CN105969793A - Method for breeding rice - Google Patents
Method for breeding rice Download PDFInfo
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- CN105969793A CN105969793A CN201610303747.3A CN201610303747A CN105969793A CN 105969793 A CN105969793 A CN 105969793A CN 201610303747 A CN201610303747 A CN 201610303747A CN 105969793 A CN105969793 A CN 105969793A
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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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
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Abstract
The invention discloses a method for breeding rice. The method comprises the following steps: enzyme digestion is carried out for wild Zizania latifolia DNA, an encoding gene containing OsMADS57 protein and an OsABC1-2 gene with restriction enzyme, cold ethyl alcohol is used at -40 DEG C overnight, centrifugation is carried out, the genes are connected a pBin438 plasmid in order to construct an exogenous plasmid DNA; the exogenous plasmid DNA is resuspended in a plasmid DNA protective agent, in order to obtain mixed liquor; after expression and purification, an Agrobacterium toxic protein VirD1, VirD2 or VirE2 solution is obtained, the mixed liquor and liposome are added in order for mixing, a buffer is added, the solution is placed for 2 hours at 4 DEG C and taken out, a pollen tube path way method is used for introducing a target gene into rice, and mutation materials are selected for carrying out breeding. Plant height, grain number per spike, and grain weight are increased, tillering is properly reduced, coordination between individual and colony is emphasized, lodging resistance is improved, and colony yield is increased to the largest extent.
Description
Technical field
The present invention relates to rice breeding field, concrete a kind of rice breeding method.
Background technology
Oryza sativa L. is one of most important cereal crops, has supported more than half population of the whole world.Along with population
Growth and growth in the living standard, no matter rice is all proposed more and more higher from yield or quality by people
Requirement.It is estimated that, to 2025, population in the world was by the population more than 8,000,000,000, with rice as staple food
Quantity will increase to 3,500,000,000.By the most conservative estimation, cause the increase of rice demand owing to population increases
At least up to 300,000,000 tons.The raising of rice yield, relies on the potentiality expanding cultivated area the most limited.Owing to being subject to
Water resource and the restriction of cultivated area, add the propelling of economic development and urbanization, many water producing rice country
Rice cultivated area is actually existing to be declined.To this end, will depend primarily upon the raising of yield per unit area from now on
Improve rice yield further.
Grain shape traits includes that grain length, grain are wide, grain is thick, grain is heavy and length-width ratio etc., is the important structure of rice yield
Become the factor, and plant plant type is one of key factor affecting crop yield, especially for rice crop.
Know at present the tillering number of Oryza sativa L., tillering angle, plant height, Leaf angle, the size of Oryza sativa L. tassel and
How many Oryza sativa L. small ear branches has together decided on plant type of rice, and the shape of the tiller number of Oryza sativa L. and tassel is certainly
Determine the most important factor of rice yield.For rice tillering is grown, tillering stage is vegetative growth of rice plants
Main period, including the formation from the differentiation of tiller bud to children's fringe.General rice varieties, growing environment bar
Part (temperature, moisture, nutriture) and planting density thereof also can affect the growth of tiller.Growth cycle is long
Typically short than the growth cycle rice tillering of rice varieties fewer.Rice tillering include one-level tiller and
Two grades of tillers.According to the situation that tiller is solid, tiller is divided into again effective tillering and ineffective tillering two kinds.Effectively
Tiller number be to determine one of rice yield key factor.
Summary of the invention
For solving the problems referred to above, the invention provides a kind of rice breeding method, by suitably increase plant height,
Increase grain number per spike, increase grain weight, suitably reduce tiller, focus on the individual coordination with colony, add anti-fall
Ability, makes population yield reach maximum.
For achieving the above object, the technical scheme that the present invention takes is:
A kind of rice breeding method, comprises the steps；
S1, operate on ice, by dense for Agrobacterium toxalbumin VirD1, VirD2, the VirE2 after expression and purification
Degree is separately adjusted to angularly 100 μ g/ μ l；
S2, with digestion with restriction enzyme wild wild rice DNA, encoding gene containing OsMADS57 albumen
After the DNA of OsABC1-2 gene, with at cold ethanol-40 DEG C overnight, centrifugal, be connected to pBin438
Exogenous plasmid dna is built on plasmid；
S3, gained exogenous plasmid dna is resuspended in plasmid DNA protective agent, obtains exogenous plasmid dna
Concentration is the mixed liquor of 1.2 μ g/ μ L；
S4, one or more taken in step S1 gained solution totally 2.1 μ l, be sequentially added into the step containing 6-8 μ l
The mixed liquor of rapid S3 gained, after the mixing of 6-9 μ l liposome, adds in the buffer of 30-40 μ l, puts for 4 DEG C
After putting 2 hours, take out, by pollen tube passage method by genes of interest Introduced into Rice, select mutative material
Carry out breeding.
