CN107760712A - A kind of method of the rapid induction hairy root in rape and identification transformation efficiency - Google Patents
A kind of method of the rapid induction hairy root in rape and identification transformation efficiency Download PDFInfo
- Publication number
- CN107760712A CN107760712A CN201711342945.1A CN201711342945A CN107760712A CN 107760712 A CN107760712 A CN 107760712A CN 201711342945 A CN201711342945 A CN 201711342945A CN 107760712 A CN107760712 A CN 107760712A
- Authority
- CN
- China
- Prior art keywords
- hairy root
- rape
- liquid
- transformation efficiency
- infect
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of method of rapid induction hairy root in rape and identification transformation efficiency, including：(1) infected by sterile mustard type rape explant with wild type and containing gus reporter gene A4 Agrobacteriums；(2) explant after infecting is co-cultured；(3) explant after co-culturing, which moves to, carries out Fiber differentiation in inducing culture；(4) subculture and amplification cultivation will be carried out by hairy root caused by induction.(5) and to there is the hairy root of gus gene to carry out GUS dyeing transformation efficiency is probed into.The present invention invades the explant preculture, the time infected, co-cultured and the thalline that influence mustard type rape hairy root induction efficiency the induced efficiency that dye liquor concentration significantly improves hairy root.And the hairy root to turning gus gene carries out dyeing identification, transformation efficiency is probed into by color, rape hairy root is realized and fast and efficiently induces.The inventive method is applied to the induction of a variety of rape variety hairy roots, have the advantages that convenient material drawing, induced velocity soon, inheritance stability and cost-effective.
Description
Technical field
The present invention relates to agriculture Applied Biotechnology field, more particularly to a kind of to rape hairy root rapid induction and right
The genetic transformation of regulation and control economical character provides method later.
Background technology
Research shows that rape is in the method for carrying out gene function checking, typically using arabidopsis or callus induction in life, life
Long period is long and transformation efficiency is shorter than the relatively low but growth of hair root cycle, is easy to cultivate, and is easy to conversion, conversion ratio
The advantages that high.At present, yellow seed rape breeding is the focus of current rapeseed breeding.Clone and gene function checking to seed coat gene
With regard to particularly important, thus it is very necessary to rape hairy root induction and genetic transformation efficiency, can be by being transferred in hairy root
Gus gene, and GUS dyeing is carried out to it to identify conversion effect, to being of great significance using rape hairy root.
Leaf Stems from Brassica juncea is induced with agrobacterium rhizogenes, and wherein agrobacterium rhizogenes strain is key substance.A4 is root of hair agriculture bar
Bacterium is a kind of, and existing document report can induce plant rooting.We are in research A4 agrobacterium rhizogenes to rape induction time and dense
Degree, incubation time etc. and constructs the A4 agrobacterium rhizogenes with gus reporter gene of conversion, and to the hairy root of conversion
GUS dyeing is carried out.By experimental studies have found that wild type and conversion A4 agrobacterium rhizogenes when OD values are 0.8, can be to oil
Dish efficiently induces hairy root, and can dye blueness with gus gene.
The content of the invention
It is an object of the invention to provide a kind of method of hairy root induction directly perceived, economic, easy and transgene efficiency, with
Just it is applied to rape hairy root induction, and constructs the A4 agrobacterium rhizogenes with gus reporter gene of conversion, and to conversion
Hairy root carried out that GUS dyeing is fixed to be used for probing into transformation efficiency.
To achieve the above object, the technical solution adopted by the present invention：One kind rapid induction hairy root and expansion in rape is numerous
Method, it is characterized in that the aseptic seedling of 2 weeks to 3 weeks, be separately added into infect liquid A and infect and 5-10 minutes, Ran Houjin infected in liquid B
Row is co-cultured, and after 24-48 hours, is transferred to screening and culturing and is cultivated, and fast growing is cut from explant after one week
Hairy root, is transferred on subculture medium, is transferred to after two weeks on fluid nutrient medium and is enlarged culture, after one month
To a large amount of hairy roots, and with B infect the hairy root that liquid induces and carry out GUS dyeing and probe into transformation efficiency.
Described infecting liquid A and infect liquid B is respectively wild type Agrobacterium rhizogenesA4 and is transferred to the A4 hairs of gus reporter gene
Root Agrobacterium.
