CN101691583A - Agrobacterium-mediated cotton transgenic method independent of tissue culture - Google Patents
Agrobacterium-mediated cotton transgenic method independent of tissue culture Download PDFInfo
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- CN101691583A CN101691583A CN200910035360A CN200910035360A CN101691583A CN 101691583 A CN101691583 A CN 101691583A CN 200910035360 A CN200910035360 A CN 200910035360A CN 200910035360 A CN200910035360 A CN 200910035360A CN 101691583 A CN101691583 A CN 101691583A
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Abstract
The invention relates to an agrobacterium-mediated cotton transgenic method independent of tissue culture, belonging to the biotechnology field. The method comprises the following steps: dispensing agrobacterium-sucrose solution with 0.05% of surfactant Silwet L-77 and 40mg of 1-1acetosyringone on stylus in the afternoon of cotton fertilization or in the morning on the next day; preserving moisture for two days; harvesting seeds and performing resistance authentication; extracting DNA and performing PCR and Southern blot detection; and obtaining transgenic plants by multiple treatments. The transformation ratio ranges from 0.05% to 0.65%, thus proving the repeatability and reliability of transformation systems of agrobacterium-dipped cotton stylus. Compared with the traditional transgenic method dependent on tissue culture, the transformation method of the agrobacterium-dipped cotton stylus is simpler, more convenient, more economical and efficient, and simultaneously can avoid problems of limitation by cotton genetype and easy occurrence of somaclonal variation and the like in the process of tissue culture.
Description
(1) technical field:
The present invention relates to a kind of agriculture bacillus mediated cotton transgenic method that does not rely on tissue culture, be exclusively used in the seed selection of farm crop (cotton) genetic transformation and transgenosis new variety, belong to biological technical field.
(2) background technology:
Cotton is one of main textile raw material.Through systematic breeding for many years, the hereditary basis of cotton variety is quite narrow.By transgenic method useful foreign gene being imported acceptor material and makes its stably express, is to increase one of cotton genetic diversity some effective.The report of cotton genetic transformation starts from 1987, and transgene cotton also enters in nineteen nineties and commercially produces.So far, transgene cotton is that one of the most successful crop is promoted in commercialization.But the genetic transformation of cotton is still a difficult problem so far.The agriculture bacillus mediated cotton genetic transformation that depends on tissue culture is present most widely used cotton transgenic method, but this method is subject to the cotton gene type.Up to the present, have only the cotton variety genotype of minority to regenerate by tissue culture.Cultivating transgene cotton can only be easy to the foreign gene importing in the tissue culture regenerated cotton variety earlier, obtains the middle kind that foreign gene isozygotys, and hybridizes the cultivation transgene cotton with middle kind and commercial variety then.This method needs cross-breeding, has prolonged the cultivating process of transgene cotton, and because the linkage of characters, some bad economical characters of middle kind itself enter among the cultivation kind with the external source goal gene.The other method that the cotton genetic transformation is commonly used is a pollen tube passage method, promptly with microsyringe DNA is injected ovary in young age behind the chasmogamous 20-24h.This method is mainly China investigator and adopts, and to obtain certain achievement.But this method operation of this method is empirical very strong, needs certain skill, if operation is unskilled, can has influence on transformation efficiency even be difficult to obtain transformed plant.Because its repeatability is undesirable, exogenous origin gene integrator and hereditary mechanism are indeterminate, still exist more dispute.