Wherein, described buffer comprises 10-18mmol/L trishydroxymethylaminomethane-hydrochloric acid,
0.5-0.7mmol/L diaminoethanes tetraacethyl, 10-15mmol/L sodium oxide, 0.035 μ g μ l-1-0.075
μg·μl-1Ethylmethane sulfonate, 10-20mmol/LpH value are the ethylenediaminetetraacetic acid of 8.0.
Wherein, plasmid DNA protective agent include aluminum hydroxide sol protective agent, calcium phosphate protective agent and
Tween20 protective agent.
Wherein, described aluminum hydroxide sol protective agent, calcium phosphate protective agent and Tween20 is protectant presses
Volume ratio is 3: 2: 1.
The method have the advantages that
By suitably increasing plant height, increasing grain number per spike, increase grain weight, suitably reduce tiller, focus on individual with
The coordination of colony, adds capacity for the resistance to lodging, makes population yield reach maximum.
Detailed description of the invention
In order to make objects and advantages of the present invention clearer, below in conjunction with embodiment, the present invention is carried out
Further describe.Should be appreciated that specific embodiment described herein only in order to explain the present invention,
It is not intended to limit the present invention.
In following example, the plasmid DNA protective agent used includes aluminum hydroxide sol protective agent, phosphorus
Acid calcium protective agent and Tween20 protective agent are 3: 2: 1 mixing gained by volume.
Embodiment 1
S1, operate on ice, by dense for Agrobacterium toxalbumin VirD1, VirD2, the VirE2 after expression and purification
Degree is separately adjusted to angularly 100 μ g/ μ l；
S2, with digestion with restriction enzyme wild wild rice DNA, encoding gene containing OsMADS57 albumen
After the DNA of OsABC1-2 gene, with at cold ethanol-40 DEG C overnight, centrifugal, be connected to pBin438
Exogenous plasmid dna is built on plasmid；
S3, gained exogenous plasmid dna is resuspended in plasmid DNA protective agent, obtains exogenous plasmid dna
Concentration is the mixed liquor of 1.2 μ g/ μ L；
S4, one or more taken in step S1 gained solution totally 2.1 μ l, be sequentially added into the step containing 6 μ l
The mixed liquor of S3 gained, after 6 μ l liposome mixing, adds in the buffer of 30 μ l, places 2 hours for 4 DEG C
After, take out, by pollen tube passage method by genes of interest Introduced into Rice, select mutative material to carry out breeding,
Wherein, the buffer used comprises 10mmol/L trishydroxymethylaminomethane-hydrochloric acid, 0.5mmol/L bis-
Aminoethane tetraacethyl, 10mmol/L sodium oxide, 0.035 μ g μ l-1Ethylmethane sulfonate,
10-20mmol/LpH value is the ethylenediaminetetraacetic acid of 8.0.
Embodiment 2
S1, operate on ice, by dense for Agrobacterium toxalbumin VirD1, VirD2, the VirE2 after expression and purification
Degree is separately adjusted to angularly 100 μ g/ μ l；
S2, with digestion with restriction enzyme wild wild rice DNA, encoding gene containing OsMADS57 albumen
After the DNA of OsABC1-2 gene, with at cold ethanol-40 DEG C overnight, centrifugal, be connected to pBin438
Exogenous plasmid dna is built on plasmid；
S3, gained exogenous plasmid dna is resuspended in plasmid DNA protective agent, obtains exogenous plasmid dna
Concentration is the mixed liquor of 1.2 μ g/ μ L；
S4, one or more taken in step S1 gained solution totally 2.1 μ l, be sequentially added into the step containing 8 μ l
The mixed liquor of S3 gained, after 9 μ l liposome mixing, adds in the buffer of 40 μ l, places 2 hours for 4 DEG C
After, take out, by pollen tube passage method by genes of interest Introduced into Rice, select mutative material to carry out breeding,
Wherein, the buffer used comprises 18mmol/L trishydroxymethylaminomethane-hydrochloric acid, 0.7mmol/L bis-
Aminoethane tetraacethyl, 15mmol/L sodium oxide, 0.075 μ g μ l-1Ethylmethane sulfonate,
20mmol/LpH value is the ethylenediaminetetraacetic acid of 8.0.
Embodiment 3
S1, operate on ice, by dense for Agrobacterium toxalbumin VirD1, VirD2, the VirE2 after expression and purification
Degree is separately adjusted to angularly 100 μ g/ μ l；
S2, with digestion with restriction enzyme wild wild rice DNA, encoding gene containing OsMADS57 albumen
After the DNA of OsABC1-2 gene, with at cold ethanol-40 DEG C overnight, centrifugal, be connected to pBin438
Exogenous plasmid dna is built on plasmid；
S3, gained exogenous plasmid dna is resuspended in plasmid DNA protective agent, obtains exogenous plasmid dna
Concentration is the mixed liquor of 1.2 μ g/ μ L；
S4, one or more taken in step S1 gained solution totally 2.1 μ l, be sequentially added into the step containing 7 μ l
The mixed liquor of S3 gained, after 7.5 μ l liposome mixing, adds in the buffer of 35 μ l, and 4 DEG C of placements 2 are little
Shi Hou, takes out, and by pollen tube passage method by genes of interest Introduced into Rice, selects mutative material to educate
Kind, wherein, the buffer used comprises 14mmol/L trishydroxymethylaminomethane-hydrochloric acid, 0.6mmol/L
Diaminoethanes tetraacethyl, 12.5mmol/L sodium oxide, 0.05 μ g μ l-1Ethylmethane sulfonate,
15mmol/LpH value is the ethylenediaminetetraacetic acid of 8.0.