Described infects liquid A by 50mlOD values (600）0.8 Agrobacterium A4 is centrifuged to be resuspended with isometric MS liquid
Obtain.
Described infects in liquid B by 50mlOD values (600）0.8 Agrobacterium A4 carries out centrifugation use etc. with gus reporter gene
Volume MS liquid is resuspended to obtain.
Comprise the following steps that：
(1) sterile mustard type rape explant with wild type and is infected liquid containing gus reporter gene A4 Agrobacteriums and infected；
(2) explant after infecting is co-cultured；
(3) cotyledon and hypocotyl after co-culturing move to and Fiber differentiation are carried out in inducing culture；
(4) squamous subculture and amplification cultivation will be carried out by rape hairy root caused by induction.
(5) and to there is the hairy root of gus reporter gene to carry out GUS dyeing transformation efficiency is probed into.
The present invention starts with from approach such as the biology of A4 agrobacterium rhizogenes and transformation efficiency, have extensively studied hairy root induction
Condition and transformation efficiency, it is found that the OD values of strain and position are key factors during rape hairy root induction.Invent herein
With chemical dyeing method, GUS dyeing is carried out, so as to simplicity, intuitively probes into out transformation efficiency；Simple to operate, analyze speed is fast,
It is cheap using reagent price.
Embodiment
A kind of rapid induction hairy root in rape method numerous with expansion, its step are:Contain GUS report bases with wild and conversion
Agrobacterium rhizogenes (Agrobacterium rhizogenes) A4 of cause is in purple leaf mustard and Sichuan Huang seed rape hypocotyls and son
Leaf is induced.By dark treatment, screening and culturing, squamous subculture, 25 °C are added in MS culture mediums, light culture 48h.Light culture
After end, shifting the MS solid mediums containing 100mg/ml cephalos, hypocotyl and cotyledon were differentiated to form later in two weeks to three weeks
Hairy root.Cut off 2-3CM hairies root and carry out screening and culturing on 100mg/ml cephalo MS solid mediums are added.Sieved after two weeks
Select the fast rape hairy root of the speed of growth and carry out squamous subculture on the MS solid mediums of the cephalo containing 50mg/ml.Subculture is trained
After supporting 2-3 times, the rape hairy root of detoxification is obtained.2-3CM hairies root is cut off to be enlarged on MS fluid nutrient mediums are added
Culture.Fast, the rape hairy root more than lateral root branch so as to grow.By experiment above, we are drawn when OD values are
When 0.8, hypocotyl inductivity is apparently higher than blade.And simultaneously indifference is for purple leaf mustard and Sichuan Huang seed hypocotyl inductivity
80%-90%.And purple leaf mustard seed leaf inductivity is 25% Sichuan Huang seed cotyledon 47%.Illustrate Sichuan Huang seed cotyledon apparently higher than purple leaf mustard
The inductivity of cotyledon.And gus reporter gene is transferred to, carries out GUS dyeing, conversion effect is probed into by shade and position
Rate.
Embodiment 1:
1. infect liquid A：By 50mlOD values (600）0.8 Agrobacterium A4 is centrifuged to be resuspended with isometric MS liquid and mixed
It is even standby.
2. infect liquid B：50mlOD values (600）0.8 Agrobacterium A4 carries out the isometric MS of centrifugation with gus reporter gene
Liquid be resuspended to obtain mix it is standby.
3. take the black seed mustard type rape purple leaf mustard of two bottle of 2 week old.
4. one group is added to 50 milliliters and infects liquid A, kind of a skin is completely soaked in a liquid, infect 5 minutes.Another set adds
Enter to 50 milliliters and infect in liquid B.
5 add in MS culture mediums 25 °C, light culture 48h.
After 6. light culture terminates, embryo under shifting the MS solid mediums containing 100mg/ml cephalos after two weeks to three weeks
Axle and cotyledon are differentiated to form hairy root.
7. cut off 2-3CM hairies root carries out screening and culturing on 100mg/ml cephalo MS solid mediums are added.Two weeks with
The fast rape hairy root of the speed of growth is filtered out afterwards carries out squamous subculture on the MS solid mediums of the cephalo containing 50mg/ml.After
Be commissioned to train it is foster 2-3 times after, obtain the rape hairy root of detoxification.
