CN102217472A - Method for screening transgenic radish in field by using kanamycin leaf smearing method - Google Patents
Method for screening transgenic radish in field by using kanamycin leaf smearing method Download PDFInfo
- Publication number
- CN102217472A CN102217472A CN 201110065447 CN201110065447A CN102217472A CN 102217472 A CN102217472 A CN 102217472A CN 201110065447 CN201110065447 CN 201110065447 CN 201110065447 A CN201110065447 A CN 201110065447A CN 102217472 A CN102217472 A CN 102217472A
- Authority
- CN
- China
- Prior art keywords
- kan
- blade
- kanamycin
- plant
- screening
- 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.)
- Granted
Links
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a method for screening transgenic radish in field by using a kanamycin leaf smearing method. The experimental material is transgenic radish of neomycin phosphotransferase II (NPT II)-labeled genes, the optimal concentration of the field screening of Kan leaf smearing is 1,000mg/L, and the observation time is preferably 12 days; and verified by polymerase chain reaction (PCR) detection, the positive detection rate of Kan screened resistance seedlings reaches 93.8 percent. According to the invention, the simple and convenient method for screening a large amount of transgenic radish in field is researched and established, and has important value on breading of the transgenic radish material.
Description
Technical field
The present invention is genetically modified crops breeding field, is specifically related to a kind of method of utilizing kanamycin blade semar technique field screening transgenosis radish
Background technology
Radish (Raphanus sativus L.), the another name radish, reed Fu belongs to the Cruciferae Rhaphanus, and root is main carnivorous organ, and is nutritious, and cultivation is all arranged all over the world.
The transgenosis work of radish also is in the starting stage, and for the screening and the evaluation of converting material, not only workload is big, cost is high to use traditional molecular detecting method, and not easy to operate.Simultaneously, also will study its offspring's genetic development for the plant that obtains, these all need the screening and the evaluation work of a large amount of transgenic progeny.Therefore, the method for quick that utilizes easy marker gene to carry out transfer-gen plant more and more comes into one's own.Kanamycin (Kanamycin, write a Chinese character in simplified form Kan) be to be widely used in the selectable marker gene that plant genetic transforms at present, whether kanamycin is used for the tissue culture stage more transformed plant is carried out Preliminary screening, utilize method testing goal genes such as PCR, Southern blot hybridization to be incorporated in the Plant Genome again.
The method of kanamycin field rapid screening transfer-gen plant, existing report in transgene cotton, soybean, tomato, paddy rice, wheat, but do not appearing in the newspapers as yet aspect the screening of transgenosis radish plant.The effect that the kanamycin field is selected fast is because of floristics, and corresponding smear concentration, observation time and alter a great deal.For this reason; this test is a material with the transgenosis radish that has NPT II marker gene; utilizing variable concentrations kanamycin blade to carry out blade smears; to determine its suitable concentration and observation time smeared; and the reliability of kanamycin smearing method carried out the PCR checking, its result for the scale that realizes radish field transfer-gen plant select fast, to accelerate breeding process significant.
Summary of the invention
The invention provides a kind of method of utilizing kanamycin blade semar technique field screening transgenosis radish, determined that the Kan blade smears optium concentration, the observing time of field screening; Through the PCR detection validation, the positive detection rate of Kan screening resistance seedling reaches 93.8%.A kind of short-cut method that the transgenosis radish is screened in a large number in the field has been set up in research, has important value for the seed selection of transgenosis radish material.
The technical solution used in the present invention is: the method for a kind of kanamycin blade semar technique field screening transgenosis radish is characterized in that carrying out according to the following steps:
(1) material is prepared
Test material is that the transgenosis radish of NPT II marker gene (is the commercially available prod, buys in Crop Institute, Hunan Academy of Agricultural Sciences; It is gene constructed in plasmid pCMIaN also can to obtain Bt by conventional technique for gene engineering, and selected marker is a NPT II gene, and agrobacterium strains transforms)
(2) the blade kanamycin is smeared determining of suitable concentration and observation time
Kanamycin is mixed with 0mg/L respectively with the distilled water of additional 0.2% polysorbas20,250mg/L, and 500mg/L, 750mg/L, 1000mg/L, 1500mg/L, 2000mg/L, 8 processing of 2500mg/L concentration, every processing repeats 4 times, adopts randomised block design; Kan is applied in writing brush on the intermediate blade of two true leaves that launch fully of plant, moistening getting final product.Respectively in the response situation of smearing the 3rd, 6,9,12, the 15 day observed and recorded blade in back, to determine the situation of turnip leaves, kanamycin screening concentration and observing time.
