CN103667343A - Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method - Google Patents
Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method Download PDFInfo
- Publication number
- CN103667343A CN103667343A CN201310652991.7A CN201310652991A CN103667343A CN 103667343 A CN103667343 A CN 103667343A CN 201310652991 A CN201310652991 A CN 201310652991A CN 103667343 A CN103667343 A CN 103667343A
- Authority
- CN
- China
- Prior art keywords
- agrobacterium rhizogenes
- transgenosis
- genetic transformation
- sweet stevia
- culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention relates to an Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method. The method comprises: pre-cultured Stevia rebaudiana stem apex explant is immersed into an Agrobacterium rhizogenes solution of pRI101-ANDNA plasmid carrying target gene, treatments such as infection, co-culture and sterilization are performed, the hairy root is cultured in a kanamycin-containing screening culture medium, the anti-kanamycin hairy root is formed on the explant through induction, the hairy root is induced to form callus, differentiation of adventitious bud is performed, the adventitious bud continuously grows to form the test-tube plantlet, and a PCR amplification method is adopted to identify the transgene plant carrying out the target gene. With the technical scheme, the target gene can be transformed into the Agrobacterium rhizogenes, such that the steps of separation from the protoplast and culture are eliminated compared with genetic transformation of polyethylene glycol and other chemical methods; and compared with genetic transformation of gene gun and other physical methods, the method of the present invention has characteristics of no requirement of expensive equipment, and simple operation technology.
Description
Technical field
The present invention relates to the plant gene engineering technology field in Plant Biotechnology, the method for the transgenosis sweet Stevia genetic transformation of the transgenosis Agrobacterium rhizogenes mediation of the Agrobacterium rhizogenes mediation mediating in particular to a kind of Agrobacterium rhizogenes.
Background technology
The genetic transformation of vegetable cell is a key link of plant genetic engineering.From (1986) such as Abel, tobacco mosaic virus (TMV) (TMV) capsid protein cDNA is imported to tobacco cell, obtain after the transgenic tobacco plant of anti-TMV virus, successively developed many plant genetic transformation methods and technology.
Sweet Stevia is introduced China from 1978 Nian You the Chinese Academy of Agricultural Sciences from Japan, has spreaded all over so far the whole nation, and its blade contains natural sweeteners steviol glycoside (steviol glycosides, SGs), is a kind of novel sugar crop.China has become maximum in the world sweet Stevia producing country and steviol glycoside export State.At present, the sweet Stevia in the whole nation is produced and all to be had following problem: due to Japan cultivate to keep No. 3, Ji Shou field, No. 2, field Glycosides Contents higher, in sweet Stevia Cultivar, there is for a long time ascendancy, once monopolized national sweet Stevia plantation.The independent intellectual property rights sweet Stevia new variety that have that seed selection is good have become the problem of needing solution badly.The report that not yet has at present sweet Stevia transgenosis system made.
Summary of the invention
The present invention directly uses sweet Stevia shoop-tips as explant, draws materials conveniently.Setting up on the basis of efficient tissue culture fast-propagation system, set up and take the method for transgenosis sweet Stevia genetic transformation of transgenosis Agrobacterium rhizogenes mediation of Agrobacterium rhizogenes mediation of the Agrobacterium rhizogenes mediation that stem apex is explant, reduce the expense of institute's use instrument, experimental implementation technology is easily carried out in common lab, for utilizing from now on transgenic technology to carry out genetic improvement to sweet Stevia, provide technical support.
MS (Murashige and Skoog, 1962) substratum, is the conventional substratum of plant cell engineering, is widely used for the tissue culture of plant, respond well.The present invention's plant culture used all be take MS as minimum medium, has added the hormone of different concns.
The present invention is achieved by the following technical solutions:
(1) extract the restructuring pRI 101 AN-DNA plasmids that carry goal gene
The intestinal bacteria DH-5 α that contains restructuring pRI 101 AN-DNA plasmids is inoculated in the LB liquid nutrient medium containing 50mg/L kantlex to incubated overnight under 37 ℃, the condition of 220rpm; After alkaline lysis method of extracting restructuring pAHC25 plasmid DNA, enzyme is cut and is identified restructuring pRI 101 AN-DNA plasmids.