The mechanism of agriculture bacillus mediated genetic transformation is clear relatively, is the prefered method of plant genetic conversion.The present invention is by the integrated mechanism of agrobacterium-mediated transformation conversion and the convenient means of pollen tube passage method, set up the whole strain live body transformation system that Agrobacterium is dipped the cotton style, the i.e. that afternoon after the cotton self-pollination and morning next day, drip with Agrobacterium and to be coated with the cotton style, repeatedly independent experiment all obtains transfer-gen plant, proves the feasibility of this transformation system.This method for transformation utilizes the principle of natural propagation process and pollen tube channel, by agriculture bacillus mediated T-DNA integration characteristic and advantage, having avoided the traditional pollen tube passage method of cotton is that the ovary injection adopts naked DNA to transform and shortcoming such as unclear, the hereditary mechanism of integration mechanism is not clear.Up to the present, do not see that taking Agrobacterium-sucrose solution to drip is coated with the report that the cotton style carries out genetic transformation after the cotton pollination.
(3) summary of the invention
Technical problem the objective of the invention is to set up the new cotton genetic conversion system that does not rely on tissue culture, thereby breaks through the bottleneck that the cotton genetic transformation is subjected to the genotype restriction, for cotton transgenic breeding and cotton molecular biology research are set up technology platform.
Technical scheme:
A kind of agriculture bacillus mediated cotton transgenic method that does not rely on tissue culture comprises:
1) at cotton pollination 17:00-19:00 that afternoon or pollination 9:00-11:00 in morning next day, with additional volume ratio 0.05% tensio-active agent Silwet L-77 and 40mg l
-1Syringylethanone, the genetically engineered Agrobacterium-sucrose solution that contains goal gene drip and are coated with the cotton style;
2) the bagging shading was preserved moisture two days;
3) carry out transgenosis Function Identification and Molecular Detection after the seed maturity results;
4) PCR and Southern hybridization checking goal gene has been integrated in the target cotton gene group, thereby obtains transgenic cotton plant.
Aforesaid method is used for the method for anti-herbicide gene converting cotton, it is characterized in that:
1) select nasal mucus cotton No. 3,7, cotton flowering period in August, the bud of selecting will bloom next day is done the usefulness of selfing in order to conversion;
2) the Agrobacterium strain is EHA105, and plasmid is pKF111, and this plasmid T-DNA contains in the district bar gene of Basta Herbicid resistant, and the engineering Agrobacterium that contains the bar gene is stored in additional 50mg l
-1Kantlex and 20mg l
-1On the solid LB substratum of Rifampin, clone in containing 50mg l for picking engineering Agrobacterium 2-3
-1Among the liquid LB of kantlex, 28 ℃ of overnight incubation, the centrifugal 5min of 4000rpm room temperature abandons supernatant, and is resuspended and engineering agrobatcerium cell concentration transferred to OD600=2.0 with mass volume ratio 10% sucrose; In Agrobacterium-sucrose solution, add 0.05% volume ratio Silwet L-77 and 40mg l
-1Syringylethanone is standby;
3) 9:00-11:00 in morning next day of blooming erases style, and is long-pending than 0.05% tensio-active agent Silwet L-77 and 40mg l with above-mentioned episome
-1Agrobacterium-the sucrose solution of Syringylethanone drips and is coated with cotton style wound; Or 17:00-19:00 that afternoon of blooming, Agrobacterium-sucrose solution drips and is coated with column cap; Or 17:00-19:00 that afternoon of blooming, extract column cap, Agrobacterium-sucrose solution drips and is coated with the style wound; Hua Heyou bagging in age shading after the processing is preserved moisture;
4) the results seed is containing the germination of volume ratio 0.05%Basta weedicide substratum, the seedling replanting and the normal management of performance Herbicid resistant through the sterilization back; After 3 months,, select the normal phytocide cotton plant that shows green no weedicide injury symptom with volume ratio 0.15% and 0.3%Basta herbicide treatment;
5) the phytocide cotton plant is extracted DNA, and the PCR, the Southern blot that carry out the bar gene detect, and the PCR product all contains 430bp bar gene fragment through 1% agarose gel electrophoresis proof Herbicid resistant plant.The Herbicid resistant plant that obtained changeed bar gene nasal mucus cotton No. 3, belongs to upland cotton (Gossypium hirsutum), was preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 9th, 2009, and culture presevation number is CGMCC NO.3275.