8. cut off 2-3CM hairies root is enlarged culture on MS fluid nutrient mediums are added.Fast, the side so as to grow
Rape hairy root more than root branch.
9. couple B, which infects the black seed mustard type rape purple leaf mustard that liquid infects and cultivated, which carries out GUS dyeing, can catch color, send out
Now become blue.
Embodiment 2:
1. infect liquid A：By 50mlOD values (600）0.8 Agrobacterium A4 is centrifuged to be resuspended with isometric MS liquid and mixed
It is even standby.
2. infect liquid B：50mlOD values (600）0.8 Agrobacterium A4 carries out the isometric MS of centrifugation with gus reporter gene
Liquid be resuspended to obtain mix it is standby.
3. take the Sichuan Huang seed of two bottle of 2 week old.
4. one group is added to 50 milliliters and infects liquid A, kind of a skin is completely soaked in a liquid, infect 5 minutes.Another set adds
Enter to 50 milliliters and infect in liquid B.
5 add in MS culture mediums 25 °C, light culture 48h.
After 6. light culture terminates, embryo under shifting the MS solid mediums containing 100mg/ml cephalos after two weeks to three weeks
Axle and cotyledon are differentiated to form hairy root.
7. cut off 2-3CM hairies root carries out screening and culturing on 100mg/ml cephalo MS solid mediums are added.Two weeks with
The fast rape hairy root of the speed of growth is filtered out afterwards carries out squamous subculture on the MS solid mediums of the cephalo containing 50mg/ml.After
Be commissioned to train it is foster 2-3 times after, obtain the rape hairy root of detoxification.
8. cut off 2-3CM hairies root is enlarged culture on MS fluid nutrient mediums are added.Fast, the side so as to grow
Rape hairy root more than root branch.
9. couple B, which infects the Sichuan Huang seed that liquid infects and cultivated, which carries out GUS dyeing, can catch color, find to become blue.
Embodiment 3:
1. infect liquid A：By 50mlOD values (600）0.8 Agrobacterium A4 is centrifuged to be resuspended with isometric MS liquid and mixed
It is even standby.
2. infect liquid B：50mlOD values (600）0.8 Agrobacterium A4 carries out the isometric MS of centrifugation with gus reporter gene
Liquid be resuspended to obtain mix it is standby.
3. take two bottle of 2 week old NILA.
4. one group is added to 50 milliliters and infects liquid A, kind of a skin is completely soaked in a liquid, infect 5 minutes.Another set adds
Enter to 50 milliliters and infect in liquid B.
5 add in MS culture mediums 25 °C, light culture 48h.
After 6. light culture terminates, embryo under shifting the MS solid mediums containing 100mg/ml cephalos after two weeks to three weeks
Axle and cotyledon are differentiated to form hairy root.
7. cut off 2-3CM hairies root carries out screening and culturing on 100mg/ml cephalo MS solid mediums are added.Two weeks with
The fast rape hairy root of the speed of growth is filtered out afterwards carries out squamous subculture on the MS solid mediums of the cephalo containing 50mg/ml.After
Be commissioned to train it is foster 2-3 times after, obtain the rape hairy root of detoxification.
8. cut off 2-3CM hairies root is enlarged culture on MS fluid nutrient mediums are added.Fast, the side so as to grow
Rape hairy root more than root branch.
9. couple B, which infects the NILA that liquid infects and cultivated, which carries out GUS dyeing, can catch color, find to become blue.
Claims (4)
1. a kind of method of rapid induction hairy root in rape and identification transformation efficiency, it is characterized in that 2 weeks to 3 weeks sterile
Seedling, it is added separately to infect liquid A and infect to infect 5-10 minutes in liquid B, is then co-cultured, after 24-48 hours, be transferred to
Screening and culturing is cultivated, and is cut the hairy root of fast growing after one week from explant, is transferred on subculture medium, two
Zhou Yihou, which is transferred on fluid nutrient medium, is enlarged culture, obtains a large amount of hairy roots after one month, and infect liquid with B and lure
Derived hairy root carries out GUS dyeing and probes into transformation efficiency.
