CN117158324A - In-vitro preservation method of coconut germplasm resources - Google Patents
In-vitro preservation method of coconut germplasm resources Download PDFInfo
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- CN117158324A CN117158324A CN202311451920.0A CN202311451920A CN117158324A CN 117158324 A CN117158324 A CN 117158324A CN 202311451920 A CN202311451920 A CN 202311451920A CN 117158324 A CN117158324 A CN 117158324A
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- 235000013162 Cocos nucifera Nutrition 0.000 title claims abstract description 129
- 244000060011 Cocos nucifera Species 0.000 title claims abstract description 129
- 238000000034 method Methods 0.000 title claims abstract description 62
- 238000004321 preservation Methods 0.000 title claims abstract description 36
- 238000000338 in vitro Methods 0.000 title claims abstract description 33
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 111
- 239000007640 basal medium Substances 0.000 claims abstract description 31
- 230000018044 dehydration Effects 0.000 claims abstract description 30
- 238000006297 dehydration reaction Methods 0.000 claims abstract description 30
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims abstract description 21
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 18
- JLIDBLDQVAYHNE-YKALOCIXSA-N (+)-Abscisic acid Chemical compound OC(=O)/C=C(/C)\C=C\[C@@]1(O)C(C)=CC(=O)CC1(C)C JLIDBLDQVAYHNE-YKALOCIXSA-N 0.000 claims abstract description 14
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims abstract description 14
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims abstract description 14
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims abstract description 14
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 14
- 239000001963 growth medium Substances 0.000 claims abstract description 14
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000007789 sealing Methods 0.000 claims abstract description 12
- 230000001954 sterilising effect Effects 0.000 claims abstract description 12
- 229930003231 vitamin Natural products 0.000 claims abstract description 9
- 235000013343 vitamin Nutrition 0.000 claims abstract description 9
- 239000011782 vitamin Substances 0.000 claims abstract description 9
- 229940088594 vitamin Drugs 0.000 claims abstract description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 7
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 claims abstract description 7
- 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 abstract description 7
- 239000004471 Glycine Substances 0.000 claims abstract description 7
- 229910021586 Nickel(II) chloride Inorganic materials 0.000 claims abstract description 7
- 235000019270 ammonium chloride Nutrition 0.000 claims abstract description 7
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000004327 boric acid Substances 0.000 claims abstract description 7
- 239000001110 calcium chloride Substances 0.000 claims abstract description 7
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 7
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229910000365 copper sulfate Inorganic materials 0.000 claims abstract description 7
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims abstract description 7
- FCRACOPGPMPSHN-UHFFFAOYSA-N desoxyabscisic acid Natural products OC(=O)C=C(C)C=CC1C(C)=CC(=O)CC1(C)C FCRACOPGPMPSHN-UHFFFAOYSA-N 0.000 claims abstract description 7
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 claims abstract description 7
- 239000011790 ferrous sulphate Substances 0.000 claims abstract description 7
- 235000003891 ferrous sulphate Nutrition 0.000 claims abstract description 7
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims abstract description 7
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims abstract description 7
- 235000019341 magnesium sulphate Nutrition 0.000 claims abstract description 7
- 229940099596 manganese sulfate Drugs 0.000 claims abstract description 7
- 239000011702 manganese sulphate Substances 0.000 claims abstract description 7
- 235000007079 manganese sulphate Nutrition 0.000 claims abstract description 7
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims abstract description 7
- 235000019799 monosodium phosphate Nutrition 0.000 claims abstract description 7
- QMMRZOWCJAIUJA-UHFFFAOYSA-L nickel dichloride Chemical compound Cl[Ni]Cl QMMRZOWCJAIUJA-UHFFFAOYSA-L 0.000 claims abstract description 7
- 239000001103 potassium chloride Substances 0.000 claims abstract description 7
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 7
- 239000004323 potassium nitrate Substances 0.000 claims abstract description 7
- 235000010333 potassium nitrate Nutrition 0.000 claims abstract description 7
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims abstract description 7
- 239000011684 sodium molybdate Substances 0.000 claims abstract description 7
- 235000015393 sodium molybdate Nutrition 0.000 claims abstract description 7
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims abstract description 7