Beneficial effect:
The present invention serve as to test acceptor cotton No. 3 with Yangtze valley commercial variety nasal mucus, discovers that the antiweed transgene cotton that is obtained can resist volume ratio 0.3%Basta weedicide, and under the contrast, the non-transgenic cotton is not resisted volume ratio 0.05%Basta weedicide.PCR and Southern hybridization checking prove that also the bar gene of antiweed has been integrated in these phytocide cotton genomes.Repeatedly independent processing has obtained transfer-gen plant, and transformation efficiency from 0.05% to 0.65% does not wait, and has proved that Agrobacterium dips the repeatability and the reliability of the transformation system of cotton style.In two treatment time sections, pollination 17:00-19:00 that afternoon carries out transformation ratio pollination 9:00-11:00 in morning next day and transforms easier acquisition transgenic cotton flower seed, and carries out transformation ratio again and do not remove the efficient that column cap directly transforms and exceed several times again after pollination 17:00-19:00 that afternoon removes the cotton column cap.
Method provided by the present invention can a plurality of cotton varieties of genetic transformation, not limited by the cotton gene type, and can obtain transgenic seed at this season of cotton growth, compare with traditional transgenic method that depends on tissue culture, transformation system of the present invention is more easy, economical and efficient, has also avoided the problems such as somaclonal variation that produce in the middle of the tissue culture procedures simultaneously.
(4) description of drawings
Position synoptic diagram and field that Fig. 1 Agrobacterium is dipped column cap transform exploded view
(a) (B: the morning next day 9:00-11:00 of blooming erases to drip behind the style and is coated with Agrobacterium-sucrose solution position Agrobacterium-sucrose solution site of administration synoptic diagram, C: pollination 17:00-19:00 that afternoon drips and is coated with Agrobacterium-sucrose solution position, D: drip behind the pollination 17:00-19:00 that afternoon excision column cap be coated with Agrobacterium-sucrose solution position); (b, c) the field bagging situation of preserving moisture.
Screening of Fig. 2 antiweed plant and evaluation
(a) non-transformed the seed is in the situation (from left to right be followed successively by volume ratio 0%, 0.0005%, 0.0025%, 0.005%, 0.01%, 0.015%, 0.025%, 0.035%, 0.05%, 0.075%, 0.1%Basta) of Basta weedicide seedling culture medium culturing after 7 days.Volume ratio 0.05%Basta effectively screens concentration.(b) the resistance seedling that on volume ratio 0.05%Basta substratum, screens.(c) blade applied volume than the performance of 0.3%Basta after seven days (be followed successively by from left to right: use Basta non-transgenic contrast blade, do not use the non-transgenic contrast blade of Basta, anti-Basta plant leaf).
The PCR of Fig. 3 Basta resistant plant bar gene and Southern hybridization detect
(a) PCR detects Basta resistant plant bar gene; M:DL2000plus markers; P: plasmid contrast; C: non-transgenic contrast; The 1-13:Basta resistant plant.The resistant plant that is detected all has the band that meets goal gene 430bp size, but not does not detect this specific band in the transfer-gen plant.
(b) Southern detects Basta resistant plant bar gene; M:lambda DNA/ (EcoRI+HindIII) markers; P: plasmid contrast; C: non-transgenic contrast; The 1-13:Basta resistant plant.EcoRI enzyme with tool single endonuclease digestion site in the plasmid vector T-DNA section is cut genomic dna, and hybridization probe is the 430bp bar gene fragment of digoxigenin labeled.The resistant plant that is detected all shows the bar gene of having integrated one or more copies, but not transfer-gen plant does not have hybridization signal
(5) embodiment
1) with Yangtze valley commercial variety nasal mucus cotton No. 3 serve as the test acceptor, 7, flowering period in August, the bud of selecting will bloom next day is done the usefulness that conversion is prepared against in selfing.