2. the method for the rapid induction hairy root according to claim 1 in rape and identification transformation efficiency is fast in rape
The method of speed induction hairy root and identification transformation efficiency method of rapid induction hairy root and identification transformation efficiency in rape, its
Be characterized in, it is described infect liquid A and infect in liquid B be respectively wild type Agrobacterium rhizogenesA4 and be transferred to the A4 of gus reporter gene
Agrobacterium rhizogenes.
3. the method for the rapid induction hairy root according to claim 1 in rape and identification transformation efficiency, it is characterized in that,
Described infects liquid A by 50mlOD values (600）0.8 Agrobacterium A4 is centrifuged to be resuspended to obtain with isometric MS liquid.
4. the method for the rapid induction hairy root according to claim 1 in rape and identification transformation efficiency, it is characterized in that,
Described infects in liquid B by 50mlOD values (600）0.8 Agrobacterium A4 carries out the isometric MS liquid of centrifugation with gus reporter gene
Body is resuspended to obtain.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711342945.1A CN107760712A (en) | 2017-12-14 | 2017-12-14 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711342945.1A CN107760712A (en) | 2017-12-14 | 2017-12-14 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107760712A true CN107760712A (en) | 2018-03-06 |
Family
ID=61278027
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711342945.1A Pending CN107760712A (en) | 2017-12-14 | 2017-12-14 | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107760712A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913716A (en) * | 2018-08-01 | 2018-11-30 | 成都大学 | A kind of rapid induction quinoa hairy method |
| CN109576302A (en) * | 2018-11-14 | 2019-04-05 | 甘肃农业大学 | A kind of method of hairy of rapid induction Ethiopia rape |
| CN109628482A (en) * | 2019-01-14 | 2019-04-16 | 甘肃农业大学 | A kind of take root hairy root induction and expanding propagation method rapidly and efficiently |
| CN111183899A (en) * | 2020-01-19 | 2020-05-22 | 贵州大学 | Method for rapidly inducing safflower hairy roots |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6051409A (en) * | 1995-09-25 | 2000-04-18 | Novartis Finance Corporation | Method for achieving integration of exogenous DNA delivered by non-biological means to plant cells |
| WO2002098911A2 (en) * | 2001-06-07 | 2002-12-12 | Syngenta Limited | Insecticidal proteins and synergistic combinations thereof |
| KR100756889B1 (en) * | 2006-09-29 | 2007-09-07 | 한국생명공학연구원 | Marker free transgenic potato hairy roots using agrobacterium rhizogenes |
| CN102827861A (en) * | 2012-08-20 | 2012-12-19 | 湖南科技大学 | Method for increasing proanthocyanidin content in escherichia coli by cotransformation of brassica juncea gene ANS and brassica carinata BAN |
| WO2015124640A1 (en) * | 2014-02-18 | 2015-08-27 | Knud Jepsen A/S | Method for obtaining plants with intermediate height |
| CN106497971A (en) * | 2017-01-13 | 2017-03-15 | 扬州大学 | A kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently |
| CN107099547A (en) * | 2017-05-08 | 2017-08-29 | 北京交通大学 | Turn the method that gshB genes improve black nightshade hairy root Cd enrichments |
| CN107119070A (en) * | 2017-05-18 | 2017-09-01 | 中国医学科学院药用植物研究所 | It is a kind of to improve Bupleurum Chinese hairy root induction efficiency and the method and its application of genetic transformation efficiency |
| CN109576302A (en) * | 2018-11-14 | 2019-04-05 | 甘肃农业大学 | A kind of method of hairy of rapid induction Ethiopia rape |
-
2017
- 2017-12-14 CN CN201711342945.1A patent/CN107760712A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6051409A (en) * | 1995-09-25 | 2000-04-18 | Novartis Finance Corporation | Method for achieving integration of exogenous DNA delivered by non-biological means to plant cells |
| WO2002098911A2 (en) * | 2001-06-07 | 2002-12-12 | Syngenta Limited | Insecticidal proteins and synergistic combinations thereof |
| KR100756889B1 (en) * | 2006-09-29 | 2007-09-07 | 한국생명공학연구원 | Marker free transgenic potato hairy roots using agrobacterium rhizogenes |
| CN102827861A (en) * | 2012-08-20 | 2012-12-19 | 湖南科技大学 | Method for increasing proanthocyanidin content in escherichia coli by cotransformation of brassica juncea gene ANS and brassica carinata BAN |