(3) transgenosis T
0Smear screening test for the radish kanamycin
The offspring T of 60 strain systems of transfer-gen plant
0For planting seed in the bed of growing seedlings, every strain is 40～150 seeds, when plant height reaches 20～25cm, carrying out blade with the Kan of 1000mg/L concentration according to the method described above smears, 12 days " Invest, Then Investigate " results, with into treatment sites blade face color normally is Kan resistant transgenic plant, and the plant that the chlorisis spot appears in the blade face is the responsive negative plant of Kan.
(4) Kan smears result's PCR detection validation
For detecting the reliability that Kan smears the result, 68 transformed plants and 1 negative contrast of unconverted plant of 4 grades of random choose, method extracting DNA in a small amount, and utilize primer the P1:5 '-AGCCGTTTGTTAGTGCCT-3 ' of BtCry1Ia gene; P2:5 '-ACTTGGATGCGGATGGAC-3 ' carries out pcr amplification, and amplified band is 769bp, and amplified production detects with 1.0% agarose gel electrophoresis.
The optium concentration that described Kan blade is smeared the field screening is 1000mg/L, and be advisable with 12 days observing time.
The beneficial effect that the present invention obtains:
Through the PCR detection validation, the positive detection rate of Kan screening resistance seedling reaches 93.8%.A kind of short-cut method that the transgenosis radish is screened in a large number in the field has been set up in research, has important value for the seed selection of transgenosis radish material.
Embodiment
Below the present invention is done a detailed description:
Embodiment:
1 materials and methods
1.1 test material
1.1.1 vegetable material
(1) material is prepared
Test material is that the transgenosis radish of NPT II marker gene (is the commercially available prod, buys in Crop Institute, Hunan Academy of Agricultural Sciences; It is gene constructed in plasmid pCMIaN also can to obtain Bt by conventional technique for gene engineering, and selected marker is a NPT II gene, and agrobacterium strains transforms)
1.2 the blade kanamycin is smeared determining of suitable concentration and observation time
Kanamycin is mixed with 0mg/L respectively with the distilled water of additional 0.2% polysorbas20,250mg/L, and 500mg/L, 750mg/L, 1000mg/L, 1500mg/L, 2000mg/L, 8 processing of 2500mg/L concentration, every processing repeats 4 times, adopts randomised block design; Kan is applied in writing brush on the intermediate blade of two true leaves that launch fully of plant, moistening getting final product.Respectively in the response situation of smearing the 3rd, 6,9,12, the 15 day observed and recorded blade in back, to determine the situation of turnip leaves, kanamycin screening concentration and observing time.
1.3 transgenosis T0 smears screening test for the radish kanamycin
The offspring T0 of 60 strains of transfer-gen plant system for planting seed in the bed of growing seedlings, every strain is 40～150 seeds, when plant height reaches 20～25cm, carrying out blade with the Kan of 1000mg/L concentration according to the method described above smears, 12 days " Invest, Then Investigate " results, with into treatment sites blade face color normally is Kan resistant transgenic plant, and the plant that the chlorisis spot appears in the blade face is the responsive negative plant of Kan.
1.4 Kan smears result's PCR detection validation
For detecting the reliability that Kan smears the result, 68 transformed plants of 4 grades of random choose and 1 unconverted plant (negative contrast), method extracting DNA in a small amount, and utilize primer (P1:5 '-AGCCGTTTGTTAGTGCCT-3 '; P2:5 '-ACTTGGATGCGGATGGAC-3 ') carry out pcr amplification, amplified band is 769bp, and amplified production detects with 1.0% agarose gel electrophoresis.
