CN102308748A - Optimized method for screening transgenic crops by coating herbicide - Google Patents
Optimized method for screening transgenic crops by coating herbicide Download PDFInfo
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- CN102308748A CN102308748A CN2011101019552A CN201110101955A CN102308748A CN 102308748 A CN102308748 A CN 102308748A CN 2011101019552 A CN2011101019552 A CN 2011101019552A CN 201110101955 A CN201110101955 A CN 201110101955A CN 102308748 A CN102308748 A CN 102308748A
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Abstract
The invention provides an optimized method for screening transgenic crops by coating herbicide, comprising the steps of: (a) marking rings with diameters of about 1 cm on the third to fifth newborn laminas of the T0-generation transgenic crops through a marker pen; (b) sucking 2 microliters of herbicide solution through a pipettor and dropwise adding the herbicide at the centers of the rings; (c) determining the plants having one or more normal laminas as transgenic positive plants after being treated by the herbicide for 5-7 days; (d) transplanting the selected transgenic positive plants in a big-arch shelter and harvesting the same. The coating selection method provided by the invention can be used for effectively and conveniently selecting the T0-generation transgenic positive plants and has an important significance in screening of the transgenic crops.
Description
Technical field
the present invention relates to plant transgenic technology field, the method for smearing the herbicide screening genetically modified crops of a kind of optimization specifically.
Background technology
import to the gene of artificial separation or modified on the organism genome, because expression of exogenous gene makes the proterties of organism that heritable change take place, this technology is referred to as transgenic technology.The species isolation has been broken in the development of transgenic technology, can the genes of interest with merit be imported in the Plant Genome, thereby make the genetic character of many plants obtain improvement.In genetically modified operating process; Have only the DNA of minority external source can be transferred in the plant cell; Further be incorporated on the plant genome DNA; Also having most plant cell is not by the foreign DNA transfection, and how rejecting these not genetically modified plant cells is one of key problems of transgenic technology.The auxiliary genetically modified screening of purpose of general using selectable marker gene at present.When genes of interest transforms, introduce selectable marker gene, give recipient plant particular screening mark, so that easy screening apace obtains the transgenic positive plant in the huge Plant Transformation offspring of number.
antibiotic is present widely used a kind of selected marker.The most of antibiotics resistant gene is as the marker gene of foliage filter screening; That the most often uses has neomycin phosphotransferase gene (NptII) and hygromix phosphotransferase (HPT) gene; The enzyme of the former gene code makes its genetically modified plants have kanamycin and neomycin resistance, and the enzyme of latter's gene code is then given the transformation receptor hygromycin resistance.Though antibiotics resistance gene has the convenient and transformation efficiency advantages of higher of screening as the selection markers gene; If but these resistance antibiotic resistances are transferred to microorganism particularly in the pathogen; To make pathogen produce antibiotic resistance, human beings'health will be produced potential safety hazard.On the other hand, antibiotic costing an arm and a leg, cost is too high as large-scale genetically modified crops industrialization is produced.The factor of this two aspect has determined that antibiotics resistance gene is restricted in the application aspect the transgenosis industrialization screening.Weed killer herbicide is a kind of selection markers that grew up in recent years, and the most frequently used weed killer herbicide is glyphosate and careless fourth phosphine.Anti-herbicide gene still is a kind of genes of interest as the selection markers gene not only, utilizes weed killer herbicide when having objective trait, can also have Herbicid resistant as selective agent render transgenic crop.On the other hand, the low price of weed killer herbicide can reduce the cost of genetically modified crops industrialization greatly.But weed killer herbicide is as a kind of emerging selection markers, and its operation system is still immature.The land for growing field crops uses weed killer herbicide generally to kill weeds with the mode of spraying, and this is the occupation mode that a kind of high selection is pressed, and is obviously improper for the screening of transgenic positive seedling for T0.Because pressing, too high selection possibly kill some transgenic positive seedlings.On the other hand, present many crops transform the live body transgenic technology of usefulness, and T0 is on behalf of chimera, and spraying can kill most T0 for plant, causes genetically modified failure.Therefore, grope the practicable screening technique of a cover and have important actual application value for promoting the use of of weed killer herbicide selected marker.
Summary of the invention
The purpose of this invention is to provide a kind of method of smearing the herbicide screening genetically modified crops of optimization, may further comprise the steps:
are (a) on 3rd ~ 5 blade of T0 for the new life of transgenic crop, with the circle of marker the about 1cm of label diameter.
The center of the herbicide solution point of 2ul at circle (b) drawn with pipettor in
.
(c) after 5 ~ 7 days, through herbicide treatment to have a slice or above blade to show normal plant be the transgenic positive plant.