Alkaline lysis can extract the plasmid of bacterium, belongs to general molecular biology routine operation.
(2) prepare the sweet Stevia Shoot tip explants that genetic transformation is used
The long stem apex of clip 1-1.5cm from the test-tube plantlet of high 5 cm left and right, be inoculated on preculture substratum, in 25 ± 1 ℃ of temperature, intensity of illumination 40001x, photoperiod, be illumination in 14 hours and under dark condition, cultivate after 1-3 days for 10 hours, as the acceptor of genetic transformation.
Preculture substratum is: mg/L NAA+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8.
(3) genetic transformation of sweet Stevia Shoot tip explants
Get activation engineering bacteria liquid and proceed in aseptic centrifuge tube, the centrifugal 5min of room temperature 4000g, removes supernatant, with the resuspended OD that is diluted to of fresh MS liquid nutrient medium
600value is 0.6, and time pours in aseptic Erlenmeyer flask, and 50 mmol/L Syringylethanones to the final concentration that adds different volumes in resuspended bacterium liquid is 100 μ mol/L;
Pre-incubated explant soaks after 30 min in bacterium liquid, takes out explant and on aseptic filter paper, blots unnecessary bacterium liquid, inoculates culture medium together), 25 ℃, under 4000 lx illumination conditions, cultivate altogether 5d;
The explant of common cultivation is transferred to except carrying out degerming cultivation on bacterium culture medium, be forwarded to continuously new removing in bacterium culture medium, to remove Agrobacterium rhizogenes.
Culture medium is altogether: mg/L NAA+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8.
Except bacterium culture medium is: mg/L NAA+100mg/L Cef+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8.
(4) screening and culturing transgenosis is sent out shape root, induction of resistance callus forms and plant regeneration
The vigorous length of growth is reached to 2cm, and completely degerming, fast growth, branch are many sends out a shape root and transfers in screening culture medium and screen, and cultivates in a large number;
The tip of a root of sending out shape root through screening culture medium screening is cut into the fragment of 1cm left and right, moves in callus inducing medium, Calli Differentiation produces indefinite bud, and indefinite bud continued growth forms test-tube plantlet.
(5) pcr amplification is identified transfer-gen plant
Adopt CTAB method to extract the DNA of transfer-gen plant, as tested template, according to known array design primer, adopt PCR (Polymerase Chain Reaction) method to identify candidate plant.
The full name of CTAB method is that hexadecyl trimethyl ammonium bromide method can be extracted plant genome DNA, belongs to general molecular biology routine operation.PCR is called again " polymerase chain reaction ", is to well known to a person skilled in the art experimental technique, and according to PCR primer and PCR reaction conditions, those skilled in the art can determine PCR reaction system according to general knowledge, amplify goal gene.
Advantage of the present invention is:
The genetic transformation system of the Agrobacterium rhizogenes mediation that sweet Stevia shoop-tips sets up as explant is usingd in the present invention, can select at short notice sweet Stevia transgenosis new variety; Easily obtain transgenic regenerated plant; Required equipment is simple, and operative technique is easily grasped.
Accompanying drawing explanation
Fig. 1 is the product that Sal I and EcoR I double digestion carry the restructuring pRI 101 AN-DNA plasmids of goal gene, wherein M:DNA Marker Lane1:pRI 101 AN-DNA:::UGT76G1; Lane2-3:pRI 101 AN-DNA:::UGT76G1:::UGT76G1;
Fig. 2 is for sending out callus and the regeneration plant of shape root induction.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Test method described in following embodiment, if no special instructions, is ordinary method; Described reagent and material, if no special instructions, all can obtain from commercial channels, or can ordinary method preparation.
After activation, choose single bacterium colony and contain in the LB liquid nutrient medium of 50 mg/mL Kan in 5 ml, 220 r/min, 37 ℃ of overnight incubation.Adopt conventional alkaline lysis method of extracting restructuring pRI 101 AN-DNA plasmids, obtain after the restructuring pRI 101 AN-DNA plasmids of purifying, with ultraviolet spectrophotometer, measure the concentration of plasmid DNA solution, the concentration of plasmid DNA solution is adjusted into 1.0 μ g/L.
