CN112458114A - Method for rapidly identifying functions of genes related to fruit color development of pepper fruits - Google Patents
Method for rapidly identifying functions of genes related to fruit color development of pepper fruits Download PDFInfo
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- CN112458114A CN112458114A CN202011561108.XA CN202011561108A CN112458114A CN 112458114 A CN112458114 A CN 112458114A CN 202011561108 A CN202011561108 A CN 202011561108A CN 112458114 A CN112458114 A CN 112458114A
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- 235000013399 edible fruits Nutrition 0.000 title claims abstract description 114
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 53
- 235000002566 Capsicum Nutrition 0.000 title claims abstract description 48
- 239000006002 Pepper Substances 0.000 title claims abstract description 45
- 235000016761 Piper aduncum Nutrition 0.000 title claims abstract description 45
- 235000017804 Piper guineense Nutrition 0.000 title claims abstract description 45
- 235000008184 Piper nigrum Nutrition 0.000 title claims abstract description 45
- 238000000034 method Methods 0.000 title claims abstract description 24
- 244000203593 Piper nigrum Species 0.000 title 1
- 241000722363 Piper Species 0.000 claims abstract description 50
- 239000013612 plasmid Substances 0.000 claims abstract description 32
- 108091008146 restriction endonucleases Proteins 0.000 claims abstract description 13
- 230000008859 change Effects 0.000 claims abstract description 12
- 239000003223 protective agent Substances 0.000 claims description 15
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 10
- 239000011259 mixed solution Substances 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 240000008574 Capsicum frutescens Species 0.000 claims description 5
- 235000002568 Capsicum frutescens Nutrition 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 102000004190 Enzymes Human genes 0.000 claims description 5
- PLUBXMRUUVWRLT-UHFFFAOYSA-N Ethyl methanesulfonate Chemical compound CCOS(C)(=O)=O PLUBXMRUUVWRLT-UHFFFAOYSA-N 0.000 claims description 5
- IPPKPBRKIKWIPN-UHFFFAOYSA-N acetic acid ethane-1,1-diamine Chemical compound CC(N)N.CC(O)=O.CC(O)=O.CC(O)=O.CC(O)=O IPPKPBRKIKWIPN-UHFFFAOYSA-N 0.000 claims description 5
- 239000001506 calcium phosphate Substances 0.000 claims description 5
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 5
- 235000011010 calcium phosphates Nutrition 0.000 claims description 5
- 239000002502 liposome Substances 0.000 claims description 5
- 102000004169 proteins and genes Human genes 0.000 claims description 5
- 239000000377 silicon dioxide Substances 0.000 claims description 5
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- 239000001728 capsicum frutescens Substances 0.000 claims 1
- 108700001094 Plant Genes Proteins 0.000 abstract description 3
- 238000003976 plant breeding Methods 0.000 abstract description 2
- 241000208293 Capsicum Species 0.000 description 3
- 239000001390 capsicum minimum Substances 0.000 description 3
- 235000021466 carotenoid Nutrition 0.000 description 3
- 150000001747 carotenoids Chemical class 0.000 description 3
- 241000208292 Solanaceae Species 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- 240000004160 Capsicum annuum Species 0.000 description 1
- 235000002567 Capsicum annuum Nutrition 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229930002877 anthocyanin Natural products 0.000 description 1
- 235000010208 anthocyanin Nutrition 0.000 description 1
- 239000004410 anthocyanin Substances 0.000 description 1
- 150000004636 anthocyanins Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 229930003935 flavonoid Natural products 0.000 description 1
- 150000002215 flavonoids Chemical class 0.000 description 1
- 235000017173 flavonoids Nutrition 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N hydrochloric acid Substances Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
-
- 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
- C12N15/8209—Selection, visualisation of transformants, reporter constructs, e.g. antibiotic resistance markers
- C12N15/821—Non-antibiotic resistance markers, e.g. morphogenetic, metabolic markers
Abstract
The invention relates to the field of plant breeding, in particular to a method for rapidly identifying functions of genes related to development of fruit color of pepper fruits, which comprises the following steps: digesting the DNA related to fruit color development by using restriction enzyme, and connecting to pBin438 plasmid to construct exogenous plasmid DNA; in the full-bloom fruit setting stage of the pepper, a target gene is introduced into a pepper plant by a pollen tube channel method; and setting a normal control group, observing and recording the phenotypic change of the fruit color of the pepper fruits, and judging the functions of the corresponding fruit color development related genes based on the phenotypic change of the fruit color of the pepper fruits. The invention can realize the rapid identification of the functions of the genes related to the development of the fruit color of the pepper fruit, and is a method for rapidly and effectively identifying the functions of the plant genes.
Description
Technical Field
The invention relates to the field of plant breeding, in particular to a method for rapidly identifying functions of genes related to development of fruit color of pepper fruits.