- 150000003722 vitamin derivatives Chemical class 0.000 claims abstract description 7
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims abstract description 7
- 229910000368 zinc sulfate Inorganic materials 0.000 claims abstract description 7
- 229960001763 zinc sulfate Drugs 0.000 claims abstract description 7
- 229930006000 Sucrose Natural products 0.000 claims description 19
- 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 19
- 239000005720 sucrose Substances 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 238000004140 cleaning Methods 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 7
- 230000008014 freezing Effects 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000008223 sterile water Substances 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 4
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims description 4
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 3
- 239000003599 detergent Substances 0.000 claims description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 3
- 239000008399 tap water Substances 0.000 claims description 3
- 235000020679 tap water Nutrition 0.000 claims description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 2
- GHOKWGTUZJEAQD-UHFFFAOYSA-N Chick antidermatitis factor Natural products OCC(C)(C)C(O)C(=O)NCCC(O)=O GHOKWGTUZJEAQD-UHFFFAOYSA-N 0.000 claims description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 2
- 229930003451 Vitamin B1 Natural products 0.000 claims description 2
- 229930003537 Vitamin B3 Natural products 0.000 claims description 2
- 229930003571 Vitamin B5 Natural products 0.000 claims description 2
- 229930003756 Vitamin B7 Natural products 0.000 claims description 2
- FAPWYRCQGJNNSJ-UBKPKTQASA-L calcium D-pantothenic acid Chemical compound [Ca+2].OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O.OCC(C)(C)[C@@H](O)C(=O)NCCC([O-])=O FAPWYRCQGJNNSJ-UBKPKTQASA-L 0.000 claims description 2
- 229960002079 calcium pantothenate Drugs 0.000 claims description 2
- 229960003512 nicotinic acid Drugs 0.000 claims description 2
- DFPAKSUCGFBDDF-UHFFFAOYSA-N nicotinic acid amide Natural products NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 claims description 2
- RADKZDMFGJYCBB-UHFFFAOYSA-N pyridoxal hydrochloride Natural products CC1=NC=C(CO)C(C=O)=C1O RADKZDMFGJYCBB-UHFFFAOYSA-N 0.000 claims description 2
- 238000011084 recovery Methods 0.000 claims description 2
- 229960003495 thiamine Drugs 0.000 claims description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 claims description 2
- 235000010374 vitamin B1 Nutrition 0.000 claims description 2
- 239000011691 vitamin B1 Substances 0.000 claims description 2
- 235000019160 vitamin B3 Nutrition 0.000 claims description 2
- 239000011708 vitamin B3 Substances 0.000 claims description 2
- 235000009492 vitamin B5 Nutrition 0.000 claims description 2
- 239000011675 vitamin B5 Substances 0.000 claims description 2
- 235000019158 vitamin B6 Nutrition 0.000 claims description 2
- 239000011726 vitamin B6 Substances 0.000 claims description 2
- 235000011912 vitamin B7 Nutrition 0.000 claims description 2
- 239000011735 vitamin B7 Substances 0.000 claims description 2
- 235000019159 vitamin B9 Nutrition 0.000 claims description 2
- 239000011727 vitamin B9 Substances 0.000 claims description 2
- 229940011671 vitamin b6 Drugs 0.000 claims description 2
- 150000001875 compounds Chemical class 0.000 claims 1
- 230000035784 germination Effects 0.000 abstract description 14
- 238000004659 sterilization and disinfection Methods 0.000 abstract description 10
- 230000004083 survival effect Effects 0.000 abstract description 5
- 210000002257 embryonic structure Anatomy 0.000 description 31
- 208000005156 Dehydration Diseases 0.000 description 25
- 239000002609 medium Substances 0.000 description 10
- 239000000306 component Substances 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 239000000203 mixture Substances 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 239000002250 absorbent Substances 0.000 description 3
- 230000002745 absorbent Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000011065 in-situ storage Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000007420 reactivation Effects 0.000 description 3
- 238000003860 storage Methods 0.000 description 3
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000006870 ms-medium Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 1
- 229910052782 aluminium Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 239000003610 charcoal Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- -1 compound vitamin Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
- Y02P60/21—Dinitrogen oxide [N2O], e.g. using aquaponics, hydroponics or efficiency measures
