CN108739402A - A method of improving corn hybrid seed production efficiency - Google Patents
A method of improving corn hybrid seed production efficiency Download PDFInfo
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- CN108739402A CN108739402A CN201810656845.4A CN201810656845A CN108739402A CN 108739402 A CN108739402 A CN 108739402A CN 201810656845 A CN201810656845 A CN 201810656845A CN 108739402 A CN108739402 A CN 108739402A
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- 240000008042 Zea mays Species 0.000 title claims abstract description 67
- 235000002017 Zea mays subsp mays Nutrition 0.000 title claims abstract description 67
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 title claims abstract description 58
- 235000005822 corn Nutrition 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 23
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- 210000001938 protoplast Anatomy 0.000 claims abstract description 29
- 210000004027 cell Anatomy 0.000 claims abstract description 15
- 238000001556 precipitation Methods 0.000 claims abstract description 11
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims abstract description 9
- 235000009973 maize Nutrition 0.000 claims abstract description 9
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 239000006228 supernatant Substances 0.000 claims abstract description 8
- 241000196324 Embryophyta Species 0.000 claims abstract description 6
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000004113 cell culture Methods 0.000 claims abstract description 4
- 238000005119 centrifugation Methods 0.000 claims abstract description 4
- 239000012531 culture fluid Substances 0.000 claims abstract description 4
- 238000004161 plant tissue culture Methods 0.000 claims abstract description 4
- 229920002523 polyethylene Glycol 1000 Polymers 0.000 claims abstract description 4
- 230000008929 regeneration Effects 0.000 claims abstract description 4
- 238000011069 regeneration method Methods 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 25
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 18
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 18
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 12
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 claims description 12
- 239000012153 distilled water Substances 0.000 claims description 12
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical group [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 11
- 229920001817 Agar Polymers 0.000 claims description 9
- 239000008272 agar Substances 0.000 claims description 9
- 239000000470 constituent Substances 0.000 claims description 9
- 239000012530 fluid Substances 0.000 claims description 9
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 9
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 9
- 235000015097 nutrients Nutrition 0.000 claims description 9
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 8
- 239000001110 calcium chloride Substances 0.000 claims description 8
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 8
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 7
- 235000019796 monopotassium phosphate Nutrition 0.000 claims description 7
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 claims description 7
- 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 6
- 239000004471 Glycine Substances 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 6
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- 229930006000 Sucrose Natural products 0.000 claims description 6
- 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 6
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 6
- 239000004327 boric acid Substances 0.000 claims description 6
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 6
- 239000011790 ferrous sulphate Substances 0.000 claims description 6
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 6
- 229940064880 inositol 100 mg Drugs 0.000 claims description 6
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 6
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 6
- 229940099596 manganese sulfate Drugs 0.000 claims description 6
- 239000011702 manganese sulphate Substances 0.000 claims description 6
- 235000007079 manganese sulphate Nutrition 0.000 claims description 6
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 6
- 235000001968 nicotinic acid Nutrition 0.000 claims description 6
- 229960003512 nicotinic acid Drugs 0.000 claims description 6
- 239000011664 nicotinic acid Substances 0.000 claims description 6
- 239000004323 potassium nitrate Substances 0.000 claims description 6
- 235000010333 potassium nitrate Nutrition 0.000 claims description 6
- 239000002994 raw material Substances 0.000 claims description 6
- 229910052938 sodium sulfate Inorganic materials 0.000 claims description 6
- 235000011152 sodium sulphate Nutrition 0.000 claims description 6
- 239000007787 solid Substances 0.000 claims description 6
- 239000005720 sucrose Substances 0.000 claims description 6
- 229960000344 thiamine hydrochloride Drugs 0.000 claims description 6
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 6
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 6
- 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 6
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 6
- 229960001763 zinc sulfate Drugs 0.000 claims description 6
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 6