2) the Agrobacterium strain is EHA105, plasmid is that pKF111 is (public, Ni M, Tepperman J, Quail P.PIF3, aPhytochrome-lnteracting Factor Necessary for Normal Photoinduced Signal Transduction, Is a Novel BasicHelix-Loop-Helix Protein.Cell, 1998,95:657-668).This plasmid T-DNA contains in the district bar gene of Basta Herbicid resistant.The engineering Agrobacterium that contains the bar gene is stored in additional 50mg l
-1Kantlex and 20mg l
-1On the solid LB substratum of Rifampin.Clone in containing 50mg l for picking engineering Agrobacterium 2-3
-1Among the 50ml liquid LB of kantlex, 28 ℃ of overnight incubation.The centrifugal 5min of 4000rpm room temperature, abandon supernatant, with the resuspended Agrobacterium precipitation of 0.5ml mass volume ratio 10% sucrose, with the concentration (mass volume ratio 10% sucrose is as blank) of Agrobacterium in the spectrophotometric determination sucrose solution, until agrobatcerium cell concentration is transferred to OD600=2.0.Adding final concentration in Agrobacterium-sucrose solution (Agrobacterium concentration OD600=2.0 sucrose concentration 10% (w/v)) is 0.05% (v/v) Silwet L-77 and 40mg l
-1Syringylethanone is standby.
3) 9:00-11:00 in morning next day of blooming erases style, and is long-pending than 0.05% tensio-active agent SilwetL-77 and 40mg l with above-mentioned episome
-1Agrobacterium-the sucrose solution of Syringylethanone drips and is coated with cotton style wound (Fig. 1-a-B); Or 17:00-19:00 that afternoon of blooming, Agrobacterium-sucrose solution drips and is coated with column cap (Fig. 1-a-C); Or 17:00-19:00 that afternoon of blooming, extract column cap, Agrobacterium-sucrose solution drips and is coated with style wound (Fig. 1-a-D).Hua Heyou bagging in age shading after the processing preserve moisture (Fig. 1 b, 1c).
4) the results seed is containing the germination of 0.05% (v/v) Basta weedicide substratum, the seedling replanting and the normal management of performance Herbicid resistant through the sterilization back.After 3 months, with 0.15% and 0.3% (v/v) Basta herbicide treatment, transfer-gen plant all normally shows green no weedicide injury symptom (Fig. 2).
5) extract phytocide cotton plant DNA with the CTAB method, concrete steps are as follows:
A. get the 5g tender leaf and place mortar, go in the centrifuge tube of 50ml, add the freshly prepared DNA extraction damping fluid of 15ml (table 2) with liquid nitrogen grinding, the vortex mixing, ice bath is preserved 10min, and 4 ℃ of centrifugal 20min of 4000rpm abandon supernatant.
B. the dna cleavage damping fluid (table 3) that in precipitation, adds 65 ℃ of preheatings of 15ml, and stir loose mixing, 65 ℃ of water-bath 30min with copper wire.
C. add the 15ml chloroform: primary isoamyl alcohol (24: 1) mixed solution, upset is left standstill 10min, 4000rpm15 ℃ of centrifugal 20min more than 50 times.
D. supernatant is changed in the 50ml centrifuge tube, add isopyknic pre-cold isopropanol, slowly overturn mixing 50 times, leave standstill 10min, the DNA flocks is gone to centrifugation in the 10ml centrifuge tube, abandon supernatant, in precipitation, add the washing with alcohol of 2ml 70%, outwell ethanol, air seasoning DNA.
E. add 3ml TE damping fluid dissolving DNA, add 3 μ l RNaseA (10mg/ml) and cultivate 30～60min for 37 ℃; Add TE to 5ml, add isopyknic phenol, slowly overturn 50 times, mixing, room temperature leaves standstill 10min, and 10,000rpm, 15 ℃ of centrifugal 10min.