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides an in-vitro preservation method of coconut germplasm resources, which comprises the steps of taking coconut embryo for sterilization, immersing the sterilized coconut embryo into an improved Y3 basic culture medium, and directly sealing and preserving; or immersing the sterilized coconut embryo into an improved Y3 basal medium for 3-5 d, taking out the cultured coconut embryo for physical dehydration and sealing; the improved Y3 basal medium comprises 1800-2200 g of potassium nitrate, 1300-160 mg of potassium chloride, 400-700 mg of ammonium chloride, 200-500 mg of sodium dihydrogen phosphate, 200-500 mg of calcium chloride, 100-400 mg of magnesium sulfate, 8-13 mg of manganese sulfate, 6-10 mg of potassium iodide, 5-9 mg of zinc sulfate, 1-5 mg of boric acid, 0.05-0.45 mg of copper sulfate, 0.05-0.45 mg of cobalt chloride, 0.05-0.45 mg of sodium molybdate, 0.005-0.045 mg of nickel chloride, 30-50 mg of ferrous sulfate, 45-65 mg of disodium ethylenediamine tetraacetate, 0.1-0.5 mg of vitamin complex, 0.05-0.15 mg of glycine, 0.05-0.45 mg of abscisic acid and 1-2.5 g of activated carbon. The invention carries out reviving culture by the improved Y3 basic culture medium required by coconut reviving embryo culture, so that the coconut embryo can be revived rapidly, the survival rate and the germination rate are improved, and a proper in-vitro preservation method of coconut germplasm resources is provided.
Description
Technical Field
The invention relates to the technical field of germplasm preservation, in particular to an in-vitro preservation method of coconut germplasm resources.
Background
The plant genetic resource may be protected in its natural habitat or outside its natural growing environment (ex vivo). In situ protection refers to the maintenance of biological resources within agricultural systems under natural environmental conditions or with traditional varieties, and plants evolve in these environments. In situ protection of coconut germplasm resources has some advantages over ex vivo protection because it maintains ecological functions and processes and allows continued evolution. However, in situ protection also has several disadvantages, including that the material may be difficult to collect and send to a laboratory when genetic improvement is required, and the protected site may still be affected by insects, diseases, natural disasters or land use changes. In contrast, seed gene banks, field seed gardens, or DNA or pollen, etc. in vitro storage materials are more convenient and cost effective germplasm resource protection techniques. However, among these techniques, the most commonly used seed pool method is not suitable for ex vivo protection of coconut germplasm resources, as coconut seeds are large and extremely sensitive to dehydration, difficult to preserve. Thus, there is a need to find suitable methods for the ex vivo preservation of coconut germplasm resources.
Disclosure of Invention
In view of the above, the present invention aims to provide an in vitro preservation method of coconut germplasm resources, and an alternative method for in vitro protection of coconut germplasm resources is to use an in vitro method, which involves a certain degree of dehydration of tissues, and then ultralow temperature preservation of the tissues. The invention relates to three techniques for preserving and transporting coconut germplasm resources, which can be used selectively according to the transportation distance and time, and simultaneously need to be used together with the embryo culture technique of coconuts, in particular to the coconut germplasm resources preserved or transported by the invention need to be subjected to the coconut reviving culture technique so as to restore the activity and further continue to grow.
The germplasm resources referred to herein are specifically coconut embryos (fertilized eggs), and are the most reproductive parts of all tissues and organs of coconuts.
The technical scheme of the invention is realized as follows:
an in vitro preservation method of coconut germplasm resources comprises an in vitro preservation method of fresh coconut embryo and dried coconut embryo;
the in-vitro preservation method of the fresh coconut embryo comprises the following steps: taking coconut embryo, sterilizing, immersing the sterilized coconut embryo into an improved Y3 basic culture medium, and directly sealing and storing;
the in-vitro preservation method of the dried coconut embryo comprises the following steps: immersing the sterilized coconut embryo into an improved Y3 basal medium for 3-5 d, taking out the cultured coconut embryo for physical dehydration, and sealing;
each liter of the modified Y3 basal medium comprises the following components in parts by weight:
1800-780 mg of potassium nitrate, 1300-160 mg of potassium chloride, 400-700 mg of ammonium chloride, 200-500 mg of sodium dihydrogen phosphate, 200-500 mg of calcium chloride, 100-400 mg of magnesium sulfate, 8-13 mg of manganese sulfate, 6-10 mg of potassium iodide, 5-9 mg of zinc sulfate, 1-5 mg of boric acid, 0.05-0.45 mg of copper sulfate, 0.05-0.45 mg of cobalt chloride, 0.05-0.45 mg of sodium molybdate, 0.005-0.045 mg of nickel chloride, 30-50 mg of ferrous sulfate, 45-65 mg of disodium ethylenediamine tetraacetate, 0.1-0.5 mg of vitamin complex, 0.05-0.15 mg of glycine, 0.05-0.45 mg of abscisic acid and 1-2.5 g of activated carbon.
