CN102217472B - 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
- CN102217472B CN102217472B CN 201110065447 CN201110065447A CN102217472B CN 102217472 B CN102217472 B CN 102217472B CN 201110065447 CN201110065447 CN 201110065447 CN 201110065447 A CN201110065447 A CN 201110065447A CN 102217472 B CN102217472 B CN 102217472B
- Authority
- CN
- China
- Prior art keywords
- blade
- kan
- 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.)
- Expired - Fee Related
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 transgenic 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 screening and the evaluation of converting material, not only workload is large, 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 identification 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 the selectable marker gene that is widely used at present Genetic Transformation in Higher Plants, kanamycin is multiplex in organizing cultivation stage to carry out Preliminary screening to transformed plant, and whether the method testing goal genes such as recycling PCR, Southern blot hybridization are incorporated in Plant Genome.
The method of kanamycin field rapid screening transfer-gen plant has been reported in transgene cotton, soybean, tomato, paddy rice, wheat, but there is not yet report aspect the screening of transgenic 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 take with the transgenic radish of NPT II marker gene as material; 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 has been 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 transgenic 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 methods that in the field, transgenic radish carried out a large amount of screenings have been set up in research, have important value for the seed selection of transgenic radish material.
The technical solution used in the present invention is: a kind of method of kanamycin blade semar technique Field Screening transgenic radish is characterized in that carrying out according to the following steps:
(1) material is prepared
Test material is that the transgenic radish of NPT II marker gene (is the commercially available prod, buys in Crop Institute, Hunan Academy of Agricultural Sciences; Also can obtain Bt by conventional technique for gene engineering gene constructed in plasmid pCMIaN, selected marker is NPT II gene, and agrobacterium strains transforms)
(2) the blade kanamycin is smeared determining of suitable concentration and observation time
Kanamycin is mixed with respectively 0mg/L 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.The response situation of the 3rd, 6,9,12,15 day observed and recorded blade after smearing respectively is 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 strains of transfer-gen plant
0For planting seed in seedling bed, 40～150 seeds of every strain, 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, normally as Kan resistant transgenic plant, the plant that the chlorisis spot appears in the blade face is the responsive negative plant of Kan take treatment sites blade face color.
(4) Kan smears the PCR detection validation of result
Smear the reliability of result for detecting Kan, 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 the primer P1:5 ' of BtCry1Ia gene-AGCCGTTTGTTAGTGCCT-3 '; 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 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 methods that in the field, transgenic radish carried out a large amount of screenings have been set up in research, have important value for the seed selection of transgenic radish material.
Embodiment
The below does a detailed description to the present invention:
Embodiment:
1 materials and methods
1.1 test material
1.1.1 vegetable material
(1) material is prepared
Test material is that the transgenic radish of NPT II marker gene (is the commercially available prod, buys in Crop Institute, Hunan Academy of Agricultural Sciences; Also can obtain Bt by conventional technique for gene engineering gene constructed in plasmid pCMIaN, selected marker is 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 respectively 0mg/L 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.The response situation of the 3rd, 6,9,12,15 day observed and recorded blade after smearing respectively is 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 for planting seed in seedling bed, 40～150 seeds of every strain, 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, normally as Kan resistant transgenic plant, the plant that the chlorisis spot appears in the blade face is the responsive negative plant of Kan take treatment sites blade face color.
1.4 Kan smears the PCR detection validation of result
Smear the reliability of result for detecting Kan, 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 processed Kan
Take unconverted Bt gene radish varieties XD2-A as for the examination material, observed Kan smear the situation of change of rear blade color.According to the result of field observation, the end reaction degree after turnip leaves is processed Kan is divided into 4 grades.1 grade: smear 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 is still green; 3 grades: smear the position chlorosis, the leaf look from green to yellow, and is different obviously with the normal leaf aberration of periphery; 4 grades: the serious chlorosis for the treatment of sites, the leaf look turns white or brown.