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 claims description 5
- GIWNQJAJNLVFSW-UHFFFAOYSA-N n-nonyl-7h-purin-6-amine Chemical compound CCCCCCCCCNC1=NC=NC2=C1NC=N2 GIWNQJAJNLVFSW-UHFFFAOYSA-N 0.000 claims description 5
- 108010059892 Cellulase Proteins 0.000 claims description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 244000061456 Solanum tuberosum Species 0.000 claims description 3
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 108010079058 casein hydrolysate Proteins 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- 239000000284 extract Substances 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 239000001257 hydrogen Substances 0.000 claims description 3
- 229910052739 hydrogen Inorganic materials 0.000 claims description 3
- 229940050906 magnesium chloride hexahydrate Drugs 0.000 claims description 3
- DHRRIBDTHFBPNG-UHFFFAOYSA-L magnesium dichloride hexahydrate Chemical compound O.O.O.O.O.O.[Mg+2].[Cl-].[Cl-] DHRRIBDTHFBPNG-UHFFFAOYSA-L 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 3
- 239000001103 potassium chloride Substances 0.000 claims description 3
- 235000011164 potassium chloride Nutrition 0.000 claims description 3
- 239000011780 sodium chloride Substances 0.000 claims description 3
- -1 sulfate dihydrate disodium hydrogen Chemical class 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 239000012981 Hank's balanced salt solution Substances 0.000 claims description 2
- 108010059820 Polygalacturonase Proteins 0.000 claims description 2
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 2
- 230000035876 healing Effects 0.000 claims description 2
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 claims 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 claims 1
- 230000001476 alcoholic effect Effects 0.000 claims 1
- 239000011575 calcium Substances 0.000 claims 1
- 229910052791 calcium Inorganic materials 0.000 claims 1
- 239000000460 chlorine Substances 0.000 claims 1
- 229910052801 chlorine Inorganic materials 0.000 claims 1
- 235000009508 confectionery Nutrition 0.000 claims 1
- 238000009395 breeding Methods 0.000 abstract description 9
- 230000001488 breeding effect Effects 0.000 abstract description 9
- 238000009396 hybridization Methods 0.000 abstract description 3
- 238000009400 out breeding Methods 0.000 abstract description 2
- 230000000392 somatic effect Effects 0.000 abstract description 2
- 238000011218 seed culture Methods 0.000 abstract 1
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 6
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 4
- 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 description 4
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 4
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 2
- 239000012930 cell culture fluid Substances 0.000 description 2
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 2
- 229960001669 kinetin Drugs 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 230000010152 pollination Effects 0.000 description 2
- 239000011591 potassium Substances 0.000 description 2
- 229910052700 potassium Inorganic materials 0.000 description 2
- 230000006641 stabilisation Effects 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- UGWKCNDTYUOTQZ-UHFFFAOYSA-N copper;sulfuric acid Chemical compound [Cu].OS(O)(=O)=O UGWKCNDTYUOTQZ-UHFFFAOYSA-N 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 238000009399 inbreeding Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000006467 substitution reaction Methods 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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of methods improving corn hybrid seed production efficiency, include the following steps：S1, corn seed culture is obtained into maize calli；S2, maize calli is separated into Corn Protoplast solution；S3, supernatant that Corn Protoplast solution is left away, precipitation are resuspended；S4, PEG1000 is added in Corn Protoplast, 9.5mlHanks solution is slowly added after 90s into test tube, test tube is stood in water-bath, supernatant is abandoned in centrifugation, 1ml cell culture fluids are added into cell precipitation, gently mixing cultivates the Corn Protoplast fused cell that 30min is activated in 37 DEG C of water-baths；S5, Corn Protoplast fused cell is trained corn regeneration hybrid plant using the method for Vitro Plant tissue cultures.The present invention greatly shortens breeding cycle, and also improve the quality of breeding, reduces breeding time so that the specific aim of breeding object increases by the way that corn seed is carried out breeding using the method for somatic hybridization.
Description
Technical field
The present invention relates to field of plant variety breeding technology more particularly to a kind of sides for improving corn hybrid seed production efficiency
Method.
Background technology
The production of business seed is the complex process for being related to many steps, and for corn, inbreeding body plant must first be in
Hybridize in big Tanaka under conditions of allowing male pollen that the fringe silk on female fringe is made to be fertilized, the kind of peasant will be sold to produce
Son, but corn seed hybridization now is all the progress female flower pollination on crop field, the time of this pollination is long, produces a stabilization
Hybrid seed production cycle it is long.
Therefore, we have proposed a kind of methods improving corn hybrid seed production efficiency for solving the above problems.
Invention content
The purpose of the present invention is to solve the growth cycle of the corn hybrid seed of stabilization existing in the prior art is long
The shortcomings that, and a kind of method of the raising corn hybrid seed production efficiency proposed.
A method of corn hybrid seed production efficiency is improved, is included the following steps：
S1, by two kinds of character complementations, the corn seed of function admirable is cultivated in MS solid mediums, obtains corn healing
Tissue；
S2, maize calli is placed into MS fluid nutrient mediums, the addition mixing enzyme solutions into MS fluid nutrient mediums, to
Maize calli is separated into cell mass to be further continued for being separated into Corn Protoplast solution；
S3, Corn Protoplast solution is transferred in the round bottom centrifuge tube of 10ml, under conditions of centrifuging temperature is 4 DEG C
100rpm/min centrifuges 5min, and supernatant is removed on the basis of not contacting Corn Protoplast precipitation, then uses physiological saline will
Corn Protoplast precipitation is resuspended so that Corn Protoplast is counted as 2 × 105-5×105cells/ml；
S4, take 10ml in test tube Corn Protoplast, and it is 50% that 0.5ml mass concentrations, which are added, into test tube
PEG1000 is slowly stirred using glass bar, is slowly added 9.5mlHanks solution after 90s into test tube, gently in test tube
After piping and druming uniformly, test tube is stood into 5min in 37 DEG C of water-baths, supernatant is abandoned after 15000rpm centrifugations 5min, into cell precipitation
1ml cell culture fluids are added, gently mixing, the Corn Protoplast fused cell that 30min is activated is cultivated in 37 DEG C of water-baths；
S5, Corn Protoplast fused cell is trained corn regeneration hybrid plant using the method for Vitro Plant tissue cultures
Strain.
Preferably, the constituent of the MS solid mediums is potassium nitrate 1.9g/L, ammonium nitrate 1.6g/L, biphosphate
Potassium 0.17g/L, magnesium sulfate 0.37g/L, calcium chloride 4.4g/L, sucrose 30g/L, agar 7g/L, potassium iodide 0.83mg/L, boric acid
6.2mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, sodium sulphate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride
0.025mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L,
Thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L, benzyladenine 0.4mg/l, 6- Furfurylamino
Purine 0.4mg/l, agar 2%, surplus are distilled water.
Preferably, the constituent of the MS fluid nutrient mediums is potassium nitrate 1.9g/L, ammonium nitrate 1.6g/L, biphosphate
Potassium 0.17g/L, magnesium sulfate 0.37g/L, calcium chloride 4.4g/L, sucrose 30g/L, agar 7g/L, potassium iodide 0.83mg/L, boric acid
6.2mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, sodium sulphate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride
0.025mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L,
Thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L, benzyladenine 0.4mg/l, 6- Furfurylamino
Purine 0.4mg/l, surplus are distilled water.
Preferably, the mixing enzyme solutions include the raw material of following weight percent：Cellulase solution 6%, pectase is molten
Liquid 3%, calcium chloride solution 3% and mannitol solution 12%.
Preferably, the constituent of the Hanks solution is sodium chloride 80g/L, epsom salt 1g/L, potassium chloride 4g/
L, magnesium chloride hexahydrate 1g/L, calcium chloride 1.4g/L, ten sulfate dihydrate disodium hydrogen 1.5g/L, potassium dihydrogen phosphate 0.6g/L, glucose
10g/L, surplus are distilled water.
Preferably, the cell culture fluid includes the raw material of following weight percent：Yeast extract 0.5%, brewer's wort
0.6%, casein hydrolysate 0.6%, potato extract 12%, 0.08% benzyladenine, 0.08% 6- Furfurylaminos are fast
Purine, surplus are distilled water.
The beneficial effects of the invention are as follows：The present invention, by the way that corn seed is carried out breeding using the method for somatic hybridization,
Breeding cycle is greatly shortened, and also improves the quality of breeding, reduces breeding time so that breeding object is directed to
Property increase；Whole process is simple and efficient, and is suitble to large-scale promotion and application.
Specific implementation mode
The present invention is made further to explain with reference to specific embodiment.
Embodiment
A method of corn hybrid seed production efficiency is improved, is included the following steps：
S1, by two kinds of character complementations, the corn seed of function admirable is in MS solid mediums（The constituent of MS solid mediums
For potassium nitrate 1.9g/L, ammonium nitrate 1.6g/L, potassium dihydrogen phosphate 0.17g/L, magnesium sulfate 0.37g/L, calcium chloride 4.4g/L, sucrose
30g/L, agar 7g/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, sodium sulphate
0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate
27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, niacin
0.5mg/L, benzyladenine 0.4mg/l, 6-nonylaminopurine 0.4mg/l, agar 2%, surplus are distilled water）In trained
It supports, obtains maize calli；
S2, maize calli is placed into MS fluid nutrient mediums（The constituent of MS fluid nutrient mediums is potassium nitrate 1.9g/
L, ammonium nitrate 1.6g/L, potassium dihydrogen phosphate 0.17g/L, magnesium sulfate 0.37g/L, calcium chloride 4.4g/L, sucrose 30g/L, agar 7g/
L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfate 22.3mg/L, zinc sulfate 8.6mg/L, sodium sulphate 0.25mg/L, sulfuric acid
Copper 0.025mg/L, cobalt chloride 0.025mg/L, disodium ethylene diamine tetraacetate 37.3mg/L, ferrous sulfate 27.8mg/L, inositol
100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L, benzyl gland are fast
Purine 0.4mg/l, 6-nonylaminopurine 0.4mg/l, surplus are distilled water）, the addition mixing enzyme solutions into MS fluid nutrient mediums
（Mixing enzyme solutions include the raw material of following weight percent：Cellulase solution 6%, pectinase solution 3%, calcium chloride solution 3%
With mannitol solution 12%）, it is further continued for being separated into Corn Protoplast solution to which maize calli is separated into cell mass；
S3, Corn Protoplast solution is transferred in the round bottom centrifuge tube of 10ml, under conditions of centrifuging temperature is 4 DEG C
100rpm/min centrifuges 5min, and supernatant is removed on the basis of not contacting Corn Protoplast precipitation, then uses physiological saline will
Corn Protoplast precipitation is resuspended so that Corn Protoplast is counted as 4 × 105cells/ml；
S4, take 10ml in test tube Corn Protoplast, and it is 50% that 0.5ml mass concentrations, which are added, into test tube
PEG1000 is slowly stirred using glass bar, is slowly added 9.5mlHanks solution after 90s into test tube in test tube（Hanks
The constituent of solution is sodium chloride 80g/L, epsom salt 1g/L, potassium chloride 4g/L, magnesium chloride hexahydrate 1g/L, calcium chloride
1.4g/L, ten sulfate dihydrate disodium hydrogen 1.5g/L, potassium dihydrogen phosphate 0.6g/L, glucose 10g/L, surplus is distilled water）, gently
Piping and druming it is uniform after, test tube is stood into 5min in 37 DEG C of water-baths, supernatant is abandoned after 15000rpm centrifugations 5min, to cell precipitation
Middle addition 1ml cell culture fluids（Cell culture fluid includes the raw material of following weight percent：Yeast extract 0.5%, brewer's wort
0.6%, casein hydrolysate 0.6%, potato extract 12%, 0.08% benzyladenine, 0.08% 6- Furfurylaminos are fast
Purine, surplus are distilled water）, gently mixing, cultivates the Corn Protoplast fused cell that 30min is activated in 37 DEG C of water-baths；
S5, Corn Protoplast fused cell is trained corn regeneration hybrid plant using the method for Vitro Plant tissue cultures
Strain.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (6)
1. a kind of method improving corn hybrid seed production efficiency, which is characterized in that include the following steps：
S1, by two kinds of character complementations, the corn seed of function admirable is cultivated in MS solid mediums, obtains corn healing
Tissue；
S2, maize calli is placed into MS fluid nutrient mediums, the addition mixing enzyme solutions into MS fluid nutrient mediums, to
Maize calli is separated into cell mass to be further continued for being separated into Corn Protoplast solution；
S3, Corn Protoplast solution is transferred in the round bottom centrifuge tube of 10ml, under conditions of centrifuging temperature is 4 DEG C
100rpm/min centrifuges 5min, and supernatant is removed on the basis of not contacting Corn Protoplast precipitation, then uses physiological saline will
Corn Protoplast precipitation is resuspended so that Corn Protoplast is counted as 2 × 105-5×105cells/ml；
S4, take 10ml in test tube Corn Protoplast, and it is 50% that 0.5ml mass concentrations, which are added, into test tube
PEG1000 is slowly stirred using glass bar, is slowly added 9.5mlHanks solution after 90s into test tube, gently in test tube
After piping and druming uniformly, test tube is stood into 5min in 37 DEG C of water-baths, supernatant is abandoned after 15000rpm centrifugations 5min, into cell precipitation
1ml cell culture fluids are added, gently mixing, the Corn Protoplast fused cell that 30min is activated is cultivated in 37 DEG C of water-baths；
S5, Corn Protoplast fused cell is trained corn regeneration hybrid plant using the method for Vitro Plant tissue cultures
Strain.
2. a kind of method improving corn hybrid seed production efficiency according to claim 1, which is characterized in that the MS
The constituent of solid medium is potassium nitrate 1.9g/L, ammonium nitrate 1.6g/L, potassium dihydrogen phosphate 0.17g/L, magnesium sulfate
0.37g/L, calcium chloride 4.4g/L, sucrose 30g/L, agar 7g/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfate
22.3mg/L, zinc sulfate 8.6mg/L, sodium sulphate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L, ethylenediamine
Tetraacethyl disodium 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/
L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L, benzyladenine 0.4mg/l, 6-nonylaminopurine 0.4mg/l, agar
2%, surplus is distilled water.
3. a kind of method improving corn hybrid seed production efficiency according to claim 1, which is characterized in that the MS
The constituent of fluid nutrient medium is potassium nitrate 1.9g/L, ammonium nitrate 1.6g/L, potassium dihydrogen phosphate 0.17g/L, magnesium sulfate
0.37g/L, calcium chloride 4.4g/L, sucrose 30g/L, agar 7g/L, potassium iodide 0.83mg/L, boric acid 6.2mg/L, manganese sulfate
22.3mg/L, zinc sulfate 8.6mg/L, sodium sulphate 0.25mg/L, copper sulphate 0.025mg/L, cobalt chloride 0.025mg/L, ethylenediamine
Tetraacethyl disodium 37.3mg/L, ferrous sulfate 27.8mg/L, inositol 100mg/L, glycine 2mg/L, thiamine hydrochloride 0.1mg/
L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L, benzyladenine 0.4mg/l, 6-nonylaminopurine 0.4mg/l, surplus
For distilled water.
4. a kind of method improving corn hybrid seed production efficiency according to claim 1, which is characterized in that described mixed
Synthase solution includes the raw material of following weight percent：Cellulase solution 6%, pectinase solution 3%, calcium chloride solution 3% and sweet
Reveal alcoholic solution 12%.
5. a kind of method improving corn hybrid seed production efficiency according to claim 1, which is characterized in that described
The constituent of Hanks solution is sodium chloride 80g/L, epsom salt 1g/L, potassium chloride 4g/L, magnesium chloride hexahydrate 1g/L, chlorine
Change calcium 1.4g/L, ten sulfate dihydrate disodium hydrogen 1.5g/L, potassium dihydrogen phosphate 0.6g/L, glucose 10g/L, surplus is distilled water.
6. a kind of method improving corn hybrid seed production efficiency according to claim 1, which is characterized in that described thin
Born of the same parents' culture solution includes the raw material of following weight percent：Yeast extract 0.5%, brewer's wort 0.6%, casein hydrolysate 0.6%,
Potato extract 12%, 0.08% benzyladenine, 0.08% 6-nonylaminopurine, surplus are distilled water.
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| CN109988740A (en) * | 2019-02-20 | 2019-07-09 | 合肥师范学院 | A kind of plant protoplast culture method |
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