CN104263753A - Method for improving conversion rate of transforming wheat by agrobacterium-mediated method - Google Patents
Method for improving conversion rate of transforming wheat by agrobacterium-mediated method Download PDFInfo
- Publication number
- CN104263753A CN104263753A CN201410576583.2A CN201410576583A CN104263753A CN 104263753 A CN104263753 A CN 104263753A CN 201410576583 A CN201410576583 A CN 201410576583A CN 104263753 A CN104263753 A CN 104263753A
- Authority
- CN
- China
- Prior art keywords
- agrobacterium
- wheat
- mediated
- plant
- transformation
- 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
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the field of the genetic breeding of crops, and particularly relates to a method for improving the conversion rate of transforming wheat by an agrobacterium-mediated method. The method comprises the steps: transforming the wheat by the agrobacterium-mediated method, and controlling the concentration of liquid of agrobacterium in an infection process to OD600=0.5. According to the invention, when the OD600 value of the liquid of the agrobacterium during infection is 0.5, the transformation efficiency is obviously the highest, and relatively, when the OD600 value used at present is between 0.6 and 0.8, the highest infection efficiency is different. Through the situation, the transformation efficiency of transforming the wheat by the agrobacterium-mediated method is improved, a high-efficiency test method for breeding a new resistant variety is provided, and the method is convenient to improve the yield of the wheat and solve the problem of food.
Description
Technical field
The invention belongs to field of crop genetic breeding.Be specifically related to a kind of method improving the transformation efficiency of Agrobacterium-mediated transformation wheat.
Background technology
1, the breeding objective of wheat
Wheat (Triticum aestivum) is that ultimate production is only second to the second largest food crop of corn in the world, is one of staple food of the mankind.Along with the sharp increase of population, the minimizing of cultivated area, raising wheat yield has become the Tough questions that the whole world faces.Common wheat (AABBDD, 2n=42) be allohexaploid plant, belong to Gramineae Triticum common wheat kind (Triticum aestivum L.), the major objective of current wheat breeding remains high yield, degeneration-resistant and high-quality, molecular marker assisted selection, the application of external source excellent genes, multiple gene polymerization are modern wheat breeding study hotspot (He Zhonghu etc., 2006).The traditional breeding method cycle is long, germplasm resource discovers and uses not, and breeding method, means rely on empirical mode to a great extent, and pass through genetic engineering means, import the foreign gene of resistance to cultivated plant, develop into the new way that improvement Genes For Plant Tolerance is coerced.60 to the seventies of 20th century, people just imitate bacterial transformation approach and have carried out the trial of plant transgene.Nineteen eighty-three, Zambryski obtains first case transfer-gen plant with agrobacterium tumefaciens-mediated transformation first, has started the beginning of plant transgene, has indicated the arrival (Zambryski et al., 1983) in plant transgene epoch.1985, Horsch etc. founded agriculture bacillus mediated " leaf disk method " transgenic system, and it enormously simplify the transformation system in the past utilizing protoplastis to be acceptor, had the meaning (Horsch et al., 1985) of milestone.Sanford in 1987 etc. have invented particle gun, and this laboratory Klein uses it for plant transgene research (Klein et al., 1987) first.Which overcome the restriction being subject to genotype and acceptor specy of agrobacterium-mediated transformation at that time, start the frontier of plant transgenic method.
2, Wheat Transformation method
At present, plant transgenic technology has become the strong means of molecular biology of plants research, the experimental tool that functional genome research is indispensable especially.Wheat is allohexaploid plant, genome is large, gene regulating difficulty, the callus ubiquity of being induced by wheat explant becomes more the problems such as rate is low, callus embryo is poor, differentiation rate is low, transformant regeneration difficulty, genotype-independent are large, is the raise crop of the most difficult universally acknowledged conversion.At present, in Efficiency of Wheat Transformation system, method used comprises agrobacterium-mediated transformation, Gene Knock-out Mice, pollen tube passage method, microinjection, Laser microbeam puncture, PEG method and electric shocking method etc.But along with the continuous improvement of particle bombardment and agrobacterium co-cultivation, they become the main method of Wheat Transformation gradually.
3, Agrobacterium-mediated Transformation method
Agrobacterium is a kind of gram negative bacterium be prevalent in soil, can infect the injury of most of dicotyledons in chemotaxis ground under field conditions (factors), and induction produces crown-gall nodule or Hairy root.In the development of plant genetic engineering, investigation and application is more clearly and be successfully the genetic transformation that agrobacterium tumefaciens (Agrobacterium tumefaciens) mediates.
There is a kind of ring-type in Agrobacterium, plasmid (tumor-inducing plasmid that size is about 150 ~ 200kb, Ti-plasmids), it having one section of T-DNA (transfer-DNA region), is a kind of naturally occurring genetic conversion system.T-DNA due to Ti-plasmids carries the genes such as growth hormone, phytokinin and synthesis opine, the plant tissue by agrobacterium tumefaciens was infected is made to produce crown-gall nodule, and synthesize a large amount of opine, opine promotes the breeding of agrobacterium tumefaciens and the transfer of Ti-plasmids conversely, thus scope is infected in expansion.According to the kind of crown-gall nodule that vegetable cell of its induction produces, 3 types can be divided into: agropine-type (octopine type), nopaline (nopaline type) and agropine type (amber alkaline) (agropine type).Ti-plasmids there are two main region, i.e. T-DNA region and vir district (vir-region), in addition, also there is Plasmid replication origins and opine catabolic enzymes gene locus.T-DNA district, also known as T district (T-region), is the DNA fragmentation that Ti-plasmids can be integrated into Plant Genome, determines the form of crown-gall nodule and the synthesis of opine.The left and right sides of the T-DNA in nopaline agrobacterium tumefaciens Ti-plasmids is the tumor-necrosis factor glycoproteins of one section of 25bp, constitute the border sequence (border sequence) of T-DNA, be respectively left margin (left border, and right margin (right border LB), RB), T-DNA relies on them the foreign gene carried can be shifted and be integrated in Plant Genome.Because shift direction is that right margin has vital role to the identification of DNA binding site in T-DNA integrates, and therefore right margin is even more important by a left side from right.Vir district and toxicity district, also known as tumorigenesis region, length is about 35kb.It controls agrobacterium tumefaciens and is attached to vegetable cell and Ti-plasmids enters cell relevant portion, relevant with the formation of crown-gall nodule.Vir district is positioned on the left of T-DNA district, comprises 6 toxicity genetic locuses: virA, virB, virC, virD, virE and virG.Vir gene determines processing and the transfer process of T-DNA.Onc gene (carcinogenic), it is coding for cytokinin and plant hormone in vegetable cell, cause the vegetable cell infinite multiplication infected, in addition, because T-DNA does not promote the gene of self transfer material containing coding, therefore by unnecessary sequence knockouts such as Onc genes, the goal gene needing to transform can be replaced, reach the object of improvement plant.Foreign gene is realized to the transfer of vegetable cell and integration by infecting of Agrobacterium, then by cell and tissue culture technology, regenerate transfer-gen plant, this i.e. Agrobacterium-mediated genetic transformation (Agrobacterium-mediated transfermation).
After vegetable cell is injured, cell wall rupture, the wound-induced molecule containing high density in secretory product, as phenolic compounds such as glycoloyl syringone (OH-AS), Syringylethanones (AS).Agrobacterium tumefaciens has chemotaxis to this kind of phenolic compound, to be attached after surface of Plant callus cell, and the vir gene of its Ti-plasmids is activated.VirA genes encoding experiences albumen, is positioned at bacterial cell membrane hydrophobic region, can signaling molecule in environment of accepting, is activated expression at first.After virA gene is activated, have activated again the expression of virG gene, virG albumen transfers active state to through phosphorylation, and then activates the expression of other genes of vir district.In addition, the carbohydrate of some small-molecular-weight, as semi-lactosi, glucose etc., also can the expression of induced activation vir district gene.VirD gene product virD1 albumen makes DNA change relaxed type state into from superhelix, the T-DNA that virD2 Protein cleavage has relaxed, and make both bounded sides produce breach, T-DNA fragment is released.In addition, free virE2 albumen is combined with free virD1/virD2-strand T-DNA nucleic acid-protein mixture, forms T-complex body, and T-DNA is not by the nuclease degradation in vegetable cell in protection.T-complex body, through preformed virB protein channel, passes cell walls and the cytolemma of agrobacterium tumefaciens and vegetable cell successively, and under the guide effect of virD albumen, enters recipient cell karyon, be finally integrated in Plant Genome.
Nineteen eighty-three, the first strain is with the appearance of agriculture bacillus mediated transgene tobacco (Zambryski et al., 1983; Analogy is cultivated oneself according to a religious doctrine, and 2010).Agrobacterium-mediated Transformation method is simple, cheap and efficient, becomes the predominant methods that dicotyledon gene transforms very soon.But because wheat is monocotyledons, not the natural host of Agrobacterium, therefore the method can be introduced into the study hotspot that the monocotyledonss such as wheat become the nineties in last century.Cheng etc. (1997) utilize Agrobacterium-mediated transformation with wheat immature embryo, preculture rataria and embryo callus first for acceptor material, obtain that there is molecular biology evidence, stable transgenic wheat, and tentatively establish a set of technical system being acceptor with wheat immature embryo and embryo callus.Subsequently, agriculture bacillus mediated Wheat Transformation obtains successfully one after another in other laboratory: Xia etc. (1999) for acceptor with wheat immature embryo and embryo callus, utilize agrobacterium-mediated transformation, successfully obtain Transgenic plant of wheat; Wang Yongqin etc. (2002) then adopt 3 kinds of different agrobacterium strains carrying the binary expression vector of gus and (or) bar gene to transform the rataria of agricultural university 170 and agricultural university 146 and embryo callus, be there is callus and the plant of PPT (glufosinates) resistance in a large number, wherein, the GUS stained positive rate of kanamycin-resistant callus tissue is up to 50%-60%, antagonism plant carry out PCR and Southern detect prove, foreign gene Successful integration in Plant Genome; Wang Cuiting etc. (2003) utilize the super malicious Agrobacterium EHA105 carrying pC3301 plasmid to transform Yangmai No.158,5 strain seedlings are obtained from 294 wheat immature embryo explants, analyze through PCR and Southern blot, wherein 2 strain seedlings incorporate foreign DNA, and transformation efficiency is 0.68%.
The advantage of agrobacterium-mediated transformation: (1) success ratio is high, effective.This conversion system imitates or is referred to as to utilize natural conversion carrier system, no matter is the wound using the overall plant of inoculation, or directly infect Cells In Vitro, usually can obtains satisfied conversion; (2) in all conversion systems, Ti-plasmids conversion system is that study mechanism obtains the most clearly, and method is the most ripe, applies also the most extensive; (3) the T-DNA district of Ti-plasmids can hold the insertion of sizable DNA fragmentation, the allogeneic dna sequence reaching 50kb is intactly transferred in vegetable cell by T-DNA at present; (4) T-DNA is upper containing the sequence guiding DNA transfer and integrate, and can, by the function on of higher plant cell re-reading system identification and transcription signal, the foreign gene being inserted into T-DNA district be expressed together in vegetable cell in company with T-DNA; (5) according to the promotor needing connection different of people, foreign gene can be enable specific expressed in the various organs of regeneration plant, such as, expresses in fruit, express in leaf, even only express in root, can control artificially; (6) foreign gene that Agrobacterium Ti plasmid conversion system transforms copies as majority with single, and genetic stability is good, and majority meets mendelian inheritance, and therefore transfer-gen plant can preferably for breeding provides middle seed selection material.
4, the influence factor of transformation efficiency
Nearly twenties years, the research of Efficiency of Wheat Transformation achieved significant progress, but successful at present that good several genes is proceeded to wheat transformation efficiency is always very low.Affect Agrobacterium-mediated Transformation efficiency because have recipient genotypes, the type of explant and physiological status thereof, agrobacterium strains type, the principal elements such as medium component and Dual culture condition, transformation efficiency can be significantly improved controlling while principal element some auxiliary strategies in addition well, such as in Dual culture process, ultrasonication 7.5min and Negative pressure 7.5min is carried out to callus, transformation efficiency can be increased to 8.16% (Chen Liguo, 2007) (Chen Liguo, rear violent, Wang Yuhai etc., the Study on Genetic Transformation [J] of Agrobacterium-mediated Wheat Mature Embryos callus, wheat crops journal, 2007, 27 (2): 188-192.), add antioxidant in the medium, when the content of DTT in substratum reach 0.10% ~ 0.15%, PVPP content reach 1.00% time, agriculture bacillus mediated Wheat Transformation has obvious promoter action (Yu Huimin, 2005) (Yu Huimin, summer is photosensitive, Hou Bingkai, improves several factors [J] of agriculture bacillus mediated genetic transformation efficiency of wheat, journal of Shandong university, in December, 2005, the 40th volume, the 6th phase), expression vector proceeds to nuclear matrix attachment sequence, utilize promotor upstream and the terminator downstream expression vector all containing TM2 rice transformation and tobacco respectively, the transformation efficiency of paddy rice improves 1.5 times than control vector, the transformation efficiency of tobacco improves 1.7-2.1 doubly (Zhang M M than contrast, 2007) (Zhang M M, Ji L S, Xue H, Yang Y T, Wu C A, Zheng C C.High transformation frequency of tobacco and rice via Agrobacterium-mediated gene transfer by flanking a tobacco matrix attachment region.Physiologia Plantarum, 2007, 129:644-651.) also can improve the transformation efficiency of agrobacterium-mediated transformation (leaf is made the country prosperous infecting in liquid to add tensio-active agent and carry out the strategy such as drying treatment to callus in Dual culture engineering, 2012) (leaf is made the country prosperous, Wang Xinmin, Wang Ke etc., improve plant Agrobacterium-mediated Transformation efficiency and assist Strategy Research Progress [J], Scientia Agricultura Sinica, 2012, 45 (15): 3007-3019).
Summary of the invention
In order to overcome the lower deficiency of current Agrobacterium-mediated transformation Wheat Transformation rate, when the present invention is infected by research, Agrobacterium bacterial concentration is for the impact of transformation efficiency, provide a kind of method of transformation efficiency when improving Agrobacterium-mediated transformation wheat, efficiently solve prior art Problems existing.
Improving a method for the transformation efficiency of Agrobacterium-mediated transformation wheat, is by Agrobacterium-mediated transformation wheat, controls Agrobacterium bacterial concentration at OD600=0.5 in infection processs.
Of the present invention by Agrobacterium-mediated transformation wheat, refer to that the goal gene that will transform is connected in super malicious carrier T, proceed in Agrobacterium EHA105, utilize Agrobacterium-mediated transformation wheat.
The present invention mainly to have studied when infecting Agrobacterium bacterial concentration for the impact of transformation efficiency.By controlling the concentration of bacterium liquid when infecting, to obtain a bacterial concentration infected the most applicable be the value of OD600 when being 0.5, and the transformation efficiency transforming gained regeneration plant is the highest.Plasmid pYN203 proceeds in Agrobacterium EHA105 by the present invention, utilize Agrobacterium-mediated transformation wheat, by control variate method, control other possible factors affecting transformation efficiency and be consistent, only change Agrobacterium concentration when infecting, that determines the best infects concentration.By the interpretation of result of the positive identification in later stage, when when finding to infect, the value of the OD600 of bacterium liquid is 0.5, transformation efficiency is obviously the highest, be between 0.6 to 0.8, infect most effective different (He Jie relative to the value of the OD600 used at present, 2011) (He Jie, Hu Haiyan, Zhou Yan etc., the research [J] of agriculture bacillus mediated Efficiency of Wheat Transformation influence factor, Henan Agricultural Sciences, 2 phases in 2011).This finds that there is and helps to improve our transformation efficiency when using Agrobacterium-mediated transformation wheat, provides a kind of high efficiency experimental technique for cultivating the resistant variety made new advances, and solves food problem provide convenience for improving wheat yield.
The present invention effectively can improve transformation efficiency during Agrobacterium-mediated transformation wheat, accelerates the speed that agrobacterium-mediated transformation breeds high grade wheat.
Accompanying drawing explanation
Fig. 1. agrobacterium co-cultivation gene transfer wheat plant regenerative process
(A: callus induction; B: Dual culture; C: screening and culturing; D: differentiation culture; E: root culture; F: become transplantation of seedlings; G: ripe sowing).
Fig. 2. turn the PCR detected result of ScPRX1 Gene Partial plant
(M:Marker; +: plasmid;-: raise wheat 14; 1-38: the some positive transfer-gen plant of acquisition).
Embodiment
Embodiment 1: experiment material and instrument
Raise wheat 14 (Y14, namely raises 0-139, Lixiahe District institute of agricultural sciences Yangmai No.158 and Yang Mai No. 6 cross breedings) to be provided by the lane housing institute of agricultural sciences of Jiangsu Province, have commercially available); Containing the EHA105 bacterial strain of plasmid pYN203 (Gao Yingying, the research [D] of resistance related gene transformed wheat, Yangzhou University, 2013), bacterial strain has commercially available; Spectrophotometer.
Embodiment 2: draw materials and sterilizing
Get 12-16 days prematurity seeds (rataria size 1.0-1.2mm) after wheat flower, first use 70% alcohol surface sterilization 5min, then use 0.1% mercuric chloride (HgCl
2) sterilizing 20min, finally use sterile water wash 3-5 time.Embodiment 3: strip wheat immature embryo, callus induction
On aseptic operating platform, seed is fixed by left hand tweezers, right hand scalper chooses rataria from top to bottom after the wheat prematurity seed of sterilising treatment cuts embryo point, scultellum is (namely seed coat one side is affixed on substratum) upwards, be inoculated in inducing culture (inducing culture: MS+2,4-D 2 mg/L+ caseinhydrolysate 500mg/L+ sucrose 30g/L+ plant gel 2.6g/L) in, about every ware 60 embryos, then 7-10 days is cultivated under being placed in incubator 25 DEG C of dark conditions, induction rataria callus is formed, and is ready for use on Agrobacterium and infects conversion.
Embodiment: 4 Agrobacterium bacterium solution preparations
Infect and from-70 DEG C of refrigerators, take out the Agrobacterium bacterial classification EHA105 containing plasmid YN203 preserved in first four days, LB solid medium containing 50mg/L kantlex activates and cultivates 48h, then picking list colony inoculation incubated overnight in the 2ml LB liquid nutrient medium containing 50mg/L kantlex, 28 DEG C, 200rpm/min light culture is about 12-16h.This is cultivated on a small quantity bacterium liquid and be placed in 50ml centrifuge tube, add LB liquid nutrient medium to 25ml, expand and shake about 7h, make the value of bacterium liquid OD600 be between 0.4 to 1.2.
Embodiment 5: Agrobacterium infects wheat immature embryo callus
Super clean bench excises young shoot unnecessary in callus, centrifugal 15min under the Agrobacterium bacterium liquid having surveyed concentration is placed in 4000rpm, then 25ml Dual culture liquid medium (MS+2 is used, 4-D 2mg/L+ caseinhydrolysate 500mg/L+ Syringylethanone 50mg/L+ sucrose 30g/L) resuspended, wheat callus is placed in bacterium liquid, 20-30min is infected under room temperature, then callus is transferred on aseptic filter paper and sucks unnecessary bacterium liquid, explant is transferred to CCM solid medium (MS+2 again, 4-D 2mg/L+ caseinhydrolysate 500mg/L+ Syringylethanone 50mg/L+ sucrose 30g/L+ plant gel 2.6g/L) in, light culture 3 days at 25 DEG C.
Embodiment 6: tissue culture screening, differentiation obtain regeneration plant
The callus of Dual culture after three days is forwarded on aseptic filter paper, suck unnecessary bacterium liquid, then screening culture medium (MS+2 is proceeded to, 4-D 2mg/L+ caseinhydrolysate 500mg/L+ Totomycin 25mg/L+ cephalo 500mg/L+ sucrose 30g/L+ plant gel 2.6g/L) in, 25 DEG C, dark condition screening and culturing.About ten days subcultures once, subculture 1-2 time.After embryo callus to be grown, callus through screening and culturing is proceeded to (MS+ phytokinin 2mg/L+ naphthylacetic acid 0.5mg/L+ caseinhydrolysate 500mg/L+ Totomycin 50mg/L+ cephalo 500mg/L+ zeatin 2mg/L+ sucrose 30g/L+ plant gel 2.6g/L) in division culture medium, 25 DEG C of illumination cultivation, photoperiod be daytime/night 16h/8h, until when growing complete plantlet, proceed in root media (MS+ caseinhydrolysate 500mg/L+ cephalo 250mg/L+ paclobutrazol 5mg/L+ sucrose 30g/L+ plant gel 2.6g/L) again, carry out strengthening seedling and rooting cultivation, treat that root growth is to about 2cm acclimatization and transplants.Experimentation refers to Fig. 1.
Embodiment 7: the detection of transfer-gen plant
The extraction of 7.1 regeneration plant genomic dnas
(1) get two panels young leaflet tablet (about 0.1g), shred in the centrifuge tube loading 2ml, be placed in liquid nitrogen and cool, smash to pieces to Powdered with chopsticks;
(2) centrifuge tube room temperature is slightly placed, and adds the buffer A (buffer A) of 700 μ l, gently after mixing, and 65 DEG C of water-bath 30min (every 5min turns upside down mixing once);
(3) taking-up is slightly cooled to room temperature, adds isopyknic phenol/chloroform (each 350 μ l), turns upside down, treat without proper respect, fully mixes, extracting 5min;
(4) 4 DEG C, 12000rpm, centrifugal 10min, draw supernatant in a new centrifuge tube (2ml);
(5) add isopyknic chloroform (about 750 μ l), turn upside down, fully mix, extracting 5min;
(6) 12000rpm, centrifugal 10min, draw supernatant in a new centrifuge tube (1.5ml);
(7) add the Virahol (about 560 μ l) of 0.7 times of volume, mix gently, place more than 10min, visible flocks for-20 DEG C;
(8) 12000rpm, centrifugal 10min, supernatant discarded, adds the washing with alcohol twice of 70% of 500 μ l, each 2-3min, dries under room temperature;
(9) TER adding 20 μ l dissolves, and 37 DEG C of temperature bath 45min, 1% agarose gel electrophoresis detectable level ,-20 DEG C save backup.
The PCR of 7.2 regeneration plants detects
According to goal gene ScPRX1 distinguished sequence, design primer by software Primer Premier 5.0, ScPRX1-F:TGGTAGATGCCGAAGGAT (SEQ ID NO.1); ScPRX1-R:GGTGGAAGGGTAGGTAAA (SEQ ID NO.2), object clip size is 244bp, carries out PCR detection to the regeneration plant obtained.PCR reaction system and program are in table 1 and table 2.
Table 1 PCR reaction system
| Composition | System (25 μ l) |
| ddH 2O | 17.3μl |
| 10×PCR?buffer | 2.5μl |
| 2.5mM?dNTP | 2μl |
| Primer ScPRX1-F | 1μl |
| Primer ScPRX1-R | 1μl |
| Taq enzyme | 0.2μl |
| Template DNA | 1μl(100ng) |
Table 2 PCR response procedures
After amplification, the agarose electrophoresis detection of 1% of PCR reaction product, the results are shown in Figure 2.By experimental result, known to control time of infection when infecting be under the situation of 30min, when value transformation efficiencies after 1.0 of bacterium liquid OD600 are 0, when value transformation efficiency 0.5 time of bacterium liquid OD600 obviously increases.Detailed experimental result is in table 3.
Wheat 14 rataria experimental result is raised in the agriculture bacillus mediated ScPRX1 gene transformation of table 3.
Embodiment 8: the application of the method on other plasmids
Utilize the method, according to the test method of embodiment 2 to embodiment 7, by plasmid YM7268, (YG8198 carrier is that structure forms on the basis of carrier pCAMBIA-1304 (having commercially available).It is respectively insert the one section of attachment of the chromatin from tobacco sequence in the multiple clone site both sides of pCAMBIA-1304 that carrier YG8198 builds, i.e. SAR sequence (Su Ruibo, Chen Ming, Xu Zhaoshi etc., the stability [J] of the minimum expression cassette transgene expression of wheat is improved by matrix attachment region (SAR) sequence, Acta Agronomica Sinica, 01 phase in 2014), such energy render transgenic region forms a relatively independent section on host chromosome, is conducive to the expression of goal gene and stable heredity.By AtNHX1 (N.Tian
a, J.Wang
b, Z.Q.Xu
a,overexpression of Na+/H+antiporter gene AtNHX1 from Arabidopsis thaliana improves the salt tolerance of kiwifruit (Actinidia deliciosa), South African Journal of Botany 77 (2011) 160 – 169) gene insert YG8198 multiple clone site region obtain plasmid called after YM7268) proceed in Agrobacterium EHA105, infect the callus of raising wheat 14 IMMATURE EMBRYOS CULTURE and obtaining, after seedling, detected result is in table 4.
Claims (1)
1. improve a method for the transformation efficiency of Agrobacterium-mediated transformation wheat, be by Agrobacterium-mediated transformation wheat, it is characterized in that, control Agrobacterium bacterial concentration in infection processs at OD600=0.5.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410576583.2A CN104263753A (en) | 2014-10-24 | 2014-10-24 | Method for improving conversion rate of transforming wheat by agrobacterium-mediated method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410576583.2A CN104263753A (en) | 2014-10-24 | 2014-10-24 | Method for improving conversion rate of transforming wheat by agrobacterium-mediated method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104263753A true CN104263753A (en) | 2015-01-07 |
Family
ID=52155331
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410576583.2A Pending CN104263753A (en) | 2014-10-24 | 2014-10-24 | Method for improving conversion rate of transforming wheat by agrobacterium-mediated method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104263753A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543278A (en) * | 2016-02-02 | 2016-05-04 | 安徽农业大学 | Dangshan pear genetic transformation method |
| CN105594598A (en) * | 2016-03-29 | 2016-05-25 | 扬州大学 | Embryonic callus culture method of Yangmai 14 mature embryos |
| CN108997484A (en) * | 2017-06-07 | 2018-12-14 | 中国农业科学院作物科学研究所 | Wheat TaWox5 gene is improving the application in Wheat Transformation efficiency |
-
2014
- 2014-10-24 CN CN201410576583.2A patent/CN104263753A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105543278A (en) * | 2016-02-02 | 2016-05-04 | 安徽农业大学 | Dangshan pear genetic transformation method |
| CN105594598A (en) * | 2016-03-29 | 2016-05-25 | 扬州大学 | Embryonic callus culture method of Yangmai 14 mature embryos |
| CN108997484A (en) * | 2017-06-07 | 2018-12-14 | 中国农业科学院作物科学研究所 | Wheat TaWox5 gene is improving the application in Wheat Transformation efficiency |
| CN108997484B (en) * | 2017-06-07 | 2020-07-24 | 中国农业科学院作物科学研究所 | Application of wheat TaWox5 gene in improving wheat transformation efficiency |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102154364A (en) | Method for agrobacterium tumefaciens-mediated genetic transformation of sugarcane | |
| CN103826439A (en) | Plant transformation method | |
| CN111424022B (en) | Verticillium dahliae VdEG target gene fragment for pathogen-resistant bacteria, interference vector and application thereof | |
| CN104263753A (en) | Method for improving conversion rate of transforming wheat by agrobacterium-mediated method | |
| Chen et al. | Agrobacterium-mediated genetic transformation of peanut and the efficient recovery of transgenic plants | |
| CN103409445A (en) | Glyphosate-resistance gene MTP-SMG2-EPSPS and application thereof in cultivation of glyphosate-resistance corn | |
| Lei et al. | Agrobacterium-mediated transformation of cotton shoot apex with SNC1 gene and resistance to cotton Fusarium wilt in T1 generation | |
| CN110951742B (en) | Method for realizing plant gene replacement without generating DNA double-strand break | |
| CN1277928C (en) | Improved wheat shoot apex transformation method induced by agrobacterium | |
| CN104388462B (en) | A kind of double super poisonous carriers of T of Wheat Transformation and application | |
| CN114854785A (en) | Preparation method and application of antiviral potato plant | |
| CN102229947B (en) | Method for directly transforming cotton seed embryos by utilizing agrobacterium tumefaciens | |
| CN110699377A (en) | Peanut transgenic method | |
| Xu et al. | High-efficiency agrobacterium-mediated transformation of chrysanthemum via vacuum infiltration of internode | |
| Danilova et al. | A novel efficient method for maize genetic transformation: usage of agrobacterial monolayer | |
| CN113755519B (en) | Multi-antibody screening poplar polygene genetic transformation method | |
| Otani et al. | Genetic transformation of sweet potato (Ipomoea batatas (L.) Lam.) by Agrobacterium tumefaciens | |
| Shi et al. | Improved production of transgenic Dioscorea zingiberensis (Dioscoreaceae) by Agrobacterium tumefaciens-mediated transformation | |
| CN104341491B (en) | Drought tolerant associated protein for plant OsERF62 and its encoding gene and application | |
| CN109042297A (en) | A kind of corn inbred line SL1303 rataria method for transformation | |
| CN116004647B (en) | Tobacco NtSWEET gene and application thereof | |
| CN115074383B (en) | Agrobacterium rhizogenes mediated peach callus genetic transformation method | |
| CN104341492B (en) | Drought tolerant associated protein for plant OsERF71 and its encoding gene and application | |
| CN101575611B (en) | Genetic transformation method for agrobacterium-mediated tetraploid durum wheat Stewart | |
| CN107119069B (en) | Maize inbred line stem tip living body transformation method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20150107 |
|
| WD01 | Invention patent application deemed withdrawn after publication |