F. supernatant liquor changes in the 10ml centrifuge tube, adds the equal-volume chloroform: primary isoamyl alcohol (24: 1) mixed solution, and upset is left standstill 10min more than 50 times, and 10,000rpm, 15 ℃ of centrifugal 10min.
G. supernatant liquor changes in the 10ml centrifuge tube, the 3M sodium-acetate (pH5.2) that adds 0.1 volume, overturn 50 times after adding isopyknic pre-cold isopropanol, leave standstill 30min, 10,000rpm, 15 ℃ of centrifugal 5min, abandon supernatant liquor, add 1ml 70% ethanol and wash the little group of DNA, change in the centrifuge tube of 1.5ml, 10,000rpm, 15 ℃ of centrifugal 5min; Abandon supernatant liquor, air seasoning DNA.
H. add 200 μ l TE damping fluid dissolving DNAs (4 ℃, more than the 30min) ,-20 ℃ of preservations are standby.
Table 1DNA extracts damping fluid and forms
Table 2DNA lysis buffer is formed
Above solution is limpid, extracts damping fluid in 4 ℃ of preservations, lysis buffer room temperature preservation.Press before using and add β-Me (beta-mercaptoethanol) at 1: 100.
6) the Herbicid resistant plant carries out the PCR detection of bar gene.Bar gene PCR primer is 5-ACCATCGTCAACCACTACATC-3 and 5-GCTGCCAGAAACCCACGTCAT-3, and the purpose clip size is 430bp, and the pcr amplification program is as follows: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 40s, 72 ℃ are extended 50s, 30 circulations; 72 ℃ are extended 5min.The PCR product is through 1% agarose gel electrophoresis, proves that the Herbicid resistant plant contains all that (Fig. 3 a) with bar gene fragment specific band 430bp of the same size.
7) the Herbicid resistant plant carries out Southern blot detection (Fig. 3 b).Method is as follows: after getting 20 μ g genomic dnas and cutting 12 hours with 37 ℃ of enzymes of restriction enzyme EcoRI (Fermentas company), whether get 1 μ l detects its enzyme with 0.6% agarose gel electrophoresis and cuts complete, cut as enzyme not and then to continue enzyme entirely and cut 2-3h until the genomic dna complete degestion, cut complete as enzyme, with the equal-volume chloroform: after primary isoamyl alcohol (24: the 1) extracting, equal-volume isopropanol precipitating DNA, 70% washing with alcohol, dry up precipitation, add the dissolving of 50 μ l sterilized waters.Enzyme is cut DNA through 0.6% agarose gel electrophoresis (voltage 30v, 12h) separate the back and press Sambrook (Sambrook J, Fritsch, E.M.and Maniatis, T.Molecular cloning:a laboratory manual, 2nd edition:Cold Spring Harbor Laboratory press, New York.1989) the capillary transfer method of introducing is transferred to Hybond-N+ nylon membrane (Amersham company), and hybridization probe is the 430bp bar gene of digoxigenin labeled.The mark of probe and crossover process reference reagent box specification sheets DIG High PrimeDNA Labeling and Detection Starter Kit I (Roche company).All resistant plants all detect hybridization signal, the copy number of integrating the bar gene has single copy, as duct 1, low copy number are arranged, may be two copies as duct 13, and high copy number is also arranged, and have seven copies (Fig. 3 b) as duct 4.
8) bloom morning next day 9:00-11:00 erases to drip behind the style and is coated with 175 cotton styles, gather in the crops 148 cotton bolls, amount to 4076 seeds, screen 2 strain transgene cottons (changeing bar gene nasal mucus cotton No. 3), transformation efficiency (transgene cotton strain number/results seed number * 100%) 0.05%;
17:00-19:00 that afternoon of blooming, Agrobacterium is dripped and is coated with 245 cotton column caps, gathers in the crops 132 cotton bolls, amounts to 3349 seeds, screens 4 strain transgene cottons (changeing bar gene nasal mucus cotton No. 3), transformation efficiency 0.12%;
Bloom behind the 17:00-19:00 excision that afternoon column cap, Agrobacterium is dripped and is coated with 124 cotton styles, gathers in the crops 44 cotton bolls, amounts to 1085 seeds, screens 7 strain transgene cottons (changeing bar gene nasal mucus cotton No. 3), transformation efficiency 0.65%.