The transgenic positive plantlet of transplant that
(d) obtain above-mentioned screening is received kind in booth.
T0 is meant for transgenic crop and uses plant living body to be acceptor among
the present invention, and the expression vector that carries the weed killer herbicide gene or carry the weed killer herbicide selectable marker gene is transformed T0 that crops obtain for transfer-gen plant.With the plant living body is that the T0 that the explant conversion obtains is a chimera for transfer-gen plant; Conventional herbicide spray screening technique can kill chimera, inventor's lot of experiment results proof is provided by the invention smear screening technique can be efficiently, screening easily obtains T0 for the transgenic positive plant.
are in the herbicide screening process; In order to make herbicide solution evenly attached to the surface of crop leaf; The inventor has added surfactant in herbicide solution, it is best proportionally to be diluted in 0.02% the silwet-L77 aqueous solution screening effect through experiment proof at herbicide formulations.
Weed killer herbicide described in
the present invention is arbitrary weed killer herbicide that can be used as the foliage filter screening mark, is preferably glyphosate and careless fourth phosphine.Crop described in the present invention is wild or cultivar, strain, breeding material or the intermediate materials of soybean, wheat, corn, paddy rice or Chinese sorghum.
will combine accompanying drawing and embodiment further to annotate the present invention for the ease of understanding below.Instantiation and accompanying drawing only are in order better to set forth the present invention, not constitute limitation of the scope of the invention.Obviously those of ordinary skill in the art can explain according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.
Description of drawings
Fig. 1 smears experiment screening diagrammatic sketch as a result for T0 for the genetically engineered soybean cremart, and wherein A figure is a wild type, and B figure is the transgenic positive plant.Red circle is the blade position that cremart is smeared.
Fig. 2 is the PCR testing result of T0 for genetically engineered soybean, and wherein swimming lane M is marker ,+be with the plasmid be template over against photograph ,-be the negative contrast of wild type, 1～5 5 transgenic lines for detecting.
Fig. 3 smears experiment screening diagrammatic sketch as a result for T0 for the transgene cotton cremart, and wherein A figure is a wild type, and B figure is the transgenic positive plant.
Fig. 4 is the PCR testing result of T0 for transgene cotton, and wherein swimming lane M is marker ,+be with the plasmid be template over against photograph ,-be the negative contrast of wild type, 1～9 9 transgenic lines for detecting.
Embodiment
Method therefor is conventional method if no special instructions among
following embodiment, and concrete steps can be referring to " Molecular Cloning:A Laboratory Manual " (Sambrook, J.; Russell, David W., Molecular Cloning:A Laboratory Manual; 3rd edition; 2001, NY, Cold Spring Harbor).The primer and dna sequence dna are synthetic by Shanghai Ying Jun Bioisystech Co., Ltd.
Embodiment one, semar technique screening genetically engineered soybean
1. experiment material:
plasmid: contain the bar gene, have the pCAMBIA1300-bar carrier of hygromycin selection marker gene, have anti-Glufosinate weed killer herbicide.Pcr amplification bar gene is connected on pCAMBIA1300 (available from the Invitrogen company) carrier and obtains the pCAMBIA1300-bar carrier.
agrobacterium strains: AGL0.
supply the soybean varieties of conversion: No. 37, black farming, No. 4, magnificent boundary.
The cultivation of Agrobacterium
The frozen Agrobacterium that contains genes of interest of
picking glycerine, setting-out on the YEB flat board, 28 ℃ of dark culturing casees were cultivated 2 days.Picking list colony inoculation is in 40ml resistance YEB liquid nutrient medium, and 28 ℃ of dark shaking table 200rpm shake and spend the night, to OD600 ≈ 1.Drawing above-mentioned culture 250～300 μ l coats on the YEB solid culture medium that plate diameter is 15cm; 28 ℃ of dark culturing casees were cultivated after 2 days; Used this subsequent use fresh dull and stereotyped same day, and about 20 ℃ of room temperature dark are for use, do not continue to retain in 28 ℃ of dark culturing casees or unloading in 4 ℃ of refrigerators.
The preparation of infiltration medium
are at first according to following formulated AA medium.
Medium preparation (1L):
MgSO4 0.12209g
KCl 2.95g
NaH2PO4·2H2O 0.15g
Inositol 0.1g
Glutamic acid 0.876g
Asparatate 0.266g
Arginine 0.174g
Casein hydrolysate 0.3g
Sucrose 20g
Iron salt solutions 5ml 1. EDTA-2Na 7.46g/L transfers pH=8.0
2. add 4.5ml/L 5M NaCl again
③FeSO4·7H2O 5.56g/L
CaCl2 solution 4.5ml 25.06g/L
VB1 solution 10ml 1g/L
VB6 solution 1ml 1g/L
Nicotinic acid solution 1ml 0.1g/L
B5 trace 1ml MnSO4H2O 7.58g/L
ZnSO4·7H2O 2.0g/L
H3BO3 3.0g/L
KI 0.75g/L
Na2MoO4·2H2O 0.25g/L?
