CN109576302A - A kind of method of hairy of rapid induction Ethiopia rape - Google Patents
A kind of method of hairy of rapid induction Ethiopia rape Download PDFInfo
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- CN109576302A CN109576302A CN201811585408.4A CN201811585408A CN109576302A CN 109576302 A CN109576302 A CN 109576302A CN 201811585408 A CN201811585408 A CN 201811585408A CN 109576302 A CN109576302 A CN 109576302A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- 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
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Abstract
The method of hairy of a kind of rapid induction Ethiopia rape, comprising steps of (1) infects sterile Leaf Stems from Brassica juncea wild type and Agrobacterium K599 containing gus gene；(2) explant after infecting is put on nonreactive MS solid medium and is co-cultured under dark condition；(3) explant after co-culturing, which moves in induced medium, to be cultivated；(4) the hairy progress subculture of rape and amplification cultivation generated induction.(5) transformation efficiency is probed into the hairy progress GUS dyeing containing gus gene.The present invention is probed into the hairy root induction explant preculture of Ethiopia's rape, the time infected, co-cultured and infected liquid concentration is influenced, and identification is dyed to hairy progress GUS of transgenosis, by identifying that color probes into transformation efficiency, the quickly induction of hairy of rape is realized.The method of the present invention is suitable for a variety of rape varieties hairy induction, and materials convenience, strong applicability, induced velocity are fast.
Description
Technical field
The present invention relates to agriculture Applied Biotechnology fields, are especially to provide the side of hairy rapid induction of a kind of pair of rape
Method.
Background technique
Rape generally uses Leaf Stems from Brassica juncea callus induction regenerating system in the method for carrying out gene function verifying, should
The technology experiment period is longer and transformation efficiency is relatively low.But rape hairy has growth cycle short, convenient for culture, is easy to
The advantages that conversion, high conversion rate.Currently, yellow seed rape breeding is the hot spot of contemporary rapeseed breeding, to rape mesosperm gene
Clone and gene function verifying are just particularly important.Therefore the hairy root induction of rape and genetic transformation efficiency are very necessary, simultaneously
It can use and be transferred to hairy middle gus gene, GUS dyeing identification transformation efficiency is carried out to it, this has to using rape hairy
Highly important meaning.
Leaf Stems from Brassica juncea induces hairy with agrobacterium rhizogenes, and wherein agrobacterium rhizogenes strain is key substance.K599 is
Agrobacterium rhizogenes is a kind of, and existing document report can induce plant rooting.Agrobacterium rhizogenes induction plant generates hairy production
Life is a complicated process, and the Ri plasmid contained therewith has close relationship, mainly by Ri plasmidThe area Ori、VirArea
And the coefficient result of the area T-DNA three parts.
Summary of the invention
The purpose of the present invention is to provide a kind of hairy methods of rapid induction Ethiopia rape.
The technical solution adopted by the present invention: a kind of method of hairy of rapid induction Ethiopia rape is by 2-3 weeks
Ethiopia's rape aseptic seedling, infected 5-10 minutes, then co-cultured, 24- with infected liquid A and infected liquid B respectively
It after 48 hours, is transferred in screening and culturing medium and is cultivated, hairy for cutting fast growing after one week from explant turns
It moves on on subculture medium, is transferred on fluid nutrient medium and expands culture after two weeks, a large amount of hairs are obtained after one month
Shape root；The infected liquid A is wild type Agrobacterium rhyzogenesK599, and infected liquid B is the agrobacterium rhizogenes for being transferred to gus reporter gene
K599。
The infected liquid A is centrifuged by (600) 0.8 Agrobacterium rhyzogenesK599 of OD value carries out weight with isometric MS liquid
It is outstanding to obtain.
The infected liquid B carries out centrifugation use etc. with gus reporter gene by (600) 0.8 Agrobacterium rhyzogenesK599 of OD value
Volume MS liquid is resuspended to obtain.
The invention has the advantages that:
The present invention uses OD value to induce for 0.8 wild type Agrobacterium rhyzogenesK599 Ethiopia's rape, can be efficiently
It induces hairy a large amount of；Also, gus reporter gene is had in hairy induced, can be dyed to blue.So as to use
Chemical dyeing method is easy, intuitively probes into out the Induction Transformation efficiency of Ethiopia's rape.Inventive method is easy to operate, analysis speed
Degree is fast, cheap using reagent price.
Specific embodiment
Embodiment 1:
A kind of method of hairy of rapid induction Ethiopia rape, comprising steps of
(1) infected liquid A: being centrifuged by (600) 0.8 Agrobacterium rhyzogenesK599 of 50mlOD value, carries out weight with isometric MS liquid
It is outstanding to obtain, it mixes spare.
(2) (600) 0.8 Agrobacterium rhyzogenesK599 of infected liquid B:50mlOD value is centrifuged with gus reporter gene, with etc.
Volume MS liquid is resuspended to obtain, and is mixed spare.
(3) the Hei Zi Ethiopia rape for taking two bottle of 2 week old, cuts section.
(4) one groups are added to 50 milliliters of infected liquid A, are completely soaked explant in a liquid, infect 5-10 minutes.In addition
One group is added in 50 milliliters of infected liquid B.
(5) it is put on MS solid medium, 25 DEG C of dark culture 48h.