The transgenic cotton flower variety that present embodiment obtained (changeing bar gene nasal mucus cotton No. 3) self-fertility, the results seed has been delivered Chinese microorganism strain preservation center preservation.
In sum, the that afternoon 17:00-19:00 of blooming extracts behind the column cap Agrobacterium-sucrose solution to drip the conversion ratio that is coated with the style wound the highest, and conversion ratio is respectively that afternoon 17:00-19:00 Agrobacterium-sucrose solution of blooming and drips and be coated with column cap and bloom that Agrobacterium-sucrose solution drips 5 times and 12 times that are coated with the style wound after morning next day, 9:00-11:00 erased style. The transfer-gen plant of three conversion processing acquisitions increases the treatment samples given figure and can obtain more transfer-gen plant all in 2 strains or more than 2 strains. Compare with traditional transgenic method that depends on the tissue cultivation, Agrobacterium is dipped more easy, the economy and efficient of method for transformation of cotton style, has also avoided simultaneously the problems such as somaclonal variation that produce in the middle of the tissue culture procedures.
Claims (2)
1. agriculture bacillus mediated cotton transgenic method that does not rely on tissue culture comprises:
1) at cotton pollination 17:00-19:00 that afternoon or pollination 9:00-11:00 in morning next day, with additional volume ratio 0.05% tensio-active agent Silwet L-77 and 40mg l
-1Syringylethanone, the genetically engineered Agrobacterium-sucrose solution that contains goal gene drip and are coated with the cotton style;
2) the bagging shading was preserved moisture two days;
3) carry out transgenosis Function Identification and Molecular Detection after the seed maturity results;
4) PCR and Southern hybridization checking goal gene has been integrated in the target cotton gene group, thereby obtains transgenic cotton plant.
2. the described method of claim 1 is used for the method for anti-herbicide gene converting cotton, it is characterized in that:
1) select nasal mucus cotton No. 3,7, cotton flowering period in August, the bud of selecting will bloom next day is done the usefulness of selfing in order to conversion;
2) the Agrobacterium strain is EHA105, and plasmid is pKF111, and this plasmid T-DNA contains in the district bar gene of Basta Herbicid resistant, and the engineering Agrobacterium that contains goal gene bar gene is stored in additional 50mg l
-1Kantlex and 20mg l
-1On the solid LB substratum of Rifampin, clone in containing 50mg l for picking engineering Agrobacterium 2-3
-1Among the liquid LB of kantlex, 28 ℃ of overnight incubation, the centrifugal 5min of 4000rpm room temperature abandons supernatant, and is resuspended and engineering agrobatcerium cell concentration transferred to OD600=2.0 with mass volume ratio 10% sucrose; In Agrobacterium-sucrose solution, add volume ratio 0.05% tensio-active agent Silwet L-77 and 40mg l
-1Syringylethanone is standby;
3) 9:00-11:00 in morning next day of blooming erases style, and is long-pending than 0.05% tensio-active agent Silwet L-77 and 40mg l with above-mentioned episome
-1Agrobacterium-the sucrose solution of Syringylethanone drips and is coated with cotton style wound; Or 17:00-19:00 that afternoon of blooming, Agrobacterium-sucrose solution drips and is coated with column cap; Or 17:00-19:00 that afternoon of blooming, extract column cap, Agrobacterium-sucrose solution drips and is coated with the style wound; Hua Heyou bagging in age shading after the processing is preserved moisture;
4) the results seed is containing the germination of volume ratio 0.05%Basta weedicide substratum through the sterilization back, and the seedling of performance Herbicid resistant is transplanted and normal management; After 3 months,, select the normal phytocide cotton plant that shows green no weedicide injury symptom with volume ratio 0.15% and 0.3%Basta herbicide treatment;
5) the phytocide cotton plant is extracted DNA, the PCR, the Southern blot that carry out the bar gene detect, the Herbicid resistant plant that is obtained changes bar gene nasal mucus cotton No. 3, belong to upland cotton (Gossypium hirsutum), be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 9th, 2009, culture presevation number is CGMCC NO.3275.