| WO2015124640A1 (en) * | 2014-02-18 | 2015-08-27 | Knud Jepsen A/S | Method for obtaining plants with intermediate height |
| CN106497971A (en) * | 2017-01-13 | 2017-03-15 | 扬州大学 | A kind of cabbage type rape hairy root induction and cultural method rapidly and efficiently |
| CN107099547A (en) * | 2017-05-08 | 2017-08-29 | 北京交通大学 | Turn the method that gshB genes improve black nightshade hairy root Cd enrichments |
| CN107119070A (en) * | 2017-05-18 | 2017-09-01 | 中国医学科学院药用植物研究所 | It is a kind of to improve Bupleurum Chinese hairy root induction efficiency and the method and its application of genetic transformation efficiency |
| CN109576302A (en) * | 2018-11-14 | 2019-04-05 | 甘肃农业大学 | A kind of method of hairy of rapid induction Ethiopia rape |
Non-Patent Citations (11)
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913716A (en) * | 2018-08-01 | 2018-11-30 | 成都大学 | A kind of rapid induction quinoa hairy method |
| CN109576302A (en) * | 2018-11-14 | 2019-04-05 | 甘肃农业大学 | A kind of method of hairy of rapid induction Ethiopia rape |
| CN109628482A (en) * | 2019-01-14 | 2019-04-16 | 甘肃农业大学 | A kind of take root hairy root induction and expanding propagation method rapidly and efficiently |
| CN111183899A (en) * | 2020-01-19 | 2020-05-22 | 贵州大学 | Method for rapidly inducing safflower hairy roots |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107760712A (en) | A kind of method of the rapid induction hairy root in rape and identification transformation efficiency | |
| CN106497971B (en) | A kind of hairy root induction of cabbage type rape rapidly and efficiently and cultural method | |
| CN103966258B (en) | A kind of agriculture bacillus mediated cabbage type rape genetic transforming method | |
| CN108300735A (en) | A kind of Lilium tenuifolium efficient genetic trasformation system based on somatic embryo occur | |
| CN105960458A (en) | Method for obtaining transformed cells of plant | |
| CN103468739A (en) | Method for agrobacterium tumefaciens-mediated genetic transformation of dried radix rehmanniae | |
| CN106754925A (en) | A kind of abscisic acid rapid induction promoter and its application | |
| CN109576302A (en) | A kind of method of hairy of rapid induction Ethiopia rape | |
| CN117187294B (en) | Application of BnaC5.ACBP4 gene in improving flooding resistance of plants | |
| CN107858372A (en) | A kind of agriculture bacillus mediated cotton transient transformation methods | |
| CN103320464A (en) | Method for generating hairy roots by efficiently inducing trifoliate orange by agrobacterium rhizogenes and application of method | |
| CN104878039A (en) | Mulberry transgenic method based on agrobacterium tumefacien mediated hybernacle injection | |
| CN102337295A (en) | Agrobacterium-mediated melon seedling apex transformation method | |
| CN105063086A (en) | Molecular breeding method fast obtaining large number of transgenosis sedum lineare new species | |
| CN105695504B (en) | A kind of method that mediated by agriculture bacillus infects the Mongolian willow of conversion | |
| CN117070557A (en) | Rapid and efficient agrobacterium-mediated iris seed genetic transformation method | |
| CN104894162A (en) | Method for transforming exogenous genes into duckweeds and expressing duckweeds | |
| CN104087611A (en) | Agrobacterium tumefaciens-mediated genetic transformation method for Jatropha curcas | |
| CN101974561A (en) | Genetic transformation method for soybean cotyledonary nodes by agrobacterium | |
| CN105177039A (en) | Method for transferring exogenous genes into betula platyphylla Suks. seedlings for transient expression | |
| CN108118068A (en) | A kind of efficiently quick chrysanthemum transgenic method | |
| CN108165572A (en) | A kind of agriculture bacillus mediated needle thorn growing point transformed wheat method | |
| CN103966220B (en) | The root-specific promoter of synthetic and application thereof | |
| CN103233039A (en) | Agrobacterium tumefaciens-mediated rice seedling gene transformation method | |
| CN103146748A (en) | Agrobacterium mediated rose bud transgenosis infecting method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180306 |
|
| RJ01 | Rejection of invention patent application after publication |