Further, each liter of the modified Y3 basal medium comprises the following components in parts by weight:
1900-210mg of potassium nitrate, 1400-1500 mg of potassium chloride, 500-600 mg of ammonium chloride, 300-400 mg of sodium dihydrogen phosphate, 300-400 mg of calcium chloride, 200-300 mg of magnesium sulfate, 9-12 mg of manganese sulfate, 7-9 mg of potassium iodide, 6-8 mg of zinc sulfate, 2-4 mg of boric acid, 0.15-0.35 mg of copper sulfate, 0.15-0.35 mg of cobalt chloride, 0.15-0.35 mg of sodium molybdate, 0.015-0.035 mg of nickel chloride, 35-45 mg of ferrous sulfate, 50-60 mg of disodium ethylenediamine tetraacetate, 0.2-0.4 mg of vitamin complex, 0.05-0.15 mg of glycine and 0.15-0.35 mg of abscisic acid.
Further, each liter of the modified Y3 basal medium comprises the following components in parts by weight:
2000-2050 mg of potassium nitrate, 1450-1500 mg of potassium chloride, 500-550 mg of ammonium chloride, 300-350 mg of sodium dihydrogen phosphate, 300-350 mg of calcium chloride, 200-250 mg of magnesium sulfate, 10-12 mg of manganese sulfate, 7-9 mg of potassium iodide, 6-8 mg of zinc sulfate, 2-4 mg of boric acid, 0.20-0.30 mg of copper sulfate, 0.20-0.30 mg of cobalt chloride, 0.20-0.30 mg of sodium molybdate, 0.020-0.030 mg of nickel chloride, 40-45 mg of ferrous sulfate, 50-60 mg of disodium ethylenediamine tetraacetate, 0.2-0.4 mg of vitamin complex, 0.08-0.12 mg of glycine and 0.20-0.30 mg of abscisic acid.
Further, the compound vitamin comprises the following components in percentage by mass of 1-2: 1-2: 1-2: 1-2: 1-2: 1-2 to obtain vitamin B6, vitamin B1, vitamin B3, vitamin B5, vitamin H and vitamin Bc.
Furthermore, in the in-vitro preservation method of the dried coconut embryo, each liter of the modified Y3 basal medium further comprises 130-150 g of sucrose.
In the method for preserving the dried coconut embryo in vitro, the length of the coconut embryo is at least 8mm, and physical dehydration is carried out.
Further, the coconut embryo is physically dehydrated until the moisture content of the coconut embryo is 19-21% FW.
In the in-vitro preservation method of the dried coconut embryo, after the cultured coconut embryo is physically dehydrated, liquid nitrogen is added for quick freezing, and freezing and sealing are carried out.
Further, the coconut embryo disinfection method comprises the following steps: taking out the coconut, cleaning the surface of the embryo by adopting a detergent, cleaning by tap water, and sequentially cleaning by using 75-85% alcohol and 1-2 min and 1.5-2.5% sodium hypochlorite solution for 10-15 min, and cleaning by using sterile water.
Further, the method for recovering and culturing the sealed coconut embryo comprises the following steps: and placing the sealed coconut embryo into the improved Y3 basal medium for culturing, wherein each liter of the improved Y3 basal medium for recovering the sealed coconut embryo also comprises 50-70 g of sucrose.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the modified Y3 basal medium is adopted to directly immerse the sterilized coconut embryo into the modified Y3 basal medium for sealing, or the modified Y3 basal medium is immersed into the modified Y3 basal medium for sealing after being subjected to rapid physical dehydration, so that the purposes of Gao Fuhuo rate, high activity, low pollution and the like after the short-term in-vitro preservation of the fresh coconut embryo or the long-term in-vitro preservation of the dried coconut embryo can be achieved.