CuSO4·5H2O 0.025g/L
CoCl2·6H2O 0.025g/L
In 1L AA medium, add following ingredients:
As（100～200μM?）；?KT（0.2mg）；?2,4-D（1mg）；DTT（1mM?）；?L-Cysteine（200～400mg）；silwet-L77（100～400μl）。
suspend cultured Agrobacterium with above-mentioned infiltration medium, the OD600 value that makes suspension bacteria liquid is 0.4～0.7, and pH to 5.0～5.5 transform the infiltration inoculum preparation and finish.
The transformation experiment of soybean
utilize the above-mentioned infiltration medium for preparing to infect soybean seedling, are transplanted to booth then and cultivate.
Screening and acquisition for the transgenic positive seedling
The T0 that
above-mentioned soybean transformation obtains is a chimera for transfer-gen plant, and the part organ of plant contains purpose expresses, and has the target phenotype.Because of carrier contains genes of interest bar, if the transgenosis success, bar inserts the soybean gene group, and the part organ of render transgenic positive plant has the Glufosinate resistance.So we screen T0 for positive transfer-gen plant through the blade that cremart detects the genetically engineered soybean among the embodiment one.The blade of soybean is a three lives compound leaf; Choose the middle vanelets of compound leaf; With circle of Mark mark; Drip 2 μ l Glufosinate solution (Basta 100mg/L+silwet-L77 200 μ l/L) in the middle of circle with pipettor, the plant performance of resistance of not having after 5-6 days wilts, significantly phytotoxicity reaction such as withered and yellow.Smear soybean with said method first up to the 5th compound leaf.Five compound leaves all show the Glufosinate phytotoxicity reaction, regard as the negative plant of transgenosis, and a slice and above blade performance cremart resistance is arranged, and regard as transgenic positive plant (Fig. 1).We utilize the semar technique screening to obtain 220 strain T0 for positive plant.
PCR for transgenic seedling detects
are carried out the PCR test experience and are verified further whether transgenosis is successful the resistant plant of T0 for the cremart screening.The transgenic positive plant shows the cremart resistance because of successfully transforming the bar gene.According to the sequence of bar gene, design the pcr amplification primer, further verify the positive of transgenic line.The new blade that launches of a slice is got in every strain, and the CTAB method is extracted plant genome DNA.The genomic DNA of getting 10pg carries out the PCR reaction.According to the sequence of bar gene, pcr amplification primer sequence is following:
Primer 1 (upper reaches): 5 ' TCATCAGATTTCGGTGACGG 3 '
Primer 2 (downstream): 5 ' TCAACTTCCGTACCGAGCCG 3 '
utilize primer 1 and primer 2 respectively, with resistant plant genomic DNA template, and amplification bar genetic fragment.
Amplification system and program are:
10X?Buffer ： ?2ul；
10mM?dNTP ： ?0.5ul；
10uM primer 1: 0.5ul;
10uM primer 2: 0.5ul;
genomic?DNA?： 10pg；
Taq?DNA?Polymerase(5U/ul)：0.4ul；
ddH2O: supply 20ul.
Reaction condition is:
Sex change in advance: 95 ℃, 5min;
Sex change: 94 ℃, 20sec;
Annealing: 55 ℃, 30sec;
Extend: 72 ℃, 1min;
30 circulations;
Extend
again: 72 ℃, and 10min.
After
reaction finishes, the PCR product is carried out 1.5% agarose gel electrophoresis detect, can detect the purpose fragment (result sees Fig. 2) of 450bp.We have carried out the PCR experiment to 5 strain systems, and the result all is positive.This resistant plant that has proved that further we obtain is transgenic positive strain system.
Embodiment two, semar technique screening transgene cotton
1. the transformation experiment of cotton
Prepare the infiltration medium that contains pCAMBIA1300-bar according to the method shown in 1 ~ 3 among the embodiment one,
converting cotton is transplanted to booth then and is cultivated.
