium had the following composition: 2/3MS + PBA0.3mg/L + NAA0.8mg/L + GA30.4mg/L, KT0.3mg/L, 3% of cane sugar, 0.1% of activated carbon, 20g/L of sweet potato juice and 30g/L of coconut juice, and the pH value is 5.8.
In the step (3), the solid medium comprises the following components: MS + BA1.0mg/L + AD10mg/L + KT0.4mg/L + 3% sucrose + 0.75% agar + 0.3% active carbon +40g/L coconut milk, and the pH value is 6.3.
Example 4
This example differs from example 1 in that, in step (2), the liquid medium had the following composition: 2/3MS + PBA0.1mg/L + NAA0.7mg/L + GA30.5mg/L, KT0.3mg/L, 3% of cane sugar, 0.1% of activated carbon, 15g/L of sweet potato juice and 30g/L of coconut juice, and the pH value is 6.0.
In the step (3), the solid medium comprises the following components: MS + BA0.9mg/L + AD10mg/L + KT0.4mg/L + 3% cane sugar + 0.75% agar + 0.3% active carbon +25g/L coconut milk, and the pH value is 6.3.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. The dioscorea composita tissue culture breeding method is characterized by comprising the following steps:
(1) selecting a stem section of dioscorea composita with strong growth, no disease and insect harm, 3-4 leaves and a stem tip as an explant, cutting off the leaves, leaving the stem section with axillary buds, putting the stem section with the axillary buds into a detergent solution, stirring, washing with flowing water, soaking the washed stem section with alcohol, then soaking with NaClO, and then rinsing with sterile water to obtain a sterile explant;
(2) inoculating the sterile explant on a liquid culture medium to obtain a test-tube seedling with complete roots and leaves;
(3) transferring the test-tube plantlet obtained in the step (2) on a solid culture medium for proliferation and rooting;
(4) dividing seedlings with complete roots and leaves into individual plants before the test-tube seedlings are transplanted out of bottles, soaking the seedlings out of the bottles in a sterile solution, then transplanting the seedlings into a transplanting container with top glass, growing under the conditions that the temperature is 20-25 ℃ and the humidity is 75-85%, and indicating that the seedlings are transplanted to survive when the height of the seedlings is 2-3 cm;
(5) hardening the transplanted survival seedlings for 3-5 days under natural conditions, and then transplanting the seedlings to a field for breeding.
2. The tissue culture breeding method of Dioscorea composita according to claim 1, wherein the liquid medium comprises: 2/3MS + PBA0.1-0.8mg/L + NAA0.2-0.8mg/L + GA30.2-0.5mg/L, 0.2-0.4mg/L of KT, 3 percent of cane sugar, 0.1 percent of active carbon, 15-20g/L of sweet potato juice and 30-40g/L of coconut juice, and the pH value is 5.8-6.2.
3. The method for breeding and culturing Dioscorea composita according to claim 2, wherein the liquid medium further comprises PVP 100 mg/L.
4. The tissue culture breeding method of Dioscorea composita according to claim 1 or 2, wherein the solid medium comprises: MS + BA0.5-1.0mg/L + AD5-10mg/L + KT0.2-0.4mg/L + 3% sucrose + 0.75% agar + 0.2-0.3% active carbon +20-40g/L coconut milk, and the pH value is 6.0-6.5.
5. The method for tissue culture and propagation of Dioscorea composita according to claim 4, wherein the MS comprises the following components: 1650mg/L of ammonium nitrate, 1900mg/L of potassium nitrate, 440mg/L of calcium chloride, 370mg/L of magnesium sulfate, 170mg/L of monopotassium phosphate, 22.3mg/L of manganese sulfate, 8.6mg/L of zinc sulfate, 0.025mg/L of cobalt chloride, 0.025mg/L of copper sulfate, 6.2mg/L of boric acid, 0.25mg/L of sodium molybdate, 0.83mg/L of potassium iodide, 27.8mg/L of ferrous sulfate, 37.2mg/L of disodium ethylenediaminetetraacetate, 0.5mg/L of nicotinic acid, and 0.5mg/L of vitamin B60.5mg/L, vitamin B10.1mg/L, inositol 100mg/L and glycine 2 mg/L.
6. The tissue culture breeding method of Dioscorea composita according to claim 1, wherein the culture temperature in step (2) and step (3) is 24 ± 2 ℃, and the culture is performed under 1000-2000 lx low light environment by red light or white light and 16 hours per day.
7. The tissue culture and propagation method of Dioscorea composita according to claim 1, wherein in the step (4), the sterilizing solution is 700-1000 times of thiophanate methyl.
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