According to the in-vitro preservation method for the coconut germplasm resources, provided by the invention, different in-vitro preservation methods for the coconut embryo can be selected according to factors such as transportation distance, transportation time and the like, meanwhile, after short-term in-vitro preservation of fresh coconut embryo or long-term in-vitro preservation of dry coconut embryo is carried out, the coconut embryo is subjected to reviving culture through an improved Y3 basic culture medium required by coconut reviving embryo culture, so that the coconut embryo is revived rapidly, and the survival rate and germination rate are improved.
In the invention, the pretreatment is carried out by combining the modified Y3 basal medium with high sucrose during physical dehydration, so that the dehydration time is effectively shortened, the dehydration efficiency is improved, and the germination rate is improved.
Detailed Description
In order to better understand the technical content of the present invention, the following provides specific examples to further illustrate the present invention.
The experimental methods used in the embodiment of the invention are conventional methods unless otherwise specified.
Materials, reagents, and the like used in the examples of the present invention are commercially available unless otherwise specified.
The invention uses Hainan high coconut.
EXAMPLE 1 modified Y3 basal Medium
The composition of the medium (Y3 basal medium per liter) required for culturing coconut reviving embryos is shown in Table 1 below.
TABLE 1 formulation of coconut reviving embryo improved Y3 basal medium
Note that: "-" indicates no component.
Preparation of the culture medium: these ingredients should be mixed in the proper ratio and dissolved in 1 liter of coconut embryo culture medium. A 1 liter bottle was prepared for the basal medium, and 500 ml of sterile water was added to the bottle, followed by stirring on a stirrer, and then all ingredients (except for activated carbon and agar) were added. The pH was adjusted using a pH meter (optimum pH for coconut embryo culture was 5.7 to 5.8), the final volume was adjusted to 1 liter with sterile water before adding activated carbon and agar, the bottle cap was sealed with an autoclave tape, placed in an autoclave (20 minutes, 121 ℃ C.), and then cooled. Sterile tubes or containers (about 2.5 cm in diameter and about 8 cm in height) were prepared for embryo culture and medium was poured into the containers under sterile conditions.
Pretreatment of endosperm containing embryo: before drying and refrigerating the coconut embryo, the endosperm blocks containing the embryo were sterilized in 50% (V/V) commercial 84 liquor for 10 minutes, then rinsed in the modified Y3 basal medium of example 1 (with 60g/L sucrose and 2.5g/L charcoal) for several seconds, and finally placed in a storage vessel to obtain the pretreated coconut embryo.
EXAMPLE 2 technique for transporting fresh coconut embryo
First, the modified Y3 basal medium of example 1 was used, without sugar, but with 1g/L of activated carbon powder and 3.5g/L of Gelzan agar. Mixing the above culture medium components, shaking, selecting 1mL centrifuge tube (capable of high temperature sterilization), adding 1mL culture medium into each tube, and sterilizing at 121deg.C for 20 min. And after sterilization, shaking up again, cooling to room temperature and solidifying.
When in use, the coconut is firstly taken out from the coconut tree, the embryo is taken out, the lipid on the surface of the embryo is cleaned by using commercial detergent, and the embryo is cleaned by tap water. In an ultra-clean workbench, washing for 1 minute by using 80% alcohol by volume, pouring out the alcohol, adding a sodium hypochlorite solution with the volume fraction of 2.1%, washing for 12 minutes, and thoroughly sterilizing the explant. After sterilization, the product was washed three times with sterile water and dried in an ultra clean bench environment. Sterilized embryos were inoculated into a centrifuge tube using high temperature sterilized forceps, and the embryos were all submerged in the medium. The technical method can be used for short-distance transportation of the embryo and can keep the activity of the coconut embryo within a week.
After the coconut embryo reaches the destination, the package is opened in an ultra-clean workbench, the centrifuge tube box is taken out, and the package is opened after alcohol spraying and disinfection. Taking out the centrifuge tube, spraying alcohol for disinfection, opening the cover, directly taking out the embryo, and placing the embryo on sterilized absorbent paper in advance to absorb the liquid culture medium. After the surface of the embryo was completely dried for about five minutes, the embryo was directly put into a reviving embryo to be revived, and 60g/L sucrose, 2.5g/L activated carbon and 7g/L agar were added using the modified Y3 basal medium of example 1.