2 results and analysis
The color changeable effect after 2.1 turnip leaves is handled Kan
With unconverted Bt gene radish varieties XD2-A serves as for the examination material, has observed Kan smear rear blade change in color situation.According to the result of field observation, the end reaction degree after turnip leaves is handled Kan is divided into 4 grades.1 grade: smearing position leaf look normal; 2 grades: blade is smeared the leaf look slight flavescence (normal Ye Selve is variant with periphery, but not obvious) at position, and the leaf look still is green; 3 grades: smear the position chlorosis, the leaf look yellow by green commentaries on classics, different obviously with the normal leaf aberration of periphery; 4 grades: the serious chlorosis of into treatment sites, the leaf look changes white or brown.
In above 4 grades, the blade reaction is in 1,2 grade processing, fails to eliminate fully the limit of turnip leaves self to kalamycin resistance, in the detection of transfer-gen plant, can increase the quantity of false positive individual plant, thus the effect that influence is selected.Blade has been eliminated the negative effect that blade self is handled kanamycin basically 3 grades processing, can be used as the standard of kanamycin initial screening.
2.2 screening concentration and the observing time of Kan
When screening concentration when being 250mg/L,, mainly present 1 order reaction along with the prolongation leaf color in reaction time no change almost; When screening concentration was 500mg/L, blade was not obvious to the reaction prolongation variation in time of Kan, mainly shows as 2 order reactions, therefore if screen with above-mentioned concentration, can increase the quantity of false positive individual plant, the effect that influence is selected; When Kan concentration 〉=1000mg/L, the rapid flavescence of leaf color, in addition withered.Therefore, comparatively desirable as the critical concentration of screening with 1000mg/L, under this screening concentration, leaf chlorosis is obvious, and along with the prolongation order of reaction of screening time obviously raises, is convenient to distinguish and observe.The no significant change of prolongation blade reaction along with the time, other concentration are handled except that 250mg/L and 500mg/L processing, and order of reaction continued to increase in 1～12 day, tended towards stability substantially after 12 days, so be advisable with 12 days observing time.
2.3 T0 smears the result for transgenosis radish Kan
Carry out blade with the Kan of 1000mg/L concentration and smear, can see after 12 days that adjoining tree is smeared the position tangible chlorisis spot, transgenic line then shows as unconspicuous chlorisis.
2.4 Kan smears result's PCR detection validation
Random choose 1000mg/L concentration Kan smears 36 strains of screening transfer-gen plant, and wherein 1 order reaction PCR detection positive rate is that 100%, 2 order reaction PCR detection positive rate is that 87.5%, 3,4 order reaction PCR detection positive rate is 0.
In the plant (reacting 1～2 grade) to the high anti-and resistance of Kan performance, the degree of conformity that Kan smears testing result and PCR testing result is 93.8%.Show thus, the situation of utilizing Kan blade semar technique can screen the foreign gene of transgenosis radish plant fast and accurately, it smears testing result and the PCR detection has uniformity preferably.
Claims (3)
1. method of utilizing kanamycin blade semar technique field screening transgenosis radish is characterized in that carrying out according to the following steps:
(1) material is prepared
Test material is the transgenosis radish of NPTII marker gene;
(2) the blade kanamycin is smeared determining of suitable concentration and observation time
Kanamycin is mixed with 0mg/L respectively with the distilled water of additional 0.2% polysorbas20,250mg/L, and 500mg/L, 750mg/L, 1000mg/L, 1500mg/L, 2000mg/L, 8 processing of 2500mg/L concentration, every processing repeats 4 times, adopts randomised block design; Kan is applied in writing brush on the intermediate blade of two true leaves that launch fully of plant, moistening getting final product; Respectively in the response situation of smearing the 3rd, 6,9,12, the 15 day observed and recorded blade in back, to determine the situation of turnip leaves, kanamycin screening concentration and observing time;
(3) transgenosis T
0Smear screening test for the radish kanamycin
The offspring T of 60 strain systems of transfer-gen plant
0For planting seed in the bed of growing seedlings, every strain is 40～150 seeds, when plant height reaches 20～25cm, carrying out blade with the Kan of 1000mg/L concentration according to the method described above smears, 12 days " Invest, Then Investigate " results, with into treatment sites blade face color normally is Kan resistant transgenic plant, and the plant that the chlorisis spot appears in the blade face is the responsive negative plant of Kan;
(4) Kan smears result's PCR detection validation
For detecting the reliability that Kan smears the result, 68 transformed plants and 1 negative contrast of unconverted plant of 4 grades of random choose, method extracting DNA designs primer and carries out pcr amplification in a small amount, amplified band is 769bp, and amplified production detects with 1.0% agarose gel electrophoresis.