Background
The Capsicum (Capsicum annuum L.) belongs to the Capsicum (Capsicum) of the Solanaceae family (Solanaceae), and a herbaceous plant is grown for one or more years, is native to tropical and subtropical regions in the south and central America, is commonly cultivated all over the world at present, and is also one of the vegetable crops with the largest planting area in China.
The pepper fruits contain a plurality of pigments such as chlorophyll, carotenoid, anthocyanin and flavonoid, wherein the color relationship between the carotenoid and the pepper fruits is close, and the color diversity of the pepper fruits is caused by the difference of the relative content of each component of the carotenoid. The formation of the fruit color of the pepper is related to a plurality of genes related to pigment synthesis, however, in the face of a large number of genes related to the fruit color, how to identify the gene function becomes a problem to be solved urgently.
Disclosure of Invention
In order to solve the problems, the invention provides a method for rapidly identifying the functions of genes related to the development of the fruit color of pepper fruits.
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for rapidly identifying the functions of the genes related to the fruit color development of hot pepper fruits comprises the following steps:
s1, performing ice operation, and respectively adjusting the concentrations of the expressed and purified agrobacterium-toxic proteins VirD1, VirD2 and VirE2 to 100 mug/muL;
s2, digesting the DNA related to fruit color development of the fruit by using restriction enzyme, and connecting to pBin438 plasmid to construct exogenous plasmid DNA;
s3, resuspending the obtained exogenous plasmid DNA in a plasmid DNA protective agent to obtain a mixed solution A with the concentration of the exogenous plasmid DNA being 1 mug/muL;
s4, taking 2 mu L of one or more solutions obtained in the step S1, sequentially adding 8 mu L of mixed solution A and 6 mu L of liposome, mixing, adding into 30 mu L of buffer solution, and standing for 2 hours at 4 ℃ for later use;
s5, introducing the target gene into a pepper plant by a pollen tube channel method in the full-bloom and fruit-setting period of the pepper;
s6, setting a normal control group, observing and recording the phenotypic change of the fruit color of the pepper fruits, and judging the functions of the corresponding fruit color development related genes based on the phenotypic change of the fruit color of the pepper fruits.
Further, the buffer solution contains 5-15mmol/L of tris-hydroxymethyl-aminomethane-hydrochloric acid, 0.4-0.6mmol/L of diaminoethane tetraacetic acid, 1-10mmol/L of calcium chloride, and 0.015. mu.g. mu.L-1-0.055μg·μL-1Ethyl methanesulfonate.
Further, the plasmid DNA protective agent is prepared by mixing mesoporous silica and a calcium phosphate protective agent according to a volume ratio of 10: 1, and mixing the components in a ratio of 1.
Further, the restriction enzyme is the restriction enzyme Hind iii. + ecori enzyme.
Furthermore, if the fruit color of the experimental group is obviously different from that of the control group, the detected gene is related to the fruit color development of the pepper fruit, and then the function of the fruit color gene of the pepper fruit is further judged according to the characteristics of the obvious difference; on the contrary, if the fruit color of the fruit is not obviously different from that of the control, the detected gene is irrelevant to the development of the fruit color of the pepper or is a non-functional gene.
The invention has the following beneficial effects:
can realize the rapid identification of the functions of the genes related to the development of the fruit color of the pepper fruits, and is a method for rapidly and effectively identifying the functions of the plant genes.
Detailed Description
In order that the objects and advantages of the invention will be more clearly understood, the invention is further described in detail below with reference to examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1
A method for rapidly identifying the functions of the genes related to the fruit color development of hot pepper fruits comprises the following steps:
s1, performing ice operation, and respectively adjusting the concentrations of the expressed and purified agrobacterium-toxic proteins VirD1 and VirD2 to 100 mu g/mu L;
s2, digesting the DNA related to fruit color development of the fruit by using restriction enzyme, and connecting to pBin438 plasmid to construct exogenous plasmid DNA;
s3, resuspending the obtained exogenous plasmid DNA in a plasmid DNA protective agent to obtain a mixed solution A with the concentration of the exogenous plasmid DNA being 1 mug/muL;
s4, taking 1 mu L each of the VirD1 solution and the VirD2 solution obtained in the step S1, sequentially adding 8 mu L of the mixed solution A and 6 mu L of the liposome, mixing, adding into 30 mu L of buffer solution, and standing for 2 hours at 4 ℃ for later use;
s5, introducing the target gene into a pepper plant by a pollen tube channel method in the full-bloom and fruit-setting period of the pepper;
s6, setting a normal control group, observing and recording the phenotypic change of the fruit color of the pepper fruits, and judging the functions of the corresponding fruit color development related genes based on the phenotypic change of the fruit color of the pepper fruits.