The above is only the preferred embodiment of the present invention, it is noted that common for the art
For technical staff, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications,
These improvements and modifications also should be regarded as protection scope of the present invention.
Claims (4)
1. a rice breeding method, it is characterised in that comprise the steps:
S1, operate on ice, by dense for Agrobacterium toxalbumin VirD1, VirD2, the VirE2 after expression and purification
Degree is separately adjusted to angularly 100 μ g/ μ l；
S2, with digestion with restriction enzyme wild wild rice DNA, encoding gene containing OsMADS57 albumen
After the DNA of OsABC1-2 gene, with at cold ethanol-40 DEG C overnight, centrifugal, be connected to pBin438
Exogenous plasmid dna is built on plasmid；
S3, gained exogenous plasmid dna is resuspended in plasmid DNA protective agent, obtains exogenous plasmid dna
Concentration is the mixed liquor of 1.2 μ g/ μ L；
S4, one or more taken in step S1 gained solution totally 2.1 μ l, be sequentially added into the step containing 6-8 μ l
The mixed liquor of rapid S3 gained, after the mixing of 6-9 μ l liposome, adds in the buffer of 30-40 μ l, puts for 4 DEG C
After putting 2 hours, take out, by pollen tube passage method by genes of interest Introduced into Rice, select mutative material
Carry out breeding.
A kind of rice breeding method the most according to claim 1, it is characterised in that described buffer
Comprise 10-18mmol/L trishydroxymethylaminomethane-hydrochloric acid, 0.5-0.7mmol/L diaminoethanes tetrem
Acid, 10-15mmol/L sodium chloride, 0.035 μ g μ l-1-0.075μg·μl-1Ethylmethane sulfonate,
10-20mmol/LpH value is the ethylenediaminetetraacetic acid of 8.0.
A kind of rice breeding method the most according to claim 1, it is characterised in that plasmid DNA
Protective agent includes aluminum hydroxide sol protective agent, calcium phosphate protective agent and Tween20 protective agent.
A kind of rice breeding method the most according to claim 3, it is characterised in that described hydroxide
Alumina gel protective agent, calcium phosphate protective agent and the protectant volume ratio of Tween20 are 3: 2: 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610303747.3A CN105969793A (en) | 2016-05-10 | 2016-05-10 | Method for breeding rice |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610303747.3A CN105969793A (en) | 2016-05-10 | 2016-05-10 | Method for breeding rice |
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| CN105969793A true CN105969793A (en) | 2016-09-28 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458114A (en) * | 2020-12-25 | 2021-03-09 | 苏州农业职业技术学院 | Method for rapidly identifying functions of genes related to fruit color development of pepper fruits |
Citations (9)
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|---|---|---|---|---|
| CN1197482A (en) * | 1995-09-25 | 1998-10-28 | 诺瓦提斯公司 | Improved integration of exogenous DNA delivered to eukaryoltic cells |
| CN101092633A (en) * | 2007-03-01 | 2007-12-26 | 石河子大学 | Method for raising conversion efficiency of pollen tube chnnael method by using toxoprotein gene of Agrobacterium |
| CN102675441A (en) * | 2012-06-05 | 2012-09-19 | 中国科学院植物研究所 | Application of OsMADS57 protein or coding gene thereof to inhibiting tillering of rice |
| CN102690341A (en) * | 2012-06-05 | 2012-09-26 | 中国科学院植物研究所 | Plant tillering related protein and coding gene thereof |
| CN103451228A (en) * | 2013-09-17 | 2013-12-18 | 淮阴师范学院 | Method for regulating size and grain weight of rice seeds |
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2016
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| CN102675441A (en) * | 2012-06-05 | 2012-09-19 | 中国科学院植物研究所 | Application of OsMADS57 protein or coding gene thereof to inhibiting tillering of rice |
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| CN105164257A (en) * | 2013-03-29 | 2015-12-16 | 日本烟草产业株式会社 | Agrobacterium bacterium to be used in plant transformation method |
| CN103451228A (en) * | 2013-09-17 | 2013-12-18 | 淮阴师范学院 | Method for regulating size and grain weight of rice seeds |
| CN104830909A (en) * | 2015-06-08 | 2015-08-12 | 田杰 | Rice breeding method |
| CN105002209A (en) * | 2015-08-15 | 2015-10-28 | 石河子大学 | Method for improving transgenosis efficiency of cotton |
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| Title |
|---|
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112458114A (en) * | 2020-12-25 | 2021-03-09 | 苏州农业职业技术学院 | Method for rapidly identifying functions of genes related to fruit color development of pepper fruits |
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Application publication date: 20160928 |