In above 4 grades, the blade reaction is in the processing of 1,2 grade, fails to eliminate fully turnip leaves self to the limit of kalamycin resistance, in the detection of transfer-gen plant, can increase the quantity of false positive individual plant, thus the effect that impact is selected.Blade has eliminated the processing of 3 grades the negative effect that blade self is processed kanamycin basically, can be used as the standard of kanamycin initial screening.
2.2 the screening concentration of Kan and observing time
When screening concentration is 250mg/L, along with the prolongation leaf color in reaction time is almost unchanged, mainly present 1 order reaction; When screening concentration was 500mg/L, blade was not obvious to the reaction prolongation variation in time of Kan, and main manifestations is 2 order reactions, if therefore screen with above-mentioned concentration, can increase the quantity of false positive individual plant, the effect that impact is selected; When Kan concentration 〉=1000mg/L, the rapid flavescence of leaf color, even 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.Except 250mg/L and 500mg/L process prolongation blade reaction along with the time without significant change, other concentration are processed, and order of reaction continued to increase in 1～12 day, substantially tended towards stability after 12 days, therefore be advisable with 12 days observing time.
2.3 T0 smears result for transgenic radish Kan
Carry out blade with the Kan of 1000mg/L concentration and smear, can see after 12 days, adjoining tree is smeared the position obvious chlorisis spot, and transgenic line shows as unconspicuous chlorisis.
2.4 Kan smears the PCR detection validation of result
Random choose 1000mg/L concentration Kan smears 36 strains of screening transgenic plant, and wherein 1 order reaction PCR Positive rate is that 100%, 2 order reaction PCR Positive rate is that 87.5%, 3,4 order reaction PCR Positive rates are 0.
In plant (reacting 1～2 grade) to Kan performance high resistance and resistance, the degree of conformity that Kan smears testing result and PCR testing result is 93.8%.Show thus, utilize the Kan blade semar technique situation of the foreign gene of screening transgenic radish plant fast and accurately, it smears testing result and the PCR detection has uniformity preferably.
Claims (2)
1. method of utilizing kanamycin blade semar technique Field Screening transgenic radish is characterized in that carrying out according to the following steps:
(1) material is prepared
Test material is the transgenic radish of NPTII marker gene;
(2) the blade kanamycin is smeared determining of suitable concentration and observation time
Kanamycin is mixed with respectively 0mg/L 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; The response situation of the 3rd, 6,9,12,15 day observed and recorded blade after smearing respectively is to determine the situation of turnip leaves, kanamycin screening concentration and observing time; End reaction degree after turnip leaves is processed Kan is divided into 4 grades, 1 grade: smear position leaf look normal; 2 grades: blade is smeared the leaf look slight flavescence at position, and normal Ye Selve is variant with periphery, but not obvious, and the leaf look is still green; 3 grades: smear the position chlorosis, the leaf look from green to yellow, and is different obviously with the normal leaf aberration of periphery; 4 grades: the serious chlorosis for the treatment of sites, the leaf look turns white or brown; In above 4 grades, the blade reaction is in the processing of 1,2 grade, fails to eliminate fully turnip leaves self to the limit of kalamycin resistance, in the detection of transfer-gen plant, can increase the quantity of false positive individual plant, thus the effect that impact is selected; Blade has eliminated the processing of 3 grades the negative effect that blade self is processed kanamycin basically, can be used as the standard of kanamycin initial screening;
(3) transgenosis T0 smears screening test for the radish kanamycin
The offspring T0 of 60 strains of transfer-gen plant for planting seed in seedling bed, 40～150 seeds of every strain, 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, normally as Kan resistant transgenic plant, the plant that the chlorisis spot appears in the blade face is the responsive negative plant of Kan take treatment sites blade face color;
(4) Kan smears the PCR detection validation of result
Smear the reliability of result for detecting Kan, 68 transformed plants and 1 negative contrast of unconverted plant of 4 grades of random choose, method extracting DNA, design primer and carry out pcr amplification in a small amount, amplified band is 769bp, and amplified production detects with 1.0% agarose gel electrophoresis;
In described step (4), the design primer is specially: P1:5 '-AGCCGTTTGTTAGTGCCT-3 '; P2:5 '-ACTTGGATGCGGATGGAC-3 '.