In this example, the buffer solution contained 10mmol/L Tris-HCl, 0.5mmol/L diaminoethane tetraacetic acid, 5.5mmol/L calcium chloride, 0.035. mu.g. mu.L-1Ethyl methanesulfonate.
In this embodiment, the plasmid DNA protective agent is composed of mesoporous silica and calcium phosphate protective agent in a volume ratio of 10: 1, and mixing the components in a ratio of 1.
In this example, the restriction enzyme was HindIII. + EcoRI enzyme.
In this embodiment, if the fruit color of the experimental group is significantly different from that of the control group, it indicates that the detected gene is related to the development of the fruit color of the pepper fruit, and if the fruit color of the fruit is not significantly different from that of the control group, it indicates that the detected gene is not related to the development of the fruit color of the pepper fruit, or is a non-functional gene.
Example 2
A method for rapidly identifying the functions of the genes related to the fruit color development of hot pepper fruits comprises the following steps:
s1, performing ice operation, and respectively adjusting the concentrations of the expressed and purified agrobacterium-toxic proteins VirD1 and VirE2 to 100 mu g/mu L;
s2, digesting the DNA related to fruit color development of the fruit by using restriction enzyme, and connecting to pBin438 plasmid to construct exogenous plasmid DNA;
s3, resuspending the obtained exogenous plasmid DNA in a plasmid DNA protective agent to obtain a mixed solution A with the concentration of the exogenous plasmid DNA being 1 mug/muL;
s4, taking 1 mu L each of the VirD1 solution and the VirE2 solution obtained in the step S1, sequentially adding 8 mu L of the mixed solution A and 6 mu L of the liposome, mixing, adding into 30 mu L of buffer solution, and standing for 2 hours at 4 ℃ for later use;
s5, introducing the target gene into a pepper plant by a pollen tube channel method in the full-bloom and fruit-setting period of the pepper;
s6, setting a normal control group, observing and recording the phenotypic change of the fruit color of the pepper fruits, and judging the functions of the corresponding fruit color development related genes based on the phenotypic change of the fruit color of the pepper fruits.
In this example, the buffer contained 10mmol/L of Tris-hydroxymethyl amino groupMethane-hydrochloric acid, 0.5mmol/L diaminoethane tetraacetic acid, 5.5mmol/L calcium chloride, 0.035. mu.g. mu.L-1Ethyl methanesulfonate.
In this embodiment, the plasmid DNA protective agent is composed of mesoporous silica and calcium phosphate protective agent in a volume ratio of 10: 1, and mixing the components in a ratio of 1.
In this example, the restriction enzyme was HindIII. + EcoRI enzyme.
In the embodiment, if the fruit color of the experimental group is obviously different from that of the control group, the detected gene is related to the fruit color development of the pepper fruit, and then the function of the fruit color gene of the pepper fruit is further judged according to the characteristics of the obvious difference, wherein at least 10 groups are arranged in each experimental group; on the contrary, if the fruit color of the fruit is not obviously different from that of the control, the detected gene is irrelevant to the development of the fruit color of the pepper or is a non-functional gene.
Example 3
A method for rapidly identifying the functions of the genes related to the fruit color development of hot pepper fruits comprises the following steps:
s1, performing ice operation, and respectively adjusting the concentrations of the expressed and purified agrobacterium-toxic proteins VirD1, VirD2 and VirE2 to 100 mug/muL;
s2, digesting the DNA related to fruit color development of the fruit by using restriction enzyme, and connecting to pBin438 plasmid to construct exogenous plasmid DNA;
s3, resuspending the obtained exogenous plasmid DNA in a plasmid DNA protective agent to obtain a mixed solution A with the concentration of the exogenous plasmid DNA being 1 mug/muL;
s4, taking 1 mu L of each 0.5 mu L, VirD2 solution of the VirD1 solution and the VirE2 solution obtained in the step S1, sequentially adding 8 mu L of the mixed solution A and 6 mu L of liposome, mixing, adding into 30 mu L of buffer solution, and standing for 2 hours at 4 ℃ for later use;
s5, introducing the target gene into a pepper plant by a pollen tube channel method in the full-bloom and fruit-setting period of the pepper;
s6, setting a normal control group, observing and recording the phenotypic change of the fruit color of the pepper fruits, and judging the functions of the corresponding fruit color development related genes based on the phenotypic change of the fruit color of the pepper fruits.
In this example, the buffer solution contained 10mmol/L Tris-HCl, 0.5mmol/L diaminoethane tetraacetic acid, 5.5mmol/L calcium chloride, 0.035. mu.g. mu.L-1Ethyl methanesulfonate.
In this embodiment, the plasmid DNA protective agent is composed of mesoporous silica and calcium phosphate protective agent in a volume ratio of 10: 1, and mixing the components in a ratio of 1.
