CN1687424A - Transgene method for plant - Google Patents
Transgene method for plant Download PDFInfo
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- CN1687424A CN1687424A CN 200510016782 CN200510016782A CN1687424A CN 1687424 A CN1687424 A CN 1687424A CN 200510016782 CN200510016782 CN 200510016782 CN 200510016782 A CN200510016782 A CN 200510016782A CN 1687424 A CN1687424 A CN 1687424A
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Abstract
A transgenic method of plant, it relates a method of plant transgenic, agrobacterium medium transform to embryo just finishing fecundation, this embryo group to seed on individual plant. We filter the seeds to obtain transform element, this technology adapts to corn, soja, paddy and so on, series of fecundation plant which we can manipulate it's germen. Its character is: no organ cultivation, no liable to gene type and experiment condition, high efficiency.
Description
Technical field:
The present invention relates to a kind of plant transgenic method, relate in particular to a kind of method that goal gene is gone to recipient plant.
Background technology:
Utilizing transgenic technology can break through the boundary of species, realize the gene transmission between species, and can improve the biological character of live species, is human service to greatest extent.Moreover, transgenic technology also provides the research means of science, makes human science and technology realize that new leap becomes possibility.But plant transgenic method has a variety of, should select what method just the most effective when carrying out plant transgene, and this is a very crucial problem.The method and the technology that are used for exogenous gene transfered plant cell at present are a lot, generally speaking can be divided into directly and indirect method.
1.DNA direct guiding method.The dna direct introductory technique can be divided into: chemical substance revulsion, electroporation, liposome method, microinjection, particle bombardment (micropellet bombardment method) and pollen tube passage method or the like.Dna direct introductory technique vegetable material with the most use is a protoplastis, and available in addition vegetable material comprises organ meristematic tissue, immature embryo, callus, suspension cell, pollen, ovum or the like.
1.1 chemical substance revulsion material with the most use now is a polyoxyethylene glycol, it can merge with protoplastis, influences the permeability of protoplast membrane, causes protoplastis to absorb foreign DNA preferably.When general PEG concentration is low, can not damage, and the transfer-gen plant that obtains is from 1 cell to protoplastis, avoided producing chimeric transformant, transformed stability and good reproducibility, but the plant that protoplastis is cultivated and regeneration is difficult is difficult to utilize, and transformation efficiency is low, generally 10
-5～10
-6
1.2. electroporation is the asymmetric perforation that plant cell membrane causes under extraneous high electric field pulse, aperture, this hole less (about 8.4nm), and can in very short time, (200ns) return to 0.5nm, and so foreign gene can enter cell.The electroporation pair cell does not have harm, but needs special equipment, and different material requires is had strict parameter control.Owing to, on using, also be subjected to very big restriction to the dependence of protoplast regeneration.
Merge 1.3. liposome method is exactly electronegative liposome and the plant protoplast that will comprise foreign gene, thereby reach genetically modified purpose.
1.4. microinjection is to use kapillary (diameter of general needle point is 0.5nm), foreign DNA is injected into a kind of direct method for transformation of vegetable cell or protoplastis at microscopically.
1.5. particle bombardment claims little bullet method of high speed or micropellet bombardment method again, promptly with tungsten or goldc grains (diameter 1～2 μ m) parcel foreign DNA, then quickens this particulate with certain method and removes penetrate plant cell wall and cytolemma, makes foreign DNA can enter into vegetable cell.Particle bombardment in use has its limitation, not high as transformation efficiency, gene inserts multiple copied often, often cause genetically modified inactivation or silence, may cause the fracture of foreign gene in the bombardment process, the gene of insertion is become does not have active fragment, non-transformant occurs or chimeric possibility is higher, may cause the improper expression of some gene of plant itself, common inhibition phenomenon also might take place.
2. carrier mediated method
2.1 the agrobacterium-mediated transformation agrobacterium-mediated transformation is the common method that present dicotyledon gene shifts.It is to utilize one section T-DNA district on the Ri plasmid of the Ti-plasmids of agrobacterium tumefaciens and Agrobacterium rhizogenes to infect in the process that plant forms tumour Agrobacterium, and T-DNA can be transferred to vegetable cell and be inserted in the chromogene group.With this natural genetic transformation characteristic of Ti-plasmids, the nonessential sequence that foreign gene can be replaced among the T-DNA can make exogenous origin gene integrator obtain stable expression to being subjected to Autosome.Agrobacterium tumefaciens plasmid Ti conversion system is maximum, the sophisticated relatively path for transformation of technological method of research at present.But all be confined in the dicotyledons scope for a long time, with agriculture bacillus mediated transgenosis.Up in recent years, just obtained important breakthrough with the Agrobacterium-mediated Transformation monocotyledons.