2. the method for claim 1 is characterized in that: to smear the optium concentration of field screening be 1000mg/L to the Kan blade in the described step (2), and be 12 days observing time.
3. method as claimed in claim 1 or 2 is characterized in that: the design primer is specially in the described step (4): P1:5 '-AGCCGTTTGTTAGTGCCT-3 '; P2:5 '-ACTTGGATGCGGATGGAC-3 '.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110065447 CN102217472B (en) | 2011-03-18 | 2011-03-18 | Method for screening transgenic radish in field by using kanamycin leaf smearing method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110065447 CN102217472B (en) | 2011-03-18 | 2011-03-18 | Method for screening transgenic radish in field by using kanamycin leaf smearing method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102217472A true CN102217472A (en) | 2011-10-19 |
| CN102217472B CN102217472B (en) | 2013-05-22 |
Family
ID=44774387
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110065447 Expired - Fee Related CN102217472B (en) | 2011-03-18 | 2011-03-18 | Method for screening transgenic radish in field by using kanamycin leaf smearing method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102217472B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149157A (en) * | 2012-12-29 | 2013-06-12 | 重庆邮电大学 | Screening method of transgenosis mustard sieve marked by kanamycin resistance |
| CN112106635A (en) * | 2020-09-22 | 2020-12-22 | 云南中烟工业有限责任公司 | Method for rapidly screening label-free gene editing tobacco material seeds by using kanamycin |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU6172400A (en) * | 1999-07-27 | 2001-02-13 | Syngenta Limited | Herbicide resistant plants and methods for the production thereof |
| US20040093649A1 (en) * | 1999-02-26 | 2004-05-13 | Howe Arlene R. | Assay for the detection of selectable marker expression in plants |
| CN101236197A (en) * | 2008-03-07 | 2008-08-06 | 四川农业大学 | Method for screening transgenic rape using chanamyn seed soaking method |
| CN101241123A (en) * | 2008-03-18 | 2008-08-13 | 武汉大学 | Method for screening transgenic paddy rice seed based on antibiotic |
| JP2009065886A (en) * | 2007-09-12 | 2009-04-02 | Institute Of Physical & Chemical Research | Method for plant configuration control |
-
2011
- 2011-03-18 CN CN 201110065447 patent/CN102217472B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040093649A1 (en) * | 1999-02-26 | 2004-05-13 | Howe Arlene R. | Assay for the detection of selectable marker expression in plants |
| AU6172400A (en) * | 1999-07-27 | 2001-02-13 | Syngenta Limited | Herbicide resistant plants and methods for the production thereof |
| JP2009065886A (en) * | 2007-09-12 | 2009-04-02 | Institute Of Physical & Chemical Research | Method for plant configuration control |
| CN101236197A (en) * | 2008-03-07 | 2008-08-06 | 四川农业大学 | Method for screening transgenic rape using chanamyn seed soaking method |
| CN101241123A (en) * | 2008-03-18 | 2008-08-13 | 武汉大学 | Method for screening transgenic paddy rice seed based on antibiotic |
Non-Patent Citations (2)
| Title |
|---|
| 《棉花学报》 20001015 祝建波等 转基因棉花的田间筛选技术研究 , 第05期 * |
| 《贵州农业科学》 20101231 李彦荣等 转Chi-Glu基因野罂粟植株卡那霉素筛选技术 , 第12期 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103149157A (en) * | 2012-12-29 | 2013-06-12 | 重庆邮电大学 | Screening method of transgenosis mustard sieve marked by kanamycin resistance |
| CN112106635A (en) * | 2020-09-22 | 2020-12-22 | 云南中烟工业有限责任公司 | Method for rapidly screening label-free gene editing tobacco material seeds by using kanamycin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102217472B (en) | 2013-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101138313B (en) | Maize inbred line resistant to MRDV bred by using molecule making | |
| CN106148353B (en) | Brown planthopper resistant gene in rice Bph6 and its close linkage molecular labeling | |
| CN109536490B (en) | Transgenic insect-resistant herbicide-resistant corn CM8101 exogenous insert flanking sequence and application thereof | |