For short-distance transportation, coconut embryo must be sterilized, and the sterilizing efficiency and the growth after sterilization are important. After 12 weeks of culture, 1 embryo dies, 3 embryos are contaminated, 26 embryos germinate, the germination rate is 86.7%, and the contamination rate is 10% in the coconut embryo of example 2. .
Coconut embryos are sterilized by the method of Ashburner, G.R. et al (Ashburner, G.R., failure, M.G., franz, P.R., tomlinson, D.R., pulo, P.Burch, J.M., thompson, W.K. (1993, november) Coconut embryo culture for remote locations InACIAR PROCEEDINGS (pp. -25) Australian Centre for International Agricultural research) and then cultured for 12 weeks, the germination rate (survival rate) of the coconut embryos cultured by the method is 66.7%, and the contamination rate is 10%.
The disinfection and sterilization method of the embodiment 2 is used, and the subsequent seedling rate of coconut embryo is improved. In the subsequent seedling process, only embryos with good states and fast growth can be used for the next generation. After about 10 days of transportation, the final seedling formation was carried out in the following manner, and the survival rate of the method of example 2 (after short-distance transportation) was 69.3%, that of the Ashburner, G.R. and the like was 61.2%.
EXAMPLE 3 transportation and storage technique of dried coconut embryo
In the embodiment, through the device consisting of the dryer, the absorbent paper and the absorbent silica gel, the water content of the embryo can be reduced to 20-30% of the fresh weight only in 7 hours. Pretreatment is carried out in 140g/L sucrose before rapid physical dehydration, so that the dehydration time of the embryo is reduced, and the water content of the embryo after dehydration reaches about 20%. Embryos with length dimensions above 8mm should be used in operation to ensure survival and germination, and embryos that are too small will lose activity and cannot be revived.
The sucrose pretreatment method comprises the following steps: a Y3 basal medium was prepared according to the formulation of example 1, to which 140g/L sucrose, 2.5g/L activated carbon and 7g/L agar were added, sterilized at 121℃for 20 minutes, and then filled into sterile 20mL vials. After the embryos are sterilized, the embryos are inoculated into the medium with forceps in a sterile environment, and care is taken at this stage to completely submerge the embryo beards into the solid medium for adequate water loss. Coconut embryos need to be cultured in pretreated solid medium for 4 days. And after the culture is finished, taking out the embryo from the culture medium, and cleaning the embryo by using sterile water. After pretreatment, embryos were wiped dry on sterile filter paper, then placed in sterile aluminum foil, folded like an envelope, and placed in a dehydration apparatus containing water absorbing silica gel for 7 hours of rapid physical dehydration. The dehydrated coconut embryo is reduced in size to one third to one fifth of the previous size, taken out of the ultra-clean workbench, put into a sterile plastic PA+PE vacuum sealing bag, and the internal air is pumped out by using a vacuum air pump. By using the method, the volume of the coconut embryo is greatly reduced, hundreds of coconut embryos can be processed at one time, the dehydration treatment efficiency of the coconut embryo is effectively improved, and meanwhile, the storage time of the coconut embryo is greatly prolonged. Coconut embryos preserved by this method can remain active for 2-3 weeks. After sealing or transportation, the sealed bag is opened in a sterile environment, coconut embryo is taken out, and is put into Y3 culture medium (containing 60g/L sucrose, 2.5g/L active carbon and 7g/L agar) of example 1 for reactivation, and normal coconut embryo culture technique can be used for subculture after water absorption and reactivation.
Through experiments, the method of the embodiment 3 is adopted, the reviving rate of the coconut embryo is about 60%, and the dehydration rate of the coconut embryo before long-distance transportation can be improved by using a glass dryer and sucrose pretreatment, so that the dehydration effect is ensured; meanwhile, the activity of the revived embryo can be ensured by adopting a novel method for restoring the activity of the embryo after drying.
During the dewatering process using example 3, the coconut embryo moisture loss was subjected to two stages, with a sharp drop in moisture content from 83% to 29% during the first 4 hours. After drying for 7 hours a final moisture content of 19% is obtained, in which case long-distance transport is possible. Embryo germination was assessed after rapid physical dehydration. For rapid physical dehydration for 4 hours (moisture content 21% FW), the percentage of germinated embryos recovered was very high (> 60%) significantly lower than that of live embryos (95%). However, when embryos are rapidly dehydrated for 7 hours (moisture content 19% FW), the percentage of germinated embryos is reduced to 40%.