After Sal I and EcoR I double digestion, get 5u1 enzyme and cut liquid, electrophoresis in containing 1.5% agarose gel plate of ethidium bromide, TAE electrophoretic buffer, voltage 5V/cm.Agarose gel plate is placed under gel imaging instrument, observes the DAN fragment (Fig. 1) that two sizes are about 11Kb and 1600bp, show the goal gene that in testing, the restructuring pRI 101 AN-DNA plasmids of use carry.
Genetic transformation and the plant regeneration of embodiment 2 Agrobacterium rhizogenes mediations
(1) preculture of sweet Stevia Shoot tip explants
The long stem apex of clip 1-1.5cm from the test-tube plantlet of high 5 cm left and right, be inoculated on preculture substratum, in 25 ± 1 ℃ of temperature, intensity of illumination 40001x, photoperiod, be illumination in 14 hours and under dark condition, cultivate after 1-3 days for 10 hours, as the acceptor of genetic transformation.
Preculture substratum is: mg/L NAA+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8.
(2) genetic transformation of sweet Stevia Shoot tip explants
Get activation engineering bacteria liquid and proceed in aseptic centrifuge tube, the centrifugal 5min of room temperature 4000g, removes supernatant, with the resuspended OD that is diluted to of fresh MS liquid nutrient medium
600value is 0.6, and time pours in aseptic Erlenmeyer flask, and 50 mmol/L Syringylethanones to the final concentration that adds different volumes in resuspended bacterium liquid is 100 μ mol/L;
Pre-incubated explant soaks after 30 min in bacterium liquid, takes out explant and on aseptic filter paper, blots unnecessary bacterium liquid, inoculates culture medium together), 25 ℃, under 4000 lx illumination conditions, cultivate altogether 5d; The explant of common cultivation is transferred to except carrying out degerming cultivation on bacterium culture medium, be forwarded to continuously new removing in bacterium culture medium, to remove Agrobacterium rhizogenes.
Culture medium is altogether: mg/L NAA+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8.Except bacterium culture medium is: mg/L NAA+100mg/L Cef+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8.
(3) screening and culturing transgenosis is sent out shape root, induction of resistance callus forms and plant regeneration
The vigorous length of growth is reached to 2cm, and completely degerming, fast growth, branch are many sends out a shape root and transfers in screening culture medium and screen, and cultivates in a large number; The tip of a root of sending out shape root through screening culture medium screening is cut into the fragment of 1cm left and right, moves in callus inducing medium, Calli Differentiation produces indefinite bud (Fig. 2), and indefinite bud continued growth forms test-tube plantlet.
Claims (7)
1. the transgenosis sweet Stevia genetic transforming method of Agrobacterium rhizogenes mediation, it is characterized in that the method is that the sweet Stevia Shoot tip explants after preculture is immersed in the Agrobacterium rhizogenes bacterium liquid that contains goal gene and screening-gene plasmid, specifically comprises the following steps:
A. the long stem apex of clip 1-1.5 cm from sweet Stevia test-tube plantlet, is inoculated on preculture substratum, illumination cultivation 1-3 days, and culture temperature is that 25 ± 1 ℃, illumination are 14 h for 3000-4000 Lux, light application time by force, as the acceptor of genetic transformation;
Described preculture substratum is: mg/L NAA+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8;
B. single bacterium colony of picking Agrobacterium rhizogenes, is inoculated in the 20 mL YEP substratum that contain 50mg/mL Rifampin, at 28 ℃, after 180 rpm shaking culture 30 h, get 50 μ L bacterium liquid and be inoculated in 50mL YEP liquid nutrient medium, at 28 ℃, 180 rpm shaking culture are to OD
600be 0.6;
C. the Shoot tip explants obtaining in step a is immersed in the Agrobacterium rhizogenes bacterium liquid obtaining in step b, containing under the condition of 100 μ mol/L Syringylethanones, infecting 30min;