Cotton T0 is for the screening and the acquisition of transgenic positive seedling
The T0 that
above-mentioned converting cotton obtains is a chimera for transfer-gen plant, and the part organ of plant contains purpose expresses, and has the target phenotype.Because of carrier contains genes of interest bar, if the transgenosis success, bar inserts the cotton gene group, and the part organ of render transgenic positive plant has the Glufosinate resistance.So we screen T0 for positive transfer-gen plant through the blade that cremart detects the transgene cotton among the embodiment one.Choose the newborn blade of the cotton that has transformed; With circle of Mark mark; Drip 2 μ l Glufosinate solution (Basta 100mg/L+silwet-L77 200 μ l/L) in the middle of circle with pipettor, the plant performance of resistance of not having after 5-6 days wilts, significantly phytotoxicity reaction such as withered and yellow.Smear cotton with said method the 3rd up to the 5th young leaves.2 young leaves all show the Glufosinate phytotoxicity reaction, regard as the negative plant of transgenosis, and a slice and above blade performance cremart resistance is arranged, and regard as transgenic positive plant (Fig. 3).We utilize the semar technique screening to obtain 120 strain T0 for positive plant.
PCR for transgene cotton detects
are carried out the PCR test experience and are verified further whether transgenosis is successful the resistant plant of T0 for the cremart screening.The transgenic positive plant shows the cremart resistance because of successfully transforming the bar gene.The new blade that launches of a slice is got in every strain, and the CTAB method is extracted plant genome DNA.The genomic DNA of getting 10pg carries out the PCR reaction according to the described method of step 6 among the embodiment one.We have carried out the PCR experiment to 9 strains system, result all be positive (Fig. 4).
Claims (5)
1. the method for smearing the herbicide screening genetically modified crops of an optimization may further comprise the steps:
(a) on 3rd ~ 5 blade of T0, with the circle of marker the about 1cm of label diameter for the new life of transgenic crop;
(b) draw the center of the herbicide solution point of 2ul with pipettor at circle;
(c) after 5 ~ 7 days, through herbicide treatment to have a slice or above blade to show normal plant be the transgenic positive plant;
(d) the transgenic positive plantlet of transplant that above-mentioned screening is obtained is received kind in booth.
2. method of smearing the herbicide screening genetically modified crops according to claim 1; It is characterized in that: said T0 is meant with the plant living body to be acceptor for transgenic crop, and the expression vector that carries the weed killer herbicide gene or carry the weed killer herbicide selectable marker gene is transformed T0 that crops obtain for transfer-gen plant.
3. method of smearing the herbicide screening genetically modified crops according to claim 1 is characterized in that: said weed killer herbicide dissolves
Liquid is that herbicide formulations proportionally is diluted in 0.02% the silwet-L77 aqueous solution.
4. according to the described arbitrary claim of claim 1 ~ 3, it is characterized in that: described weed killer herbicide is the weed killer herbicide that can be used for the genetically modified plants screening, is preferably glyphosate and careless fourth phosphine.
5. according to the described arbitrary claim of claim 1 ~ 3, it is characterized in that: described crops are wild or cultivar, strain, breeding material or the intermediate materials of soybean, cotton, wheat, corn, paddy rice or Chinese sorghum.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102771384A (en) * | 2012-08-22 | 2012-11-14 | 江苏丰源种业有限公司 | Method for seed production of two-line hybrid rice |
| CN107667849A (en) * | 2017-09-25 | 2018-02-09 | 黑龙江省农垦总局九三农业科学研究所 | A kind of breeding method of genetically engineered soybean |
Citations (2)
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| CN1687424A (en) * | 2005-05-13 | 2005-10-26 | 吉林省农业科学院 | Transgene method for plant |
| CN101407817A (en) * | 2008-11-28 | 2009-04-15 | 北京市农林科学院 | Method for cultivating disease-resistant maize |
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2011
- 2011-04-22 CN CN2011101019552A patent/CN102308748A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1687424A (en) * | 2005-05-13 | 2005-10-26 | 吉林省农业科学院 | Transgene method for plant |
| CN101407817A (en) * | 2008-11-28 | 2009-04-15 | 北京市农林科学院 | Method for cultivating disease-resistant maize |
Non-Patent Citations (3)
| Title |
|---|
| ANTHONY T. ET AL: "Transformation of Medicaga truncatula via infiltration of seedings or flowering plants with Agrobacterium", 《THE PLANT JOUTNAL》, vol. 22, no. 6, 31 December 2000 (2000-12-31) * |
| 梁雪莲等: "玉米3种非组培转基因方法转化外源bar 基因研究", 《作物学报》, vol. 31, no. 12, 31 December 2005 (2005-12-31) * |
| 邹智等: "整株转化法及其在油菜上的应用与展望", 《植物学报》, vol. 44, no. 2, 31 December 2009 (2009-12-31) * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102771384A (en) * | 2012-08-22 | 2012-11-14 | 江苏丰源种业有限公司 | Method for seed production of two-line hybrid rice |
| CN107667849A (en) * | 2017-09-25 | 2018-02-09 | 黑龙江省农垦总局九三农业科学研究所 | A kind of breeding method of genetically engineered soybean |
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Application publication date: 20120111 |