CN110218738A - A kind of method of antiweed coix lacryma-jobi resource acquisition - Google Patents
A kind of method of antiweed coix lacryma-jobi resource acquisition Download PDFInfo
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- CN110218738A CN110218738A CN201910599338.6A CN201910599338A CN110218738A CN 110218738 A CN110218738 A CN 110218738A CN 201910599338 A CN201910599338 A CN 201910599338A CN 110218738 A CN110218738 A CN 110218738A
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- antiweed
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Links
- 244000077995 Coix lacryma jobi Species 0.000 title claims abstract description 34
- 235000007354 Coix lacryma jobi Nutrition 0.000 title claims abstract description 30
- 238000000034 method Methods 0.000 title claims abstract description 22
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 41
- 239000005561 Glufosinate Substances 0.000 claims abstract description 20
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 15
- IAJOBQBIJHVGMQ-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid Chemical compound CP(O)(=O)CCC(N)C(O)=O IAJOBQBIJHVGMQ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 230000035755 proliferation Effects 0.000 claims abstract description 7
- 238000012216 screening Methods 0.000 claims abstract description 7
- 108091006146 Channels Proteins 0.000 claims abstract description 6
- 238000003501 co-culture Methods 0.000 claims abstract description 6
- 230000006698 induction Effects 0.000 claims abstract description 5
- 229930006000 Sucrose Natural products 0.000 claims description 22
- 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 22
- 239000005720 sucrose Substances 0.000 claims description 22
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 19
- 238000005286 illumination Methods 0.000 claims description 18
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical compound [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 claims description 18
- 108010079058 casein hydrolysate Proteins 0.000 claims description 17
- 239000001963 growth medium Substances 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 16
- 238000005406 washing Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 241000196324 Embryophyta Species 0.000 claims description 8
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 claims description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 8
- 241000894006 Bacteria Species 0.000 claims description 6
- 229930186147 Cephalosporin Natural products 0.000 claims description 6
- 229940124587 cephalosporin Drugs 0.000 claims description 6
- 150000001780 cephalosporins Chemical class 0.000 claims description 6
- 238000003752 polymerase chain reaction Methods 0.000 claims description 6
- 238000009331 sowing Methods 0.000 claims description 6
- 239000007921 spray Substances 0.000 claims description 6
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 5
- 239000008272 agar Substances 0.000 claims description 5
- 239000006160 differential media Substances 0.000 claims description 5
- 239000011159 matrix material Substances 0.000 claims description 4
- 241000589158 Agrobacterium Species 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- TWFZGCMQGLPBSX-UHFFFAOYSA-N carbendazim Chemical compound C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims description 3
- 238000006243 chemical reaction Methods 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000007791 dehumidification Methods 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 238000007598 dipping method Methods 0.000 claims description 3
- 230000000694 effects Effects 0.000 claims description 3
- 230000035784 germination Effects 0.000 claims description 3
- IAJOBQBIJHVGMQ-BYPYZUCNSA-N glufosinate-P Chemical compound CP(O)(=O)CC[C@H](N)C(O)=O IAJOBQBIJHVGMQ-BYPYZUCNSA-N 0.000 claims description 3
- 229960002523 mercuric chloride Drugs 0.000 claims description 3
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 3
- 239000004576 sand Substances 0.000 claims description 3
- 239000008223 sterile water Substances 0.000 claims description 3
- 229910052902 vermiculite Inorganic materials 0.000 claims description 3
- 235000019354 vermiculite Nutrition 0.000 claims description 3
- 239000010455 vermiculite Substances 0.000 claims description 3
- 239000002023 wood Substances 0.000 claims description 2
- XYFCBTPGUUZFHI-UHFFFAOYSA-N Phosphine Chemical compound P XYFCBTPGUUZFHI-UHFFFAOYSA-N 0.000 claims 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 claims 1
- 244000025254 Cannabis sativa Species 0.000 claims 1
- 108010076119 Caseins Proteins 0.000 claims 1
- 108010000912 Egg Proteins Proteins 0.000 claims 1
- 102000002322 Egg Proteins Human genes 0.000 claims 1
- 239000005018 casein Substances 0.000 claims 1
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims 1
- 235000021240 caseins Nutrition 0.000 claims 1
- 235000014103 egg white Nutrition 0.000 claims 1
- 210000000969 egg white Anatomy 0.000 claims 1
- 230000007062 hydrolysis Effects 0.000 claims 1
- 238000006460 hydrolysis reaction Methods 0.000 claims 1
- 229910000073 phosphorus hydride Inorganic materials 0.000 claims 1
- 230000002363 herbicidal effect Effects 0.000 abstract description 6
- 239000004009 herbicide Substances 0.000 abstract description 6
- 238000009333 weeding Methods 0.000 abstract description 6
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 208000003643 Callosities Diseases 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000209504 Poaceae Species 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 1
- -1 ammonia Amide Chemical class 0.000 description 1
- 235000013339 cereals Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000035558 fertility Effects 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 239000002689 soil Substances 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
Abstract
The invention discloses a kind of methods of antiweed coix lacryma-jobi resource acquisition, comprising the following steps: (1) sterile plumule culture；(2) callus induction；(3) callus proliferation culture；(4) anti-glufosinate bar channel genes callus co-cultures；(5) Multiplying culture of resistant calli；(6) resistant calli is screened in glufosinate culture；(7) anti-glufosinate embryo callus breaks up again；(8) acclimatization and transplants.Pass through the accurate culture screening of multistep science, antiweed coix lacryma-jobi resource can efficiently be obtained, it has stronger resistance to antiweed, the scruple of herbicide application can preferably be released, weeding process can be simplified in coix lacryma-jobi planting process, it improves labor efficiency, increases production value, there is preferable popularizing value.
Description
Technical field
The invention belongs to technical field of plant cultivation more particularly to a kind of methods of antiweed coix lacryma-jobi resource acquisition.
Background technique
Coix lacryma-jobi field weed is many kinds of, they adaptable, and fertility is vigorous, fights for sunlight, moisture, fertilizer with coix lacryma-jobi
Material and space, negligence of management can bring Severe Reduction, and removing weeds is the important cultivation technique link in coix lacryma-jobi production.Herbicide
Component part of the weeding as modern agricultural systemof production is measure most reliable, most economical in the weeding technique of coix lacryma-jobi field, to section
It saves labour, raise labour productivity and soil protection structure is all significant.Herbicide is also right while eliminating weeds
Coix lacryma-jobi itself has harmful effect, and which limits the applications of herbicide.The application scale of coix lacryma-jobi is compared with standing grain such as rice and kernel corns simultaneously
Undergraduate course crop grass family is small, and the work of antiweed the Study on Resources is done less.Coix lacryma-jobi production is main in the remote regions, and field is hoed up weeds
It relies primarily on chemistry before broadcasting to hoe up weeds and combined with manpower weeding to complete, efficiency is lower.Therefore antiweed coix lacryma-jobi resource can be compared with
The good scruple for releasing herbicide application realizes that coix lacryma-jobi field is efficiently hoed up weeds.
Summary of the invention
The object of the invention is to remedy the disadvantages of known techniques, provides a kind of side of antiweed coix lacryma-jobi resource acquisition
Method.
In order to achieve the above purpose, the present invention the following technical schemes are provided:
A kind of method of antiweed coix lacryma-jobi resource acquisition, comprising the following steps:
(1) sterile plumule culture: removing the coix seed of involucre through 75% alcohol and impregnate 2min, and aseptic water washing 3 times, sterile water
Paper bed culture, 0.1% mercuric chloride sterilizes 8min after germination, and aseptic water washing 5-8 times cuts plumule, is connected to the sterile training of MS culture medium
It supports, 21-23 DEG C of temperature, intensity of illumination 2000lx；
(2) callus induction: after bud is up to 3cm, oblique section, switching is in culture medium, PH5.8, and 21-23 DEG C of temperature, illumination
13h, 1200lx, it is former culture medium subculture 2-3 times rear out；
(3) callus proliferation culture: after subculture 2-3 times, access culture medium, PH5.8,21-23 DEG C of temperature, illumination 13h,
1500lx, again squamous subculture 2-3 times；
(4) anti-glufosinate bar channel genes callus co-cultures: by the Agrobacterium bacterium solution culture containing bar genophore
Night is diluted OD value to 0.6-0.8, is infected the embryo callus 15min of coix lacryma-jobi, during which not with bacterium solution diluted medium
Stop shaking, blot bacterium solution extra on callus with filter paper later, access dark culture 2d on culture medium, the embryo after culture is cured
Sterile washing 2 times of injured tissue containing 500mg/L cephalosporin, with filter paper suck dry moisture；
(5) Multiplying culture of resistant calli: access culture medium, PH5.8,21-23 DEG C of temperature, illumination 13h, 1500lx, then
Secondary squamous subculture 2-3 times；
(6) resistant calli is screened in glufosinate culture: the callus switching resistance Selective agar medium after proliferation, PH5.8, temperature
21-23 DEG C, illumination 13h, 1500lx of degree, screening and culturing is screened 3 times, every 15d subculture 1 time, is grown after 3 wheel selections healthy and vigorous anti-
Property callus, is numbered；
(7) anti-glufosinate embryo callus breaks up again: resistant calli switching differential medium is broken up after number
Culture, while the DNA of resistant calli system is extracted, polymerase chain reaction (PCR) detection is carried out, conversion ratio, embryo are counted
The green point that callus occurs further differentiates adventitious bud, grows to 2-3cm to adventitious bud, transfers in root media (1/
2N6) on, 2% sucrose, PH5.8,21-23 DEG C of temperature, illumination 13h, 2000lx carry out culture of rootage；
(8) acclimatization and transplants: seedling grows to 6-7cm, selects root system to induce preferable seedling corkage hardening 5-7d, transplants after carbendazol root dipping
In matrix, 1/8N6Culture solution sprays, and by day dehumidification hardening, moves back into greenhouse within 2 weeks, then moves to crop field after surviving and normally plant
Training management, sowing, next year sowing spray glufosinate and examine changing effect, continue to employ applicable resource.
Preferably, callus induction used medium in the step 2 are as follows: N6+ 2.0mg/L 2,4-D+500mg/L
Proline+300mg/L caseinhydrolysate+1.0mg/L AgNO3+ 30g/L sucrose.
Preferably, callus proliferation culture used medium in the step 3 are as follows: N6+ 1.0mg/L 2,4-D+
500mg/L proline+300mg/L caseinhydrolysate+0.5mg/L AgNO3+ 30g/L sucrose.
Preferably, anti-glufosinate bar channel genes callus co-cultures used medium in the step 4 are as follows: N6+
500mg/L proline+300mg/L caseinhydrolysate+30g/L sucrose+20mg/L acetosyringone.
Preferably, in the step 5 resistant calli Multiplying culture used medium are as follows: N6+ 1.0mg/L 2,
4-D+500mg/L proline+300mg/L caseinhydrolysate+500mg/L glutamine+30g/L sucrose.
Preferably, resistance Selective agar medium used in glufosinate culture screening resistant calli in the step 6 are as follows: N6
+ 1.0mg/L 2.4-D+500mg/L proline+300mg/L caseinhydrolysate+500mg/L glutamine+30g/L sucrose+
500mg/L cephalosporin+15mg/L glufosinate.
Preferably, anti-glufosinate embryo callus breaks up differential medium used again in the step 7 are as follows: N6+
0.5mg/L NAA+3mg/L 6-BA+500g/L proline+300mg/L caseinhydrolysate+500mg/L glutamine+30g/L
Sucrose.
Preferably, the matrix of the step 8 is made of the 1:1:2 mixing by volume of wood sawdust, vermiculite, river sand.
The invention has the advantages that
The present invention can efficiently obtain antiweed coix lacryma-jobi resource, fight weeding by the accurate culture screening of multistep science
Agent has stronger resistance, can preferably release the scruple of herbicide application, can simplify weeding in coix lacryma-jobi planting process
Journey is improved labor efficiency, and production value is increased, and has preferable popularizing value.
Specific embodiment
Below in conjunction with specific example, technical scheme is described further:
A kind of method of antiweed coix lacryma-jobi resource acquisition, comprising the following steps:
1, sterile plumule culture
Remove involucre coix seed through 75% alcohol impregnate 2min, aseptic water washing 3 times, sterile water paper bed culture.After germination
0.1% mercuric chloride sterilizes 8min, aseptic water washing 5~8 times, cuts plumule, is connected to MS culture medium sterile culture, temperature (22 ± 1)
DEG C, intensity of illumination 2000lx.
2, callus induction
After bud is up to 3cm, oblique section is transferred in culture medium (N6+ 2.0mg/L2,4-D+500mg/L proline+300mg/L
Caseinhydrolysate+1.0mg/L AgNO3+ 30g/L sucrose), PH5.8, temperature (22 ± 1) DEG C, illumination 13h, 1200lx are cured out
Former culture medium subculture 2~3 times afterwards.
3, callus proliferation culture
After subculture 2~3 times, culture medium (N is accessed6+ 1.0mg/L2,4-D+500mg/L proline+300mg/L caseinhydrolysate
+ 0.5mg/L AgNO3+ 30g/L sucrose), PH5.8, temperature (22 ± 1) DEG C, illumination 13h, 1500lx, squamous subculture 2 again
~3 times.
4, anti-glufosinate bar channel genes callus co-cultures
By the Agrobacterium bacterium solution overnight incubation containing bar genophore, with bacterium solution diluted medium (N6+ 500mg/L proline+
300mg/L caseinhydrolysate+30g/L sucrose+20mg/L acetosyringone) OD value is diluted to 0.6~0.8, infect coix lacryma-jobi
Embryo callus 15min, during which do not stop to shake, blot bacterium solution extra on callus with filter paper later, access culture
Base (N6+ 500mg/L proline+300mg/L caseinhydrolysate+30g/L sucrose+20mg/L acetosyringone) on dark culture 2d,
Sterile washing 2 times of embryo callus containing 500mg/L cephalosporin after culture, with filter paper suck dry moisture.
5, the Multiplying culture of resistant calli
Access culture medium (N6+ 1.0mg/L2,4-D+500mg/L proline+300mg/L caseinhydrolysate+500mg/L paddy ammonia
Amide+30g/L sucrose), PH5.8, temperature (22 ± 1) DEG C, illumination 13h, 1500lx, squamous subculture 2~3 times again.
6, resistant calli is screened in glufosinate culture
Callus switching resistance Selective agar medium (N after proliferation6+ 1.0mg/L2.4-D+500mg/L proline+300mg/L
Caseinhydrolysate+500mg/L glutamine+30g/L sucrose+500mg/L cephalosporin+15mg/L glufosinate), PH5.8, temperature
(22 ± 1) DEG C, illumination 13h, 1500lx, screening and culturing.Screening 3 times.Every 15d subculture 1 time.Healthy and vigorous resist is grown after 3 wheel selections
Property callus, is numbered.
7, anti-glufosinate embryo callus breaks up again
Resistant calli switching differential medium (N after number6+ 0.5mg/LNAA+3mg/L6-BA+500g/L proline+
300mg/L caseinhydrolysate+500mg/L glutamine+30g/L sucrose) differentiation culture is carried out, while extracting kanamycin-resistant callus tissue group
The DNA for being is knitted, polymerase chain reaction (PCR) detection is carried out, counts conversion ratio.The green point that embryo callus occurs is further
Adventitious bud is differentiated, 2~3cm is grown to adventitious bud, transfers in root media (1/2N6) on, 2% sucrose, PH5.8, temperature
(22 ± 1) DEG C, illumination 13h, 2000lx carry out culture of rootage.
8, acclimatization and transplants
Seedling grows to 6,7cm, selects root system to induce preferable seedling corkage 5~7d of hardening, transplanting is in matrix (sawmilling after carbendazol root dipping
Bits, vermiculite, river sand volume ratio 1:1:2), 1/8N6Culture solution sprays, and by day dehumidification hardening, moves back into greenhouse within 2 weeks, after surviving
Then move to the normal cultivation management in crop field, sowing.Next year sowing sprays glufosinate and examines changing effect, continues to employ applicable resource.
Claims (8)
1. a kind of method of antiweed coix lacryma-jobi resource acquisition, which comprises the following steps:
(1) sterile plumule culture: removing the coix seed of involucre through 75% alcohol and impregnate 2min, and aseptic water washing 3 times, sterile water
Paper bed culture, 0.1% mercuric chloride sterilizes 8min after germination, and aseptic water washing 5-8 times cuts plumule, is connected to the sterile training of MS culture medium
It supports, 21-23 DEG C of temperature, intensity of illumination 2000lx；
(2) callus induction: after bud is up to 3cm, oblique section, switching is in culture medium, PH5.8, and 21-23 DEG C of temperature, illumination
13h, 1200lx, it is former culture medium subculture 2-3 times rear out；
(3) callus proliferation culture: after subculture 2-3 times, access culture medium, PH5.8,21-23 DEG C of temperature, illumination 13h,
1500lx, again squamous subculture 2-3 times；
(4) anti-glufosinate bar channel genes callus co-cultures: by the Agrobacterium bacterium solution culture containing bar genophore
Night is diluted OD value to 0.6-0.8, is infected the embryo callus 15min of coix lacryma-jobi, during which not with bacterium solution diluted medium
Stop shaking, blot bacterium solution extra on callus with filter paper later, access dark culture 2d on culture medium, the embryo after culture is cured
Sterile washing 2 times of injured tissue containing 500mg/L cephalosporin, with filter paper suck dry moisture；
(5) Multiplying culture of resistant calli: access culture medium, PH5.8,21-23 DEG C of temperature, illumination 13h, 1500lx, then
Secondary squamous subculture 2-3 times；
(6) resistant calli is screened in glufosinate culture: the callus switching resistance Selective agar medium after proliferation, PH5.8, temperature
21-23 DEG C, illumination 13h, 1500lx of degree, screening and culturing is screened 3 times, every 15d subculture 1 time, is grown after 3 wheel selections healthy and vigorous anti-
Property callus, is numbered；
(7) anti-glufosinate embryo callus breaks up again: resistant calli switching differential medium is broken up after number
Culture, while the DNA of resistant calli system is extracted, polymerase chain reaction (PCR) detection is carried out, conversion ratio, embryo are counted
The green point that callus occurs further differentiates adventitious bud, grows to 2-3cm to adventitious bud, transfers in root media (1/
2N6) on, 2% sucrose, PH5.8,21-23 DEG C of temperature, illumination 13h, 2000lx carry out culture of rootage；
(8) acclimatization and transplants: seedling grows to 6-7cm, selects root system to induce preferable seedling corkage hardening 5-7d, transplants after carbendazol root dipping
In matrix, 1/8N6Culture solution sprays, and by day dehumidification hardening, moves back into greenhouse within 2 weeks, then moves to crop field after surviving and normally plant
Training management, sowing, next year sowing spray glufosinate and examine changing effect, continue to employ applicable resource.
2. the method for antiweed coix lacryma-jobi resource acquisition according to claim 1, which is characterized in that in the step 2 more
Injured tissue induces used medium are as follows: N6+ 2.0mg/L 2,4-D+500mg/L proline+300mg/L caseinhydrolysate+
1.0mg/L AgNO3+ 30g/L sucrose.
3. the method for antiweed coix lacryma-jobi resource acquisition according to claim 1, which is characterized in that in the step 3 more
Injured tissue Multiplying culture used medium are as follows: N6+ 1.0mg/L 2,4-D+500mg/L proline+300mg/L caseinhydrolysate
+ 0.5mg/L AgNO3+ 30g/L sucrose.
4. the method for antiweed coix lacryma-jobi resource acquisition according to claim 1, which is characterized in that resist in the step 4
Glufosinate bar channel genes callus co-cultures used medium are as follows: N6+ 500mg/L proline+300mg/L hydrolyzes junket egg
White+30g/L sucrose+20mg/L acetosyringone.
5. the method for antiweed coix lacryma-jobi resource acquisition according to claim 1, which is characterized in that resist in the step 5
The Multiplying culture used medium of property callus are as follows: N6+ 1.0mg/L 2,4-D+500mg/L proline+300mg/L hydrolysis
Casein+500mg/L glutamine+30g/L sucrose.
6. the method for antiweed coix lacryma-jobi resource acquisition according to claim 1, which is characterized in that step 6 medium-height grass
Resistance Selective agar medium used in resistant calli is screened in fourth phosphine culture are as follows: N6+ 1.0mg/L 2.4-D+500mg/L proline
+ 300mg/L caseinhydrolysate+500mg/L glutamine+30g/L sucrose+500mg/L cephalosporin+15mg/L glufosinate.
7. the method for antiweed coix lacryma-jobi resource acquisition according to claim 1, which is characterized in that resist in the step 7
Glufosinate embryo callus breaks up differential medium used again are as follows: N6+0.5mg/L NAA+3mg/L 6-BA+500g/L
Proline+300mg/L caseinhydrolysate+500mg/L glutamine+30g/L sucrose.
8. the method for antiweed coix lacryma-jobi resource acquisition according to claim 1, which is characterized in that the base of the step 8
Matter is made of the 1:1:2 mixing by volume of wood sawdust, vermiculite, river sand.
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| CN110199878A (en) * | 2019-07-04 | 2019-09-06 | 安徽省农业科学院棉花研究所 | A kind of abductive approach of the embryo callus of coix lacryma-jobi |
| CN110656129A (en) * | 2019-11-05 | 2020-01-07 | 贵州大学 | Genetic transformation method of agrobacterium-mediated coix lacryma-jobi |
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