The dehydration speed of coconut embryo can be improved by adopting sucrose pretreatment. In the control treatment without sucrose, embryos with a moisture content of 20% FW were not reached until 7 hours after drying. However, when the embryo is pre-cultured in sucrose solution, the same moisture content can be achieved only for 4 hours. After rapid physical dehydration, the germination capacity (73%) of large embryos was still high even though the moisture content of the embryos was reduced to 20% FW. If the dehydration treatment is performed for 1 hour (to 19% FW), the germination rate is reduced to 40%. Although the embryos reached a moisture content of 22% FW after 4 hours of drying, their germination rate was poor (37%) compared to the large embryos.
The above demonstrates that the rapid physical dehydration protocol used in example 3 in combination with sucrose pretreatment is effective in increasing the dehydration rate of embryos prior to cryopreservation.
EXAMPLE 4 Freeze transport preservation technique of coconut embryo
Embryos were surface sterilized and washed by the method of example 2 and then rapidly physically dehydrated (to reduce their moisture content to about 19% FW) by the method of example 3. And (3) putting the dried coconut embryo into a 2 milliliter liquid nitrogen freezing tube, then directly immersing the coconut embryo into a liquid nitrogen bottle for quick freezing, taking out the coconut embryo after 15 seconds, and directly putting the coconut embryo into a-80 ℃ refrigerator for preservation. A liquid nitrogen bottle or a dry ice box is required to be used as a container during transportation. Restoring the cryopreserved coconut embryos requires thawing the tube in room temperature water for 3 minutes, then carefully removing the embryos in an ultra clean bench sterile environment and placing them in culture medium for germination (dark culture).
The test shows that the recovery rate of coconut embryo (from dried embryo to young seedling) is about 40% by the method of example 4.
The germination percentage of the coconut embryo after the freeze preservation is about 26% and the germination percentage before the freeze preservation is about 60% by directly adopting physical dehydration without pretreatment of sucrose.
The above shows that the coconut embryo adopts sucrose pretreatment to carry out rapid physical dehydration, so as to improve the germination rate of the coconut embryo after freezing preservation.
Comparative example 1-MS plant Medium
TABLE 2 formulation of MS plant Medium for coconut reviving embryo
Note that: "-" indicates no component.
Preparation of the culture medium: these components should be mixed in a proper ratio and dissolved in 1 liter of medium. In addition to inorganic salts, 30g sucrose, 2.5g activated carbon, 3g Gelzan agar (Sigma) were added per liter of MS medium, and the mixture was sterilized at 121℃for 20 minutes and allowed to stand still for cooling.
According to the treatment of live coconut embryos of example 2, the modified Y3 basal medium was replaced with MS medium, and the following data were obtained through experiments:
TABLE 3 Effect of formulations of different coconut reactivation embryo cultures on in vitro preservation of coconut embryo
The above shows that the modified Y3 basal medium is more suitable for the in vitro preservation medium of coconut germplasm resources.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. An in vitro preservation method of coconut germplasm resources is characterized by comprising an in vitro preservation method of fresh coconut embryo and dried coconut embryo;
the in-vitro preservation method of the fresh coconut embryo comprises the following steps: taking coconut embryo, sterilizing, immersing the sterilized coconut embryo into an improved Y3 basic culture medium, and directly sealing and storing;
the in-vitro preservation method of the dried coconut embryo comprises the following steps: immersing the sterilized coconut embryo into an improved Y3 basal medium for 3-5 d, taking out the cultured coconut embryo for physical dehydration, and sealing;
each liter of the modified Y3 basal medium comprises the following components in parts by weight:
1800-780 mg of potassium nitrate, 1300-160 mg of potassium chloride, 400-700 mg of ammonium chloride, 200-500 mg of sodium dihydrogen phosphate, 200-500 mg of calcium chloride, 100-400 mg of magnesium sulfate, 8-13 mg of manganese sulfate, 6-10 mg of potassium iodide, 5-9 mg of zinc sulfate, 1-5 mg of boric acid, 0.05-0.45 mg of copper sulfate, 0.05-0.45 mg of cobalt chloride, 0.05-0.45 mg of sodium molybdate, 0.005-0.045 mg of nickel chloride, 30-50 mg of ferrous sulfate, 45-65 mg of disodium ethylenediamine tetraacetate, 0.1-0.5 mg of vitamin complex, 0.05-0.15 mg of glycine, 0.05-0.45 mg of abscisic acid and 1-2.5 g of activated carbon.