2.2 virus-mediated method virus vector is nearest emerging a kind of carrier that is used for Plant Transformation.Plant virus transgenosis system is inserted into foreign gene in the viral genome, by virus to the infection of vegetable cell and with exogenous gene transfered plant cell.Virus vector is smaller, be convenient in experiment, operate, and this class carrier is as long as cultivate altogether just can be than highland infection plant cell with vegetable cell, foreign gene can be in vegetable cell quick copy and high level expression.But the finite capacity of virus vector can not be packed big segmental foreign DNA gene, and host range is narrow, duplicates poor stability, transcribes and duplicate the mechanism complexity.So the evidence that does not also have so far to determine shows whether plant virus can be integrated in the genome of vegetable cell.Someone thinks that the possibility of the foreign gene genetic stability that changes over to is little.Therefore, the plant viral vector system also is not used widely at present.
3. additive method also has electrophoretic method, pollen tube channel and the seed infusion method etc. of ion-beam mediated method, ultrasonic-mediated method, laser microbeam perforation method, germinating seed except that some above-mentioned main method.They respectively have its relative merits and the scope of application, for specific plant or be proved to be effective under certain conditions.
The dna direct introductory technique, for stable plant or be proved to be under certain conditions effectively, but its a main weak point is to exist changeable DNA to integrate mode, and it is more that the dna direct introductory technique imports the copy number of foreign gene, and it is low to change frequency over to.And the arrangement mode of the foreign gene of multiple copied on the recipient plant genome is series connection mostly, inherited character take the form of individual gene, sometimes, the foreign gene of multiple copied may be interspersed within the genomic different loci of recipient plant, make the integration mode very complicated, also bring difficulty for determining effectively to integrate.The frequency that these variations or modification take place is far longer than the carrier mediated method of soil Agrobacterium.Since nineteen ninety-three, agriculture bacillus mediated gene transformation is succeedd on some important cereal crops in succession, the Agrobacterium-mediated Transformation method has remarkable advantages, show as: mostly the foreign gene that changes over to is that single copy or low copy number insert, and insert the active region earlier, the transformation efficiency height, the foreign gene that changes over to is the Mendelian inheritance of expection usually, the plant of transformation tissue culture normally can educate, and method is simple, effective.Therefore, agriculture bacillus mediated conversion more and more becomes the main method of various plant transgenes, is used widely.The acceptor of agrobacterium mediation converted comprises callus that rataria, rataria produce, stem, leaf, shoot apical meristem, cotyledonary node etc.
Above-mentioned method all depends on tissue culture, all needs to set up the system of a cover plant high frequency regeneration, like this, has a variety of materials to exist genotype to rely on, and a variety of plants and kind can't successfully be set up this individual system, also just can't do conversion with these technology.A lot of work of these methods will be operated in testing laboratory, need the certain condition support.
Summary of the invention:
The object of the present invention is to provide a kind of new plant transgenic method that does not rely on tissue culture.
Technical scheme of the present invention is achieved in that elder generation causes wound at the ovary position that plant closes on the zygotic embryo of after fertilization formation; Use the During Agrobacterium wound again, allow genophore plasmid in the Agrobacterium transform zygophase rataria on the live body plant; The rataria that transforms is grown up into seed on the live body plant; Seed is screened, thereby obtain transfer-gen plant.
Specifically can adopt following steps to carry out:
1, the recipient plant that desire is transformed is cultivated to blooming;
2, before conversion operation, the Agrobacterium that will contain genophore is cultured to logarithmic phase, transforms with culture medium is resuspended to 0.D600=0.5-1.5 concentration altogether, as transformed bacteria liquid with plant tissue culture;
3, make the ovary upper end cause wound at recipient plant after fertilization zygophase;
4, transformed bacteria liquid is contacted with wound on the ovary, on plant, cultivate altogether with ovary;
5, nurse is cultivated to growing up to seed;
6, the results seed screens seed, obtains transfer-gen plant.
Advantage of the present invention is:
The present invention does not rely on genotype without tissue culture, does not rely on laboratory condition, is applicable to that various pollinations are solid, the artificial plant of wound of ovary, comprises corn gramineous, paddy and wheat; The soybean of pulse family, pea, cowpea, broad bean, mung bean, red bean, Kidney bean; The tomato of Solanaceae, eggplant, capsicum; Cucumber cucurbitaceous, watermelon, pumpkin, wax gourd; The radish of Cruciferae, rape, Chinese cabbage, wild cabbage, leaf mustard, turnip and cotton can carry out the plant that artificial wound is handled to ovary.
Embodiment:
The present invention according to the difference of recipient plant, adopts different methods to make ovary cause wound, and with different methods such as being coated with, smearing, being stained with, dripping transformed bacteria liquid is contacted according to the floral organ characteristics with wound on the ovary when concrete the application.When seed was screened, the screening-gene according to used carrier contains screened on substratum or in the nutrition pot the seed seedling, after the selective agent primary election, with PCR, Southern blot Screening and Identification.Describe below in conjunction with specific embodiment:
Embodiment 1:
Corn transforms.Material: self-mating system 8902; Gene and carrier: pinellia agglutinin (PTA) pCAMBIA3300PTA, bacterial classification EHA101.In maize planting self-mating system 8902 in spring, wait to grow to the phase of being pollinated, 10:00 pollination in the morning, second day afternoon, 4:00 carried out conversion operation.The day before yesterday is prepared bacterium liquid in operation, draws from flat board and gets single bacterium colony, cultivates a night, and before the operation in second day, culture medium is resuspended altogether with Plant Transformation, and concentration 0.D600=1.0-1.5 is as conversion fluid.During conversion, earlier female fringe is handled, strip off Bao Ye, with filigree, promptly the gynoecium of Huaing is vertically from top to bottom pulled apart from root, is meant female fringe tip from top to bottom to the fringe bottom, dips in the fine, soft fur brush then and gets the brushing of bacterium liquid.Afterwards, the Bao Ye that closes, good with plastic pocket, put paper bag outward again, gather in the crops seed autumn.Altogether 2255 seeds, be sowed in the little nutrition pot, one in an alms bowl is inserted it in dish that can be filled with water again, and nutrition soil is once soaked into, and waits for germinateing.When germination grew two leaves, 100-200ppm evenly sprayed with Basta weedicide (carrier contains the Bar gene), sprayed once after several days again, and unconverted seedling death finally obtains 109 strains seedling alive, transplants in the field.After treating slow seedling, get blade and be PCR and carry out further evaluation and screening, therefrom obtain transformant.
Embodiment 2:
Soybean transforms.Material: the soybean varieties Ji is educated No. 25; Gene and carrier pBI121pta. wait to grow to the phase of being pollinated, 8:00--10:00 self-pollination in the morning, and afternoon, 4:00 carried out conversion operation.The day before yesterday is prepared bacterium liquid in operation, draws from flat board and gets single bacterium colony, cultivates a night, and before the operation in second day, culture medium is resuspended altogether with the plant tissue culture conversion, and concentration 0.D.600=0.5-1.0 is as conversion fluid.Draw the bacterium drop in the ovary wound with liquid-transfering gun, dip in bacterium liquid with cotton ball and cover on the inflorescence of operation, wait for results.Altogether 112 seeds, be sowed in the little nutrition pot, one in an alms bowl is inserted it in dish that can be filled with water again, and nutrition soil is once soaked into.Wait to sprout two true leaves when open and flat, 500ppm evenly sprays with kantlex (carrier contains the NPTII gene), after several days again spray once, unconverted seedling death finally obtains the 5 strains seedling of living, and transplants in flowerpot.After treating slow seedling, get blade and be PCR and carry out further evaluation and screening.Therefrom obtain transformant, it is positive that Southern blot result proves.
Claims (5)
1, a kind of plant transgenic method is characterized in that: elder generation causes wound at the ovary position that plant closes on the zygotic embryo of after fertilization formation; Use the During Agrobacterium wound again, allow genophore plasmid in the Agrobacterium transform zygophase rataria on the live body plant; The rataria that transforms is grown up into seed on the live body plant; Seed is screened, thereby obtain transfer-gen plant.
2, plant transgenic method according to claim 1 is characterized in that described conversion fluid is that the Agrobacterium that will contain genophore is cultured to logarithmic phase, transforms with culture medium is resuspended to O.D600=0.5-1.5 altogether with plant tissue culture.
3, plant transgenic method according to claim 1, it is characterized in that the desire plant transformed is cultivated to blooming, treat after fertilization zygote embryonic stage processing floral organ, wound is caused in the ovary upper end, conversion fluid is contacted with ovary upper cut place, on plant, cultivate altogether with ovary.
4, plant transgenic method according to claim 3, it is characterized in that the method for milpa is the female fringe Bao Ye of strip off, make outside its ovary is exposed to, shut down filigree vertically downward, dip in fine, soft fur brush and to get bacterium liquid and brush from top to bottom to the fringe bottom, the Bao Ye that closes puts plastics bag, overlap a paper bag again, cultivate altogether.
5, plant transgenic method according to claim 3 is characterized in that for the method for soybean plant strain for drawing the bacterium drop on the ovary wound with liquid-transfering gun, dips in bacterium liquid with cotton and is buckled in operation and takes, and cultivates altogether.
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| CNB2005100167829A CN100360677C (en) | 2005-05-13 | 2005-05-13 | Transgene method for plant |
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| CNB2005100167829A CN100360677C (en) | 2005-05-13 | 2005-05-13 | Transgene method for plant |
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| CN100360677C CN100360677C (en) | 2008-01-09 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102061309A (en) * | 2010-11-25 | 2011-05-18 | 武汉大学 | Transgenic method for agrobacterium-mediated rice living body |
| CN102308748A (en) * | 2010-05-20 | 2012-01-11 | 北京未名凯拓作物设计中心有限公司 | Optimized method for screening transgenic crops by coating herbicide |
| CN102321666A (en) * | 2011-10-10 | 2012-01-18 | 浙江大学 | Rapid preparation method of dicotyledonous plant transgenic acceptor |
| CN103497970A (en) * | 2013-09-04 | 2014-01-08 | 吉林省农业科学院 | Gene transfer method with corn ovule as receptor |
| CN105647966A (en) * | 2016-03-17 | 2016-06-08 | 山东棉花研究中心 | TRV(tobacco rattle virus)-induced gene silencing method by taking cotton immature embryo as receptor for inoculation |
| CN111454984A (en) * | 2020-03-04 | 2020-07-28 | 深圳大学 | Plant gene transformation method and application thereof |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0604662B1 (en) * | 1992-07-07 | 2008-06-18 | Japan Tobacco Inc. | Method of transforming monocotyledon |
| CN1064408C (en) * | 1995-12-13 | 2001-04-11 | 中山大学 | Method for convertion of rice by using agricultural bacillus as medium to lead exogenous gene |
| PL202067B1 (en) * | 1999-04-19 | 2009-05-29 | Biogemma S A S | Method of plant transformation |
| CN1194095C (en) * | 2001-02-26 | 2005-03-23 | 山东大学 | Process for transforming macroseed plant and its application |
-
2005
- 2005-05-13 CN CNB2005100167829A patent/CN100360677C/en not_active Expired - Fee Related
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102308748A (en) * | 2010-05-20 | 2012-01-11 | 北京未名凯拓作物设计中心有限公司 | Optimized method for screening transgenic crops by coating herbicide |
| CN102061309A (en) * | 2010-11-25 | 2011-05-18 | 武汉大学 | Transgenic method for agrobacterium-mediated rice living body |
| CN102321666A (en) * | 2011-10-10 | 2012-01-18 | 浙江大学 | Rapid preparation method of dicotyledonous plant transgenic acceptor |
| CN102321666B (en) * | 2011-10-10 | 2013-12-18 | 浙江大学 | Rapid preparation method of dicotyledonous plant transgenic acceptor |
| CN103497970A (en) * | 2013-09-04 | 2014-01-08 | 吉林省农业科学院 | Gene transfer method with corn ovule as receptor |
| CN103497970B (en) * | 2013-09-04 | 2015-06-24 | 吉林省农业科学院 | Gene transfer method with corn ovule as receptor |
| CN105647966A (en) * | 2016-03-17 | 2016-06-08 | 山东棉花研究中心 | TRV(tobacco rattle virus)-induced gene silencing method by taking cotton immature embryo as receptor for inoculation |
| CN111454984A (en) * | 2020-03-04 | 2020-07-28 | 深圳大学 | Plant gene transformation method and application thereof |
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| CN100360677C (en) | 2008-01-09 |
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Granted publication date: 20080109 Termination date: 20110513 |