D. use aseptic filter paper to draw the unnecessary bacterium liquid of explant in step c, transfer in common culture medium, cultivate altogether 5 days, culture temperature is 25 ℃ altogether;
Described culture medium is altogether: mg/L NAA+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8;
E. the explant that steps d obtains proceeds to except on bacterium culture medium, carries out degerming cultivation, cultivates in 2 weeks degerming initial stages and every 3-4 days, is transferred to the new bacterium culture medium that removes, and shifts once week about, until degerming is complete later;
The described bacterium culture medium that removes is: except bacterium culture medium is: mg/L Cef+30g/L sucrose+6, mg/L NAA+100, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8;
F. the explant of step e gained is proceeded in screening culture medium, cultivate after 30d, go in callus inducing medium, be cultured to and induce callus regenerated adventitious bud;
Described screening culture medium is: mg/L NAA+100mg/L kan+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8;
G. treat that in f, indefinite bud grows to 2-3cm, go in root media, culture temperature is that 25 ± 1 ℃, illumination are by force for 3000-4000 Lux, light application time are 14 h;
Described root media is: mg/L NAA+100mg/L kan+30g/L sucrose+6, mg/L 6-BA+0.05, MS+0.1 g/L agar, pH 5.6-5.8;
H. adopt CTAB method to extract the DNA of transgenosis sweet Stevia, as detected template, adopt PCR method to identify transfer-gen plant, Auele Specific Primer used designs according to turned goal gene.
2. the method for the transgenosis sweet Stevia genetic transformation that the transgenosis Agrobacterium rhizogenes of a kind of Agrobacterium rhizogenes mediation according to claim 1 mediates, it is characterized in that, described Agrobacterium rhizogenes contains goal gene, and the Agrobacterium rhizogenes that contains the expression vector that carries goal gene builds by the following method:
After goal gene is inserted to expression vector promotor, by freeze-thaw method, the expression vector that carries goal gene building is imported to Agrobacterium rhizogenes, through resistance screening, obtain the Agrobacterium rhizogenes that contains the expression vector that carries goal gene.
3. the method for the transgenosis sweet Stevia genetic transformation that the transgenosis Agrobacterium rhizogenes of a kind of Agrobacterium rhizogenes mediation according to claim 1 mediates, it is characterized in that, in described step c, explant is soaked in to step in the Agrobacterium rhizogenes that contains the expression vector that carries goal gene and is first the Agrobacterium rhizogenes cellar culture that contains the expression vector that carries goal gene to OD
6ooafter=0.6, adding final concentration is 100 μ mol/L Syringylethanones, then explant is soaked in this Agrobacterium rhizogenes bacterium liquid, and the time is 28-30 min.
4. the method for the transgenosis sweet Stevia genetic transformation of the transgenosis Agrobacterium rhizogenes mediation of a kind of Agrobacterium rhizogenes mediation according to claim 1, is characterized in that, described method comprises the step of carrying out callus induction through the Shoot tip explants of common cultivation.
5. the method for the transgenosis sweet Stevia genetic transformation of the transgenosis Agrobacterium rhizogenes mediation of a kind of Agrobacterium rhizogenes mediation according to claim 1, is characterized in that, described method comprise callus carry out differentiation culture step.
6. the method for the transgenosis sweet Stevia genetic transformation that the transgenosis Agrobacterium rhizogenes of a kind of Agrobacterium rhizogenes mediation according to claim 1 mediates, it is characterized in that, in described S minimum medium, add the 6-benzamido group VITAMIN B4 of different concns, naphthylacetic acid, 3% sucrose and 0. 6% agar, Cef, kan, pH is 5. 6-5. 8.