| CN103952402A (en) | SNP (single nucleotide polymorphism) site related to characters of plant root system and application thereof | |
| CN103266132A (en) | Bacillus thuringiensis cry1Ah/cry1Ie bivalent gene expression vector and application thereof | |
| CN102212552A (en) | Method for transforming genes by using efficient living body of chemical male sterilant | |
| CN117082972A (en) | Plant resistance genes and means for their identification | |
| JP2022531054A (en) | Genes of resistance to plant diseases | |
| CN110468229B (en) | Coseparation molecular marker Hxjy-1 of rice broad-spectrum high-resistance bacterial leaf blight gene Xa45(t) | |
| CN103160584A (en) | Method and special primer for screening or auxiliary screening of wheat with high pre-harvest sprouting resistance | |
| CN110358861B (en) | Molecular marker R13I14 closely linked with rice broad-spectrum high-resistance bacterial blight gene Xa45(t) | |
| CN102217472B (en) | Method for screening transgenic radish in field by using kanamycin leaf smearing method | |
| CN101948870B (en) | Method for reducing branch quantity of plant and improving chlorophyll and anthocyanin contents of plant | |
| CN115261237B (en) | Strain for promoting germination of azalea seeds and application thereof | |
| CN103789325B (en) | Cotton cells wall extensin gene GbEXPATR and application | |
| CN100413965C (en) | Phosphorus starvation induced gene promoter and its application | |
| CN105802931A (en) | CRK4 protein and application of coded gene thereof in regulating and controlling growth of plant stems and leaves | |
| CN113179884B (en) | Method for cultivating high-yield wheat with gibberellic disease resistance in Huanghuai wheat area based on polygene polymerization | |
| CN106929518B (en) | A kind of rubber tree HbAG genes and its application | |
| CN111793633B (en) | Application of OsNRT2.3b in improving yield and rice quality | |
| CN110358862B (en) | Molecular marker Hxjy-14 closely linked with rice broad-spectrum high-resistance bacterial blight gene Xa45(t) | |
| CN113249429A (en) | Large-scale rice blast resistance identification method suitable for breeding application | |
| CN107779522B (en) | Specific codominant molecular marker of brown planthopper resistant gene Bph15 of rice and application thereof | |
| CN101509006B (en) | Uses of arabidopsis thaliana gene AIK1 in regulating and controlling vegetation growth, and in stress resistance aspect | |
| Diouf et al. | Casuarinaceae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C53 | Correction of patent for invention or patent application | ||
| CB03 | Change of inventor or designer information |
Inventor after: Cui Lei Inventor after: Liu Yuhua Inventor after: Yuan Shangyong Inventor after: He Wenyuan Inventor after: Yu Binwu Inventor after: Wang Zhongyuan Inventor after: Fu Yibin Inventor after: Gao Xiang Inventor after: Mei Xuefei Inventor after: Mei Shiyong Inventor after: Yuan Weiling Inventor after: Wang Qingfang Inventor after: Gan Caixia Inventor after: He Yunqi Inventor after: Huang Laichun Inventor after: Ding Zili Inventor after: Zhang Xingzhong Inventor before: Cui Lei Inventor before: Gao Xiang Inventor before: Mei Shiyong Inventor before: Gan Caixia Inventor before: He Yunqi Inventor before: Huang Laichun Inventor before: Wang Qingfang Inventor before: Ding Zili Inventor before: Zhang Xingzhong Inventor before: Mei Xuefei |
|
| COR | Change of bibliographic data |
Free format text: CORRECT: INVENTOR; FROM: CUI LEI MEI SHIYONG GAN CAIXIA HE YUNQI HUANG LAICHUN WANG QINGFANG DING ZILI ZHANG XINGZHONG MEI XUEFEI GAO XIANG TO: CUI LEI MEI SHIYONG YUAN WEILING WANG QINGFANG GAN CAIXIA HE YUNQI HUANG LAICHUN DING ZILI ZHANG XINGZHONG LIU YUHUA YUAN SHANGYONG HE WENYUAN YU BINWU WANG ZHONGYUAN FU YIBIN GAO XIANG MEI XUEFEI |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20130522 Termination date: 20150318 |
|
| EXPY | Termination of patent right or utility model |