2. The method for the ex vivo preservation of coconut germplasm resources according to claim 1, wherein each liter of said modified Y3 basal medium comprises the following components by weight:
1900-210mg of potassium nitrate, 1400-1500 mg of potassium chloride, 500-600 mg of ammonium chloride, 300-400 mg of sodium dihydrogen phosphate, 300-400 mg of calcium chloride, 200-300 mg of magnesium sulfate, 9-12 mg of manganese sulfate, 7-9 mg of potassium iodide, 6-8 mg of zinc sulfate, 2-4 mg of boric acid, 0.15-0.35 mg of copper sulfate, 0.15-0.35 mg of cobalt chloride, 0.15-0.35 mg of sodium molybdate, 0.015-0.035 mg of nickel chloride, 35-45 mg of ferrous sulfate, 50-60 mg of disodium ethylenediamine tetraacetate, 0.2-0.4 mg of vitamin complex, 0.05-0.15 mg of glycine and 0.15-0.35 mg of abscisic acid.
3. The method for the ex vivo preservation of coconut germplasm resources according to claim 2, wherein each liter of said modified Y3 basal medium comprises the following components by weight:
2000-2050 mg of potassium nitrate, 1450-1500 mg of potassium chloride, 500-550 mg of ammonium chloride, 300-350 mg of sodium dihydrogen phosphate, 300-350 mg of calcium chloride, 200-250 mg of magnesium sulfate, 10-12 mg of manganese sulfate, 7-9 mg of potassium iodide, 6-8 mg of zinc sulfate, 2-4 mg of boric acid, 0.20-0.30 mg of copper sulfate, 0.20-0.30 mg of cobalt chloride, 0.20-0.30 mg of sodium molybdate, 0.020-0.030 mg of nickel chloride, 40-45 mg of ferrous sulfate, 50-60 mg of disodium ethylenediamine tetraacetate, 0.2-0.4 mg of vitamin complex, 0.08-0.12 mg of glycine and 0.20-0.30 mg of abscisic acid.
4. The method for in vitro conservation of coconut germplasm resources according to any one of claims 1-3, wherein the compound vitamins comprise the following components in mass ratio of 1-2: 1-2: 1-2: 1-2: 1-2: 1-2 to obtain vitamin B6, vitamin B1, vitamin B3, vitamin B5, vitamin H and vitamin Bc.
5. The method for preserving coconut germplasm resources in vitro according to claim 1, wherein in said method for preserving dried coconut embryo in vitro, each liter of said modified Y3 basal medium further comprises 130-150 g sucrose.
6. The method for preserving coconut coir resources ex vivo as recited in claim 1, wherein said method for preserving dried coconut coir is characterized in that physical dehydration is performed by a length of at least 8 mm.
7. The method for preserving coconut germplasm resources ex vivo as defined in claim 1, wherein the moisture content of the coconut embryo is 19-21% FW by physical dehydration.
8. The method for preserving coconut germplasm resources in vitro according to claim 1, wherein in the method for preserving dried coconut embryo in vitro, after physical dehydration of the cultured coconut embryo, liquid nitrogen is added for quick freezing, and freezing and sealing are carried out.
9. The method for the ex vivo preservation of coconut coir resources as recited in claim 1, wherein the method for sterilizing coconut coir comprises: taking out the coconut, cleaning the surface of the embryo by adopting a detergent, cleaning by tap water, and sequentially cleaning by using 75-85% alcohol and 1-2 min and 1.5-2.5% sodium hypochlorite solution for 10-15 min, and cleaning by using sterile water.
10. The method for the ex vivo preservation of coconut germplasm resources according to claim 1, wherein the method for the recovery culture of the preserved coconut embryo comprises: and placing the sealed coconut embryo into the improved Y3 basal medium for culturing, wherein each liter of the improved Y3 basal medium for recovering the sealed coconut embryo also comprises 50-70 g of sucrose.
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