2. the method for claim 1 is characterized in that: in described step (2), to smear the optium concentration of Field Screening be 1000mg/L to the Kan blade, and be 12 days observing time; Carry out blade with the Kan of 1000mg/L concentration and smear, can see after 12 days, adjoining tree is smeared the position obvious chlorisis spot, and transgenic line shows as unconspicuous chlorisis.
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 CN102217472A (en) | 2011-10-19 |
| CN102217472B true 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) |
Families Citing this family (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 (4)
| 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 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 (4)
| 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 |
| 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 (3)
| Title |
|---|
| JP特开2009-65886A 2009.04.02 |
| 李彦荣等.转Chi-Glu基因野罂粟植株卡那霉素筛选技术.《贵州农业科学》.2010,(第12期), * |
| 祝建波等.转基因棉花的田间筛选技术研究.《棉花学报》.2000,(第05期), * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102217472A (en) | 2011-10-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Bajwa et al. | Stable transformation and expression of GhEXPA8 fiber expansin gene to improve fiber length and micronaire value in cotton | |
| CN106148353B (en) | Brown planthopper resistant gene in rice Bph6 and its close linkage molecular labeling | |
| CN103667309B (en) | Brown planthopper resistant gene in rice Bph9 and molecule marker thereof and application | |
| CN109536490B (en) | Transgenic insect-resistant herbicide-resistant corn CM8101 exogenous insert flanking sequence and application thereof | |
| CN111206031A (en) | Nucleic acid sequence for detecting corn plant NAZ-4 and detection method thereof | |
| Bajwa et al. | Expression of Calotropis procera expansin gene CpEXPA3 enhances cotton fibre strength | |
| CN103952402A (en) | SNP (single nucleotide polymorphism) site related to characters of plant root system and application thereof | |
| CN109312350A (en) | The plant of abiotic stress tolerance and method | |
| CN105408485A (en) | Means and methods for yield performance in plants | |
| CN112280743A (en) | Corn event 2A-7 and methods for identifying same | |
| CN110305980B (en) | Breeding method and application of anti-clubroot high-oleic-acid rape | |
| CN116410977A (en) | Insect-resistant glyphosate-resistant transgenic corn event KJ1172 and detection method thereof | |
| CN117082972A (en) | Plant resistance genes and means for their identification | |
| CN102229976A (en) | Method for simply and rapidly identifying transgenic seeds and estimating copy numbers | |
| JP2022531054A (en) | Genes of resistance to plant diseases | |
| CN102217472B (en) | Method for screening transgenic radish in field by using kanamycin leaf smearing method | |
| CN107304422A (en) | Control the application of the OsSBR1 genes and its RNA interference fragments of resisting rice sheath blight | |
| CN110468229B (en) | Coseparation molecular marker Hxjy-1 of rice broad-spectrum high-resistance bacterial leaf blight gene Xa45(t) | |
| CN110358861B (en) | Molecular marker R13I14 closely linked with rice broad-spectrum high-resistance bacterial blight gene Xa45(t) | |
| CN103396477A (en) | Protein related to plant potassium ion absorption capacity, and coding gent and application thereof | |
| CN104119430A (en) | Plant drought resistance related protein, coding gene and application thereof | |
| CN113179884B (en) | Method for cultivating high-yield wheat with gibberellic disease resistance in Huanghuai wheat area based on polygene polymerization | |
| CN107779522B (en) | Specific codominant molecular marker of brown planthopper resistant gene Bph15 of rice and application thereof | |
| WO2020107816A1 (en) | Upland cotton transformation event 18c006-10-13 and specific identification method therefor | |
| CN101509006B (en) | Uses of arabidopsis thaliana gene AIK1 in regulating and controlling vegetation growth, and in stress resistance aspect |
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 |