7. the method for the transgenosis sweet Stevia genetic transformation of the transgenosis Agrobacterium rhizogenes mediation of a kind of Agrobacterium rhizogenes mediation according to claim 1, is characterized in that, present method can be applicable to the transgenosis of other dicotyledons.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310652991.7A CN103667343B (en) | 2013-12-06 | 2013-12-06 | A kind of transgenosis STEVIA REBAUDIANA genetic transforming method of agrobacterium rhizogenes mediation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310652991.7A CN103667343B (en) | 2013-12-06 | 2013-12-06 | A kind of transgenosis STEVIA REBAUDIANA genetic transforming method of agrobacterium rhizogenes mediation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103667343A true CN103667343A (en) | 2014-03-26 |
| CN103667343B CN103667343B (en) | 2017-10-31 |
Family
ID=50306112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310652991.7A Active CN103667343B (en) | 2013-12-06 | 2013-12-06 | A kind of transgenosis STEVIA REBAUDIANA genetic transforming method of agrobacterium rhizogenes mediation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103667343B (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296528A (en) * | 2015-10-21 | 2016-02-03 | 四川农业大学 | Genetic transformation method of lobelia sessilifolia |
| CN107365796A (en) * | 2017-09-11 | 2017-11-21 | 苏州大学 | The antibacterial processing method of the red palm transgenic line obtained to Agrobacterium_mediated method |
| CN109666692A (en) * | 2018-11-29 | 2019-04-23 | 南京农业大学 | A kind of method and its application of the soybean Hairy root of agrobacterium rhizogenes induction and soybean Mosaic evil system |
| WO2019113485A1 (en) * | 2017-12-07 | 2019-06-13 | Purecircle Usa Inc | Stevia cultivar '16228013' |
| CN110511956A (en) * | 2018-10-24 | 2019-11-29 | 聊城大学 | The pimento genetic transforming method of mediated by agriculture bacillus |
| CN112322653A (en) * | 2020-11-19 | 2021-02-05 | 北京林业大学 | Agrobacterium-mediated genetic transformation method for Maohua chrysanthemum |
| CN112430620A (en) * | 2020-12-18 | 2021-03-02 | 山东农业大学 | Agrobacterium tumefaciens-mediated chrysanthemum 'shenma' transgenic method |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
| CN114836466A (en) * | 2022-05-13 | 2022-08-02 | 宁夏大学 | Agrobacterium-mediated genetic transformation method for stevia rebaudiana |
| CN113755521B (en) * | 2021-07-29 | 2024-02-06 | 上海市农业科学院 | Construction method of agrobacterium-mediated strawberry 'sweet Charles' genetic transformation system |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1876669A (en) * | 2006-07-12 | 2006-12-13 | 哈尔滨绿叶科技开发有限责任公司 | High purity pharmaceutical stevioside preparation method |
-
2013
- 2013-12-06 CN CN201310652991.7A patent/CN103667343B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1876669A (en) * | 2006-07-12 | 2006-12-13 | 哈尔滨绿叶科技开发有限责任公司 | High purity pharmaceutical stevioside preparation method |
Non-Patent Citations (2)
| Title |
|---|
| 熊玲媛等: "GFP-mut2植物表达载体的构建及在甜菊愈伤组织中的表达", 《厦门大学学报（自然科学版）》 * |
| 郝再彬等: "高品质甜叶菊品系"1096"的组培快繁", 《中国糖料》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105296528A (en) * | 2015-10-21 | 2016-02-03 | 四川农业大学 | Genetic transformation method of lobelia sessilifolia |
| CN107365796A (en) * | 2017-09-11 | 2017-11-21 | 苏州大学 | The antibacterial processing method of the red palm transgenic line obtained to Agrobacterium_mediated method |
| US11284578B2 (en) | 2017-12-07 | 2022-03-29 | Purecircle Usa Inc. | Stevia cultivar ‘16228013’ |
| WO2019113485A1 (en) * | 2017-12-07 | 2019-06-13 | Purecircle Usa Inc | Stevia cultivar '16228013' |
| CN111511197A (en) * | 2017-12-07 | 2020-08-07 | 谱赛科美国股份有限公司 | Stevia cultivar "16228013" |
| CN110511956A (en) * | 2018-10-24 | 2019-11-29 | 聊城大学 | The pimento genetic transforming method of mediated by agriculture bacillus |
| CN110511956B (en) * | 2018-10-24 | 2023-12-05 | 聊城大学 | Agrobacterium-mediated sweet pepper genetic transformation method |
| CN109666692B (en) * | 2018-11-29 | 2021-05-28 | 南京农业大学 | Method for soybean hairy root and soybean mosaic virus disease system induced by agrobacterium rhizogenes and application of method |