In this example, the restriction enzyme was HindIII. + EcoRI enzyme.
In the embodiment, if the fruit color of the experimental group is obviously different from that of the control group, the detected gene is related to the fruit color development of the pepper fruit, and then the function of the fruit color gene of the pepper fruit is further judged according to the characteristics of the obvious difference, wherein at least 10 groups are arranged in each experimental group; on the contrary, if the fruit color of the fruit is not obviously different from that of the control, the detected gene is irrelevant to the development of the fruit color of the pepper or is a non-functional gene.
Experiments prove that the method can realize the rapid identification of the functions of the genes related to the development of the fruit color of the pepper fruits, has the accuracy of about 98 percent, and is a method for rapidly and effectively identifying the functions of the plant genes.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (5)
1. A method for rapidly identifying the functions of genes related to the fruit color development of pepper fruits is characterized by comprising the following steps:
s1, performing ice operation, and respectively adjusting the concentrations of the expressed and purified agrobacterium-toxic proteins VirD1, VirD2 and VirE2 to 100 mug/muL;
s2, digesting the DNA related to fruit color development of the fruit by using restriction enzyme, and connecting to pBin438 plasmid to construct exogenous plasmid DNA;
s3, resuspending the obtained exogenous plasmid DNA in a plasmid DNA protective agent to obtain a mixed solution A with the concentration of the exogenous plasmid DNA being 1 mug/muL;
s4, taking 2 mu L of one or more solutions obtained in the step S1, sequentially adding 8 mu L of mixed solution A and 6 mu L of liposome, mixing, adding into 30 mu L of buffer solution, and standing for 2 hours at 4 ℃ for later use;
s5, introducing the target gene into a pepper plant by a pollen tube channel method in the full-bloom and fruit-setting period of the pepper;
s6, setting a normal control group, observing and recording the phenotypic change of the fruit color of the pepper fruits, and judging the functions of the corresponding fruit color development related genes based on the phenotypic change of the fruit color of the pepper fruits.
2. The method as claimed in claim 1, wherein the buffer solution comprises tris (hydroxymethyl) aminomethane-hydrochloric acid 5-15mmol/L, diaminoethane tetraacetic acid 0.4-0.6mmol/L, calcium chloride 1-10mmol/L, and calcium chloride 0.015 μ g μ L-1-0.055μg·μL-1Ethyl methanesulfonate.
3. The method for rapidly identifying the gene function related to the development of the fruit color of the capsicum frutescens fruit according to claim 1, wherein the plasmid DNA protective agent is prepared from mesoporous silica and calcium phosphate protective agent according to the volume ratio of 10: 1, and mixing the components in a ratio of 1.
4. The method for rapidly identifying the functions of the genes related to the fruit color development of the pepper fruits as claimed in claim 1, wherein the restriction enzyme is restriction enzyme Hind III. + EcoRI enzyme.
5. The method as claimed in claim 1, wherein if the fruit color of the experimental group is significantly different from that of the control group, the gene to be detected is related to the fruit color development of the pepper fruit; on the contrary, if the fruit color of the fruit is not obviously different from that of the control, the detected gene is irrelevant to the development of the fruit color of the pepper or is a non-functional gene.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695537A (en) * | 2013-11-29 | 2014-04-02 | 西北农林科技大学 | Method for rapidly identifying functions of pepper fruit color development related genes |
| CN105002209A (en) * | 2015-08-15 | 2015-10-28 | 石河子大学 | Method for improving transgenosis efficiency of cotton |
| CN105349573A (en) * | 2015-12-05 | 2016-02-24 | 山西省原平市双惠种业有限公司 | Sorghum variety breeding and cultivating method |
| CN105969793A (en) * | 2016-05-10 | 2016-09-28 | 广西兆和种业有限公司 | Method for breeding rice |
-
2020
- 2020-12-25 CN CN202011561108.XA patent/CN112458114A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103695537A (en) * | 2013-11-29 | 2014-04-02 | 西北农林科技大学 | Method for rapidly identifying functions of pepper fruit color development related genes |
| CN105002209A (en) * | 2015-08-15 | 2015-10-28 | 石河子大学 | Method for improving transgenosis efficiency of cotton |
| CN105349573A (en) * | 2015-12-05 | 2016-02-24 | 山西省原平市双惠种业有限公司 | Sorghum variety breeding and cultivating method |
| CN105969793A (en) * | 2016-05-10 | 2016-09-28 | 广西兆和种业有限公司 | Method for breeding rice |
Non-Patent Citations (1)
| Title |
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| HWANG, H.H.等: "Agrobacterium-mediated plant transformation:biology and applications", 《ARABIDOPSIS BOOK》 * |
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Application publication date: 20210309 |