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| CN102174564A (en) * | 2011-02-14 | 2011-09-07 | 新疆农业科学院核技术生物技术研究所 | Method for obtaining genetically modified sea island cotton |
| CN102250948A (en) * | 2011-07-12 | 2011-11-23 | 南京农业大学 | Simple method for integrally transforming soybean under agrobacterium tumefaciens mediation |
| CN103667342A (en) * | 2013-11-29 | 2014-03-26 | 河南科技学院 | Method for preparing genetically modified cotton from axillary bud of cotton cotyledon |
| CN105695493A (en) * | 2016-04-12 | 2016-06-22 | 江苏省农业科学院 | Application of ALS mutant type genes in aspect of herbicide resistance |
| CN108374020A (en) * | 2017-12-28 | 2018-08-07 | 南京农业大学 | A kind of agriculture bacillus mediated soybean transformation in planta method of improvement |
| CN110791523A (en) * | 2019-12-13 | 2020-02-14 | 南京农业大学 | Cotton drought-resistant related gene GhRCHY1 and application thereof |
| CN111763689A (en) * | 2020-05-22 | 2020-10-13 | 浙江大学 | Method for improving transgenic efficiency of upland cotton standard line TM-1 |
| CN113632724A (en) * | 2021-07-28 | 2021-11-12 | 山西省农业科学院棉花研究所 | Method for improving gene transformation efficiency of cotton living body |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102174564A (en) * | 2011-02-14 | 2011-09-07 | 新疆农业科学院核技术生物技术研究所 | Method for obtaining genetically modified sea island cotton |
| CN102250948A (en) * | 2011-07-12 | 2011-11-23 | 南京农业大学 | Simple method for integrally transforming soybean under agrobacterium tumefaciens mediation |
| CN102250948B (en) * | 2011-07-12 | 2013-02-20 | 南京农业大学 | Simple method for integrally transforming soybean under agrobacterium tumefaciens mediation |
| CN103667342A (en) * | 2013-11-29 | 2014-03-26 | 河南科技学院 | Method for preparing genetically modified cotton from axillary bud of cotton cotyledon |
| CN103667342B (en) * | 2013-11-29 | 2015-10-28 | 河南科技学院 | A kind of method utilizing cotton cotyledon axillalry bud to prepare transgene cotton |
| CN105695493A (en) * | 2016-04-12 | 2016-06-22 | 江苏省农业科学院 | Application of ALS mutant type genes in aspect of herbicide resistance |
| CN108374020A (en) * | 2017-12-28 | 2018-08-07 | 南京农业大学 | A kind of agriculture bacillus mediated soybean transformation in planta method of improvement |
| CN110791523A (en) * | 2019-12-13 | 2020-02-14 | 南京农业大学 | Cotton drought-resistant related gene GhRCHY1 and application thereof |
| CN110791523B (en) * | 2019-12-13 | 2022-05-10 | 南京农业大学 | Cotton drought-resistant related gene GhRCHY1 and application thereof |
| CN111763689A (en) * | 2020-05-22 | 2020-10-13 | 浙江大学 | Method for improving transgenic efficiency of upland cotton standard line TM-1 |
| CN113632724A (en) * | 2021-07-28 | 2021-11-12 | 山西省农业科学院棉花研究所 | Method for improving gene transformation efficiency of cotton living body |
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