(6) it co-cultures after terminating, is transferred in the MS solid medium containing 100mg/ml cephalo, cultivate two weeks to three
Zhou Yihou, hypocotyl and cotyledon are differentiated to form hairy.
(7) 2-3cm hairy is cut off, carries out screening and culturing on 100mg/ml cephalo MS solid medium is added.Two weeks
Hairy of the fast rape of the speed of growth is filtered out later, carries out squamous subculture on the MS solid medium of the cephalo containing 50mg/ml.
After squamous subculture 2-3 times, rape hairy of detoxification is obtained.
(8) 2-3cm shape root is cut off, is expanded culture on MS fluid nutrient medium is added, obtains growth fastly, lateral root branch
More rapes hairy.
(9) Ethiopia's rape for B infected liquid being infected and being cultivated carries out GUS dyeing, and discovery becomes blue.
Embodiment 2:
A kind of method of hairy of rapid induction Ethiopia rape, comprising steps of
(1) it infected liquid A: is centrifuged by (600) 0.8 Agrobacterium K599 of 50mlOD value, be resuspended with isometric MS liquid
It arrives, mixes spare.
(2) (600) 0.8 Agrobacterium K599 of infected liquid B:50mlOD value is centrifuged with gus reporter gene, with isometric
MS liquid is resuspended to obtain, and is mixed spare.
(3) two bottle of 2 week old Huang Zi Ethiopia rape is taken, section is cut.
(4) one groups are added to 50 milliliters of infected liquid A, are completely soaked explant in a liquid, infect 5-10 minutes.In addition
One group is added in 50 milliliters of infected liquid B.
(5) it is put on MS solid medium, 25 DEG C of dark culture 48h.
(6) it co-cultures after terminating, is transferred on the MS solid medium containing 100mg/ml cephalo, cultivate two weeks to three
Zhou Yihou, hypocotyl and cotyledon are differentiated to form hairy.
(7) 2-3cm hairy is cut off, carries out screening and culturing on 100mg/ml cephalo MS solid medium is added.Two weeks
Hairy of the fast rape of the speed of growth is filtered out later, carries out squamous subculture on the MS solid medium of the cephalo containing 50mg/ml.
After squamous subculture 2-3 times, rape hairy of detoxification is obtained.
(8) 2-3cm hairy is cut off, is expanded culture on MS fluid nutrient medium is added, fast, lateral root branch must be grown
More rapes hairy.
(9) the Huang Zi Ethiopia rape for B infected liquid being infected and being cultivated carries out GUS dyeing, and discovery becomes blue.
Claims (3)
1. the method for hairy of a kind of rapid induction Ethiopia rape, it is characterized in that: by Ethiopia's oil of 2-3 week old
Dish aseptic seedling is infected 5-10 minutes with infected liquid A and infected liquid B respectively, is then co-cultured, after 24-48 hours, is transferred to
It is cultivated in screening and culturing medium, hairy for cutting fast growing after one week from explant is transferred to subculture medium
On, it is transferred in fluid nutrient medium and expands culture after two weeks, obtained after one month hairy a large amount of；Described infects
Liquid A is wild type Agrobacterium rhyzogenesK599, and infected liquid B is the Agrobacterium rhyzogenesK599 for being transferred to gus reporter gene.
2. the method for hairy of rapid induction Ethiopia according to claim 1 rape, characterized in that described invades
Dye liquor A is centrifuged by (600) 0.8 Agrobacterium rhyzogenesK599 of OD value, is resuspended to obtain with isometric MS liquid.
3. the method for hairy of rapid induction Ethiopia according to claim 1 rape, characterized in that described invades
Dye liquor B is centrifuged by (600) 0.8 Agrobacterium rhyzogenesK599 of OD value with gus reporter gene, carries out weight with isometric MS liquid
It is outstanding to obtain.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
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| CN201811349215 | 2018-11-14 | ||
| CN2018113492159 | 2018-11-14 |
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| CN109576302A true CN109576302A (en) | 2019-04-05 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760712A (en) * | 2017-12-14 | 2018-03-06 | 湖南科技大学 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
| CN111183899A (en) * | 2020-01-19 | 2020-05-22 | 贵州大学 | Method for rapidly inducing safflower hairy roots |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760712A (en) * | 2017-12-14 | 2018-03-06 | 湖南科技大学 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
-
2018
- 2018-12-24 CN CN201811585408.4A patent/CN109576302A/en not_active Withdrawn
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760712A (en) * | 2017-12-14 | 2018-03-06 | 湖南科技大学 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
Non-Patent Citations (3)
| Title |
|---|
| JOSEFINE NYMARK HEGELUND 等人: "Increasing genetic variability in oilseed rape (Brassica napus)–Genotypesand phenotypes of oilseed rape transformed by wild type Agrobacterium rhizogenes", 《PLANT SCIENCE》 * |
| 刘后利 编著: "《油菜遗传育种学》", 31 August 2000, 中国农业大学出版社 * |
| 李隆 等: "芥菜型油菜毛状根诱导体系构建及TTG1基因功能初步研究", 《作物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107760712A (en) * | 2017-12-14 | 2018-03-06 | 湖南科技大学 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
| CN111183899A (en) * | 2020-01-19 | 2020-05-22 | 贵州大学 | Method for rapidly inducing safflower hairy roots |
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