| CN109666692A (en) * | 2018-11-29 | 2019-04-23 | 南京农业大学 | A kind of method and its application of the soybean Hairy root of agrobacterium rhizogenes induction and soybean Mosaic evil system |
| CN112322653A (en) * | 2020-11-19 | 2021-02-05 | 北京林业大学 | Agrobacterium-mediated genetic transformation method for Maohua chrysanthemum |
| CN112430620A (en) * | 2020-12-18 | 2021-03-02 | 山东农业大学 | Agrobacterium tumefaciens-mediated chrysanthemum 'shenma' transgenic method |
| CN112430620B (en) * | 2020-12-18 | 2023-03-21 | 山东农业大学 | Agrobacterium tumefaciens-mediated chrysanthemum 'shenma' transgenic method |
| US11299700B1 (en) | 2021-02-19 | 2022-04-12 | Acequia Biotechnology, Llc | Bioreactor containers and methods of growing hairy roots using the same |
| CN113755521B (en) * | 2021-07-29 | 2024-02-06 | 上海市农业科学院 | Construction method of agrobacterium-mediated strawberry 'sweet Charles' genetic transformation system |
| CN114836466A (en) * | 2022-05-13 | 2022-08-02 | 宁夏大学 | Agrobacterium-mediated genetic transformation method for stevia rebaudiana |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103667343B (en) | 2017-10-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103667343A (en) | Agrobacterium rhizogenes-mediated transgene Stevia rebaudiana genetic transformation method | |
| CN100379864C (en) | Genetic transformation method for tree | |
| CN109679993A (en) | A kind of construction method for the genetically modified plants that agrobacterium rhizogenes mediates | |
| CN104450711B (en) | OsmiR156f genes are increasing paddy rice effective tillering and the application in improving paddy rice single plant yield | |
| CN103444524B (en) | Method for quickly building genetic transformation regeneration system of grapes | |
| Mendes et al. | Agrobacterium-mediated genetic transformation of'Hamlin'sweet orange | |
| CN104762314A (en) | Screening marker gene-deletable plant expression vector and use thereof | |
| Yang et al. | Factors affecting Agrobacterium-mediated transformation efficiency of kumquat seedling internodal stem segments | |
| CN105255938A (en) | Agrobacterium rhizogenes mediated genetically modified Erigeron breviscapus hairy root transformation method | |
| CN105039391A (en) | Method of establishing genetic transformation of mature embryos of agrobacterium tumefaciens mediated Dongnong winter wheat No.1 | |
| CN114150003B (en) | Application of cucumber green mottle mosaic virus gene interval region in resistant tobacco cultivation | |
| US7026529B2 (en) | Methods for Agrobacterium-mediated transformation of dandelion | |
| CN116042698A (en) | Method for establishing rapid and efficient alfalfa hairy root transformation system | |
| CN108728481A (en) | A kind of method of RHIIZOMA DIOSCOREAE from Henan of China genetic transformation | |
| CN114134169A (en) | Method for establishing tobacco hairy root genetic transformation system | |
| Georgiev et al. | Agrobacterium rhizogenes-mediated genetic transformations: a powerful tool for the production of metabolites | |
| CN105886527A (en) | Agrobacterium tumefaciens mediated transformation system for efficiently obtaining transgenic plants of paspalum vaginatum and application thereof | |
| CN107034231B (en) | Gene transformation method of Chinese chestnut | |
| Magambo et al. | Somatic embryo production and GFP genetic transformation in elite Ugandan cassava genotypes | |
| CN108841853A (en) | A method of using glyphosate as the carrot genetic transformation of selective agent | |
| CN114107371B (en) | Cucumber green mottle mosaic virus gene mediated transgenic tobacco method | |
| CN112375780B (en) | Coral vegetable PDS gene VIGS silencing system and construction method and application thereof | |
| CN102676546B (en) | Polymerase gene (PGENE) and method for expressing polymerase gene (PGENE) functional protein in tomato system | |
| CN113755519B (en) | Multi-antibody screening poplar polygene genetic transformation method | |
| CN102660575B (en) | Transformation method of eucalyptus grandis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |