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Agrobacterium-Mediated Transfer of Arabidopsis ICE1 Gene into Lemon (Citrus Limon (L.) Burm. F. cv. Eureka) 被引量:11
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作者 HUANG Jia-quan SUN Zhong-hai 《Agricultural Sciences in China》 CAS CSCD 2005年第9期714-720,共7页
The Arabidopsis ICEI (inducer of CBF expression 1) gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon (Citrus Limon (L.) Burm. F. cv. Eureka) genome using Agrobacterium-mediated tr... The Arabidopsis ICEI (inducer of CBF expression 1) gene was cloned through RT-PCR of Arabidopsis cDNAs and introduced into the lemon (Citrus Limon (L.) Burm. F. cv. Eureka) genome using Agrobacterium-mediated transformation method. Epicotyl segments from in vitro grown lemon seedlings were co-cultivated with A. tumefaciens strain EHA 105 carrying the binary plasmid pMVICE1, whose T-DNA region contain ICEI gene driven by 35S CaMV promoter. Among 320 epicotyl segments inoculated, 71 explants responded and regenerated 51 elongated shoots. These shoots were subjected to an extra month of kanamycin exposure. In this way, the number of escapes reduced. Thirteen of 31 survived shoots formed roots and 7 were tested positive using PCR technique. Southern blot analyses confirmed PCR results and demonstrated that more than two copies of the ICE1 gene were integrated into the lemon genome. 展开更多
关键词 LEMON ICE1 agrobacterium-mediated transformation REGENERATION
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Agrobacterium-Mediated Transformation of Rice: Constraints and Possible Solutions 被引量:7
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作者 Sulaiman MOHAMMED Azman Abd SAMAD Zaidah RAHMAT 《Rice science》 SCIE CSCD 2019年第3期133-146,共14页
Genetic transformation of rice(Oryza sativa L.) by introducing beneficial traits is now a central research instrument in plant physiology and a practical tool for plant improvement. Many approaches are verified for st... Genetic transformation of rice(Oryza sativa L.) by introducing beneficial traits is now a central research instrument in plant physiology and a practical tool for plant improvement. Many approaches are verified for stable introduction of foreign genes into the plant genome. The review examined the different constraints that limit the success of rice genetic transformation via Agrobacterium-mediated approach and suggested possible solutions. Explant identification, gene transfer technique and construct to tailor the integration, transgene expression without collateral to genetic damage and transformant selection are among the technical challenges affecting the rice transformation. Despite the contests, Agrobacteriummediated transformation system has been a better option for producing transgenic rice varieties because of its exact T-DNA processing and simple integration of low copy-number transgene. This information is necessary for improving the transformation system for recalcitrant rice varieties. 展开更多
关键词 RICE agrobacterium-mediated TRANSFORMATION TISSUE CULTURE gene transfer T-DNA integration
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Effect of Phytosulfokine-α on Agrobacterium-Mediated Transformation in Rice 被引量:3
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作者 CHEN De-xi Xu Zheng-jun MA Bing-tian LI Shi-gui 《Rice science》 SCIE 2005年第4期255-260,共6页
Phytosulfokine- α (PSK- α ), a biologically active peptide acting as a growth factor, plays a key role in cellular differentiation and proliferation. To test if PSK- α has some influence on agrobacterium-mediated... Phytosulfokine- α (PSK- α ), a biologically active peptide acting as a growth factor, plays a key role in cellular differentiation and proliferation. To test if PSK- α has some influence on agrobacterium-mediated transformation in rice, PSK-α at a series of concentrations was added into co-culture medium respectively. The results showed that PSK- α indeed affected the recovery of resistant calli and the transformation frequency of rice varieties Taipei 309 and Lijiangxintuanheigu, PSK- α at the concentration of 10 nmol/L could increase induction of resistant callus and efficiency of transformation, with a 11% and 4.9% top increase, respectively than the control. However, PSK- αat 200 nmol/L could inhibit the induction of the resistant calli. Further more, the effect of PSK-α on agrobacterium-mediated transformation is related with the concentration of 2, 4-D in selection medium. Higher induction rate of resistant calli was obtained from tissues treated with PSK- α plus 2 mg/L 2, 4-D. 展开更多
关键词 PHYTOSULFOKINE RICE agrobacterium-mediated transformation
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Development of Alfalfa (Medicago sativa L.) Regeneration System and Agrobacterium-Mediated Genetic Transformation 被引量:10
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作者 ZHANG He HUANG Qi-man SU Jin 《Agricultural Sciences in China》 CAS CSCD 2010年第2期170-178,共9页
The regeneration ability of four alfalfa (Medicago sativa L.) cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on reg... The regeneration ability of four alfalfa (Medicago sativa L.) cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on regeneration were compared. The better callus forming frequency obtained from hypocotyls of Xinjiang Daye is 88.5% and regeneration frequency is 9.8% in our initial experiments. To further optimize regeneration system for genetic transformation, we therefore changed concentrations of plant growth regulators and supplemented with glutamine into callus-induction and shoot-regeneration media. Callus forming frequency and shoot differentiation frequency were increased to 100%. The time taken to generate transgenic plants (16 weeks) was shorter than that for previouse procedure (25 weeks) and regeneration frequency was promoted to 15.1%. The results show that addition of glutamine is particularly important for shortening period of regeneration and promoting regeneration frequency. For study of genetic transformation of alfalfa, Agrobacterium tumefaciens-mediated transformation of Xinjiang Daye was developed based on this optimized regeneration system. The plant expression vector carrying two glutamine synthetases (GS 1 and GS2) and △1-pyrroline-5-carboxylate synthetase (P5CS) gene was used for alfalfa in vitro transformation. Six transgenic alfalfa plantlets with resistance to PPT were obtained. The introduction of foreign genes into plants was assessed in the transformants by PCR analysis and Southern hybridizations. 展开更多
关键词 alfalf glutamine synthetases △1-pyrroline-5-carboxylate synthetase agrobacterium tumefaciens-mediated transformation transgenic alfalfa
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Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.) 被引量:4
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作者 YU Gui-rong LIU Yan +8 位作者 DU Wen-ping SONG Jun LIN Min XU Li-yuan XIAO Fang-ming LIU Yong-shengKey Laboratory for Bio-Resource and Eco-Environment Ministry of Education/State Key Laboratory of Hydraulics and Mountain River Engineering College of Life Science Sichuan University 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2013年第12期2134-2142,共9页
Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested ... Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine(100 mg L-1) in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA(5 mg L-1) and a low concentration of 2,4-D(0.2 mg L-1) to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome. 展开更多
关键词 MAIZE immature embryo agrobacterium tumefaciens-mediated transformation transgenic approach glyphosate resistance
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In planta Agrobacterium-Mediated Transformation of Rice 被引量:3
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作者 Kumrop RATANASUT Weerawan ROD-IN Kawee SUJIPULI 《Rice science》 SCIE CSCD 2017年第3期181-186,共6页
The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to vari... The floral-dip transformation, the simplest technique, is no requirement of tissue culture procedure, and can directly transfer the interest gene into plant reproductive cells. It has been successfully applied to various plant species. In this study, the optimal conditions of a floral-dip method for production of transgenic rice variety RD41 were explored. The simple and effective inoculation medium was composed of Murashige and Skoog(MS) medium, 5% sucrose, 44 nmol/L benzylaminopurine, and 0.075% surfactant Tween-20 with pH 5.7. The transformation efficiencies of Agrobacterium tumefaciens strains AGL1 and EHA105 were compared with the Agrobacterium density at OD_(600) = 0.8–1.0 and the co-cultivation at 25 ℃ for 48 h. A. tumefaciens strain EHA105 gave slightly higher transformation efficiency than AGL1, with statistically non-significant difference. The floral-drop transformation using the optimal floral-dip conditions showed higher transformation efficiency than the floral-dip method, but the dropped flowers turned brown and died within 2 d. Production of transgenic rice variety RD41 by the floral-dip method was achieved using A. tumefaciens strain EHA105 with the optimal conditions. Screening for the gus A gene by PCR using the gus A specific primers in the T_0 lines, there were 4 transgenic lines from 286 T_0 lines(1.4% transformation efficiency). However, histochemical glucuronidase(GUS) assay demonstrated that only three of four transgenic lines exhibited gus A expression. These results indicated that floral-dip transformation is a potential tool for production of the transgenic rice, which can be used for molecular breeding via genetic engineering in the future. 展开更多
关键词 agrobacterium-mediated TRANSFORMATION floral-dip floral-drop TRANSFORMATION efficiency in PLANTA RICE
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Scarabaeid Larvae- and Herbicide-Resistant Transgenic Perennial Ryegrass (Lolium perenne L.) Obtained by Agrobacterium tumefaciens-Mediated Transformation of cry8Ca2, cry8Ga and bar Genes 被引量:3
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作者 WU Jin-xia ZHANG Zhi-guo +2 位作者 ZHANG Qian LANG Zhi-hong SUN Xue-hui 《Journal of Integrative Agriculture》 SCIE CSCD 2012年第1期53-61,共9页
Insect pest and weeds are two major problems for forage and turf grasses. In this study, scarab larvae- and herbicide-resistant transgenic perennial ryegrass (Lolium perenne L.) was obtained by transforming it with ... Insect pest and weeds are two major problems for forage and turf grasses. In this study, scarab larvae- and herbicide-resistant transgenic perennial ryegrass (Lolium perenne L.) was obtained by transforming it with cry and bar genes simultaneously via the Agrobacterium-mediated method. To optimize the callus induction and plant regeneration conditions, various concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine were assayed. The transformation efficiencies of different Agrobacterium suspension media, used during Agrobacterium-mediated transformation, were compared. Then, plasmids of pCAMBIA3301 containing cry gene (cry8Ca2 or cry8Ga) and bar gene, driven by ubiquitin promoter, were transformed into perennial ryegrass. The transformants were generated and confirmed by both Southern hybridization analysis and Western hybridization analysis. Further, the resistance of transgenic perennial ryegrass plants to scarab larvae and herbicide were analyzed. After 30 d of co-cultivation with scarab larvae, the damage to the root system of transgenic plants was less than that of non-transgenic control plants. Additionally, the leaves of transgenic plants were resistant to Basta, while leaves of the wild plants wilted after Basta spraying. These results show that cry gene and bar gene were successfully transferred into perennial ryegrass by the Agrobactgerium-mediated method, and convey resistance to scarab larvae and herbicide in transgenic perennial ryegrass plants. 展开更多
关键词 agrobacterium tumefaciens-mediated transformation transgenic perennial ryegrass scarab larvae resistance herbicide resistance
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Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA 被引量:3
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作者 YANG Ying-qing YANG Mei +3 位作者 LI Ming-hai LI Yong HE Xiao-xia ZHOU Er-xun 《Rice science》 SCIE 2011年第4期297-303,共7页
To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transf... To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118. 展开更多
关键词 rice sheath blight Rhizoctonia solani agrobacterium tumefaciens-mediated transformation T-DNA insertional mutagenesis METHODOLOGY
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Response of Explants of Calli Rice (<i>Oryza sativa</i>L.) Japonica cv. “Ilmi” to Gene Transformation Using <i>Agrobacterium tumefaciens</i>-Mediated 被引量:1
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作者 Fika Ayu Safitri Mohammad Ubaidillah +1 位作者 Miswar   Kyung-Min Kim 《American Journal of Plant Sciences》 2013年第4期838-843,共6页
The AtBI-1 gene encoding the Arabidopsis thaliana Bax inhibitor was introduced into Japonica cultivars of rice (“Ilmi”) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced... The AtBI-1 gene encoding the Arabidopsis thaliana Bax inhibitor was introduced into Japonica cultivars of rice (“Ilmi”) by Agrobacterium-mediated transformation, and a large number of transgenic plants were produced. The neomycin phosphotransferase II (NPTII) gene was used as a selectable marker. The activity of neomycin phosphotransferase could be successfully detected in transgenic rice calluses. Introduction of the AtBI-1 gene was also confirmed by PCR using AtBI-1 specific oligonucleotide primers in regenerated plants. Stable integration and expression of the AtbI-1 gene in plants were confirmed by GFP analysis. 展开更多
关键词 AtBI-1 agrobacterium-mediated GFP Transformation RICE
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Development of Marker-Free Transgenic Cry1Ab Rice with Lepidopteran Pest Resistance by Agrobacterium Mixture-Mediated Co-transformation 被引量:2
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作者 QI Yong-bin YE Sheng-hai +2 位作者 Lu Yan-ting JIN Qing-sheng ZHANG Xiao-ming 《Rice science》 SCIE 2009年第3期181-186,共6页
CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and ... CrylAb gene was transformed into four rice varieties, Zhejing 22, Zhejing 27, Jiahua 1 and Xiushui 63 mediated by Agrobacterium-mixture co-transformation. Rice genotype had an important effect on callus induction and transformation efficiency. Different mixtures of Agrobacterium strains (EHA105 and EHA101) contained Hpt and CrylAb genes resulted in different frequencies of resistant calli. There was no correlation between the frequency of transformants with the ratio of the Agrobacterium strain mixture contained Hpt and CrylAb genes. A total of 509 transgenic plants were obtained from the four rice varieties, and 272 T2 progenies were analyzed for CrylAb and Hpt genes. PCR analysis revealed that 412 regenerated plants were Hpt positive (80.94%), 62 plants were also CrylAb co-transformants (15.05% in total frequency), and 42 plants among the 272 T2 progenies were CrylAb positive but Hpt negative. This suggests that marker-free transgenic plants could be produced by co-transformation mediated by mixed Agrobacterium strains with the selectable marker gene and target gene Southern blot analysis of five independent marker-free T2 transgenic lines co-transformed from Zhejing 22 showed that CrylAb gene had been inserted into rice genome with a single copy. The transgenic plants showed significantly stronger resistance to lepidopteron than the non-transgenic plants under no application of insecticides against lepidopteron. 展开更多
关键词 rice (Oryza sativa) agrobacterium mixture-mediated co-transformation CrylAb gene Hpt gene gene transfer marker-free transgenic plants pest resistance
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Agrobacterium-mediated Transformation and Regeneration by Direct Shoot Organogenesis in Cotton (G. hirsutum)
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作者 G.BALASUBRAMANI J.AMUDHA +2 位作者 P.A.KUMAR A.B.DONGRE C.D.MAYEE 《棉花学报》 CSCD 北大核心 2002年第S1期87-87,共1页
Genotype independent transformation andregeneration of Indian cotton(Gossypiumhirsutum L.)cultivar was standardized with Bt-Cry 1A(b)gene by Agrobacterium-mediation.Apical meristem of elite G.hirsutum cultivarLRK-516 ... Genotype independent transformation andregeneration of Indian cotton(Gossypiumhirsutum L.)cultivar was standardized with Bt-Cry 1A(b)gene by Agrobacterium-mediation.Apical meristem of elite G.hirsutum cultivarLRK-516 and LRA 5166 were co-cultivated withA.tumefaciens LBA 4404 carrying synthetic Bt- 展开更多
关键词 COTTON agrobacterium COTTON ELITE CULTIVAR Transformation REGENERATION carrying standardized mediatION
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<i>Agrobacterium</i>-Mediated Transformation of Mexican Lime (<i>Citrus aurantifolia</i>Swingle) Using Optimized Systems for Epicotyls and Cotyledons
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作者 Maria Luiza P. de Oliveira Gloria Moore +1 位作者 James G. Thomson Ed Stover 《Advances in Bioscience and Biotechnology》 2015年第11期657-668,共12页
Transgenic Mexican lime (Citrus aurantifolia Swingle) was produced through two explant sources, each using systems previously optimized for each source. One used epicotyls segments, which was the predominant explant f... Transgenic Mexican lime (Citrus aurantifolia Swingle) was produced through two explant sources, each using systems previously optimized for each source. One used epicotyls segments, which was the predominant explant for transgenic Citrus production following co-cultivation with Agrobacterium, and has a well-established protocol. The other procedure used embryo cotyledons from mature seeds, which was developed in our lab as an alternative for stable Citrus transformation. Cotyledon transformation and regeneration protocols were optimized by comparing variables in culture medium composition on shoot regeneration and four parameters in transient transformation. The optimized protocols were compared, and frequency of regeneration, frequency of transgenic plant-recovery and stable transformation efficiency indicated the superiority of the cotyledon protocol for Agrobacterium-mediated genetic transformation in Mexican lime. The tissue choice resulted in marked improvement in shoot regeneration (14.1% of explants producing shoots in epicotyls;55.8% in cotyledons), stable transformation frequency (11.4% of epicotyls explants;40.2% in cotyledons), and frequency of transgenic plant-recovery (37.9% in epicotyl explants;92.6% in cotyledons). Thus, easy availability of explants using embryo cotyledons from mature seeds, technical simplicity, shortening of transformation time-course, and higher transformation and regeneration frequencies makes this new system an attractive alternative over the previously published Citrus transformation protocols. In the course of this project, we generated Mexican lime with a Recombinase Mediated Exchange Cassette landing pad, which was designed for stacking transgenes. 展开更多
关键词 agrobacterium TUMEFACIENS Embryo COTYLEDONS Epicotyls Segments RECOMBINASE mediated Exchange Cassette Tissue Culture
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Agrobacterium-Mediated Transformation of Embryogenic Calli of Anliucheng and Regeneration of Plants Containing the Chimaeric Ribonuclease Gene
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作者 LI Dong-dong, SHI Wei and DENG Xiu-xinNational Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University , Wuhan 430070 , P. R. China 《Agricultural Sciences in China》 CAS CSCD 2003年第2期127-131,共5页
Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cul-tivar in south of China, was transformed by the strain of Agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, whi... Anliucheng (Citrus sinensis Osbeck), a very seedy and widely spread acidless sweet orange cul-tivar in south of China, was transformed by the strain of Agrobacterium Tumefaciens EHA105 carrying pTA29-barnase gene, which will induce pollen sterility in transgenic plants. The embryogenic calli of Anliucheng were co-cultivated with Agrobacterium tumefaciens for 3 days, and then transferred to selective medium containing 50 mg I/1 basta (a kind of herbicide) for 5 weeks. The resistant calli were recovered and regenerated 118 embryoids. A total of 13 entire plants were obtained after micro-grafted on trifoliate orange. These regenerated plants were verified by PCR amplification and confirmed by PCR-Southern blotting analysis. 展开更多
关键词 CITRUS CALLI pTA-barnase Genetic transformation agrobacterium-mediated
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Female reproductive system of <i>Amaranthus</i>as the target for <i>Agrobacterium</i>-mediated transformation
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作者 Umaiyal Munusamy Siti Nor Akmar Abdullah +1 位作者 Maheran Abd Aziz Huzwah Khazaai 《Advances in Bioscience and Biotechnology》 2013年第2期188-192,共5页
Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy... Agrobacterium-mediated transformation through floral dip and rapid selection process after transgenic event had become a preference as it will overcome the difficulties faced in tissue culturing procedures and lengthy time for screening transformed progenies. Therefore, in this study, three constructs, p5b5 (14,289 bp), p5d9 (15,330 bp) and p5f7 (15,380 bp) in pDRB6b vector which has hygromycin as a selectable marker gene were introduced individually into Agrobacterium tumefaciens strain (AGL1). The cell suspension was applied to Amaranthus inflorescence by drop-by-drop technique and was left to produce seeds (T1). The T1 seeds were germinated and grown to produce seedlings under non-sterile condition. Hygromycin selection on seedling cotyledon leaves results in identification of 12 putative transformants, three from p5b5, four from p5d9 and five from p5f7. All positive putative transformants that were selected at the first stage through hygromycin spraying showed positive result in leaf disk hygromycin assay and in a construct specific polymerase chain reaction-based assay. A ~750 bp amplified hygromycin gene was further verified through sequencing. Our results suggest that Amaranthus inflorescences were able to be transformed and the transformed progenies could be verified through a combination of simple and rapid methods . 展开更多
关键词 agrobacterium-mediated Transformation AMARANTHUS Direct PCR Female Reproductive System Floral-Dip HYGROMYCIN
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Efficient Regeneration and Agrobacterium-mediated Transformation of Brassica napus Cultivar Qingza No.5
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作者 Yajing XIE Aihua WU Xianjin LIU 《Agricultural Biotechnology》 CAS 2016年第3期48-53,56,共7页
Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish... Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB + 5 mg/L thidiazuron (TDZ) +7.5 mg/L AgNO3 + 0.1 mg/L NAA + 2 mg/L proline (L-pro) + 250 mg/L casein acid hydrolysate (CH) + 3% sucrose; the appropriate growth medium was 1/2 MSB + 1 mg/L IBA + 2 mg/L L-pro + 250 mg/L CH + 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB + 0.2 mg/L IAA + 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants. 展开更多
关键词 words Brassica napus L. Efficient regeneration from hypocotyls agrobacterium-mediated transformation Insect resistance genes GUS staining Transgenicplants
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Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System for Aspergillus awamori
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作者 Feng CHEN Kun WANG +3 位作者 Chao YIN Deming LI Nan REN Junxing LI 《Agricultural Biotechnology》 CAS 2012年第3期44-48,51,共6页
[ Objective ] This study aimed to establish Agrobacterium tumefaciens-mediated genetic transformation system for Aspergillus awamori and investigate the feasibility of expressing heterologous proteins in A. awamori. [... [ Objective ] This study aimed to establish Agrobacterium tumefaciens-mediated genetic transformation system for Aspergillus awamori and investigate the feasibility of expressing heterologous proteins in A. awamori. [ Method] Appropriate A. awamori host strains were determined according to the secretory protein profile. Selectable marker was selected for genetic transformation by drug sensitivity analysis. The established A. awamori genetic transformation system was used for transformation and expression analysis of Rhizomucor miehei lipase (RML). The feasibility of using A. awamori to express heterologous proteins was investigated by identification of transformants and property analysis. [ Result~ Based on the analysis of secretory protein profile, A. awamori strains CBS115.52 and CICC2257 were determined as the host strains for heterologous protein expression ; drug sensitivity analysis shows that hygromycin B resistance gene ( HygBr ) is an effective ge- netic seleetable marker; by using Agrobacterium tumefaciens-mediatcd transformation (ATMT) method, the plasmid pHGW-amdS containing HygBr was successfully transformed into A. awamori strain CBS115.52 to establish the genetic transformation system ofA. awamorl with HygBr as selectable marker. RML was transformed into A. awamori and its expression was validated by substrate hydrolysis test, SDS-PAGE and Western blot. [ Conclusion] This study demonstrates that the genetic transformation system of A. awamori mediated by Agrobacterium tumefaciens has potential feasibility for expression of heterologous proteins. 展开更多
关键词 Aspergillus awamoH agrobacterium tumefaciens-mediated transformation (ATMT) Hygromycin B resistant gene (HygBr) Rhizomucor miehei lipase (RML)
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Transformation of dissociation transposon in japonica rice Zhonghua 11 mediated by Agrobacterium 被引量:1
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作者 ZHANG Jianjun YIN Liqin WANG Xinqi FAN Kunhua CHEN Quanqing SHEN Gezhi,Crop Res Inst,Shanghai Acad of Agri Sci,Shanghai 201107,China 《Chinese Rice Research Newsletter》 1999年第2期3-4,共2页
Dissociation (Ds) transposon is one of thetransposable elements in corn. The trans-posons can be transferred into other plantswhere the transposons were not found. Oncethe transposon was inserted into target gene ofth... Dissociation (Ds) transposon is one of thetransposable elements in corn. The trans-posons can be transferred into other plantswhere the transposons were not found. Oncethe transposon was inserted into target gene ofthese plants, it could be used as a marker todistinguish and isolate the gene. The object ofthis study is to transfer Ds transposon to riceby Agrobacterium -mediated transformation.The calli of immature embryos, mature 展开更多
关键词 gene Transformation of dissociation transposon in japonica rice Zhonghua 11 mediated by agrobacterium
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Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells 被引量:5
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作者 LUAN Bo~1,HAN Ya-ling~1,SUN Ming-yu~1,GUO Liang~1,GUO Peng~1,TAO Jie~1,DENG Jie~1,WU Guang-zhe~1,YAN Cheng-hui~1, LI Shao-hua~2 (1.Department of Cardiology,Shenyang Northern Hospital, Shenyang,China 2.Division of Vascular Surgery,Robert Wood Johnson Medical School-UMDNJ,New Jersey,USA) 《岭南心血管病杂志》 2011年第S1期186-186,共1页
Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle ce... Background The present study aimed to investigate the detailed mode and specific sites for their binding as well as the functional relevance of this binding in the phenotypic proliferation of vascular smooth muscle cells(SMCs). Methods CREG knocked-down SMCs were employed to evaluate the biological activity of wtCREG and mCREG.Expressions of SMC differentiation markers SM myosin heavy chain(SM-MHC),SM-actin,heavy caldesmon and myocardin were determined by Western blotting using specific antibodies. Cellular growth of SMCs was assessed by bromide dewuridine (BrdU) incorporation and cell cycle analysis on fluorescence-activated cell sorting(FACS).A solid-phase binding assay was used to study the binding of CREG to extracellular domains of M6P/IGF2R.The cellular co-localization of the two recombinant CREGs with M6P/IGF2R was detected on SMC surface by immunoprecipitation and immunofluorescence analysis.Results The molecular weight of wtCREG was around 30 kD while that of the mCREG was~25 kD.Treatment of wtCREG with PNGase F reduced its molecular weight from~30 kD to~25 kD,whereas PNGase F treatment had no effect on the molecular weight of mCREG.Both wtCREG and mCREG proteins enhanced SMC differentiation,inhibited BrdU incorporation,and arrested cell cycle progression when added to the culture medium.In CREG knocked-down SMCs,the amount of CREG detected by immunoblotting in M6P/IGF2R immunoprecipitates was significantly reduced when compared to normal cells.Both recombinant CREGs co-immunoprecipitated with M6P/IGF2R, although slightly reduced amount of the mutant CREG was detected in M6P/IGF2R immunoprecipitates.Immunostaining revealed that His-tagged CREGs co-localized with IGF2R on the cell surface in a glycosylation-independent manner.In vitro binding assay showed that CREGs bound to M6P/ IGF2R extracellular domains 7-10 and 11-13 in a glycosylation -dependent and -independent manner,respectively.Further blocking experiments using soluble M6P/IGF2R fragments and M6P/IGF2R neutralizing antibody indicated that the biological activities of recombinant CREGs in SMC growth and the up-regulation of SMC differentiation markers were all abolished by treatment with the M6P/IGF2R neutralizing antibody. However,although the growth inhibitory effect of wtCREG was nearly abolished by D7-10 or D11-13,the effect of mCREG was only reversed by Dll-13,indicating that the binding to domains 11-13 is required for CREG to modulate the proliferation of SMCs.Conclusions These data suggest that solubleCREG proteins can exert their biological function via binding to the extracellular domains 7-10 and 11-13 of cell surface M6P/IGF2R in both a glycosylation-dependent and -independent manner. 展开更多
关键词 CREG Glycosylation-independent binding to extracellular domains 11-13 of mannose-6-phosphate/insulin-like growth factor-2 receptor mediates the effects of soluble CREG on the phenotypic proliferation of vascular smooth muscle cells IGF
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基于农杆菌介导法将CP4-EPSPS基因转入玉米自交系B73的研究
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作者 韩平安 常悦 +6 位作者 唐宽刚 李晓东 王力伟 梁亚晖 杨静 石海波 吴新荣 《北方农业学报》 2023年第1期31-37,共7页
【目的】建立玉米遗传转化体系培育抗除草剂玉米材料,解决玉米杂草危害问题。【方法】采用农杆菌介导法在玉米自交系B73幼胚中转入抗除草剂基因(CP4-EPSPS),通过PCR、qRT-PCR鉴定转基因植株;利用ddPCR技术筛选低拷贝转基因植株,并对低... 【目的】建立玉米遗传转化体系培育抗除草剂玉米材料,解决玉米杂草危害问题。【方法】采用农杆菌介导法在玉米自交系B73幼胚中转入抗除草剂基因(CP4-EPSPS),通过PCR、qRT-PCR鉴定转基因植株;利用ddPCR技术筛选低拷贝转基因植株,并对低拷贝植株进行草铵膦抗性鉴定。【结果】在所获得的181株抗性植株中12株为阳性,转基因植株的外源抗除草剂基因(CP4-EPSPS)在转录水平上均能正常表达;转基因植株中筛选到低拷贝植株5株;抗性鉴定证明转基因植株均具有草铵膦抗性。收获T1代转基因低拷贝植株种子5份。【结论】建立了以B73为受体,基于农杆菌法介导的玉米遗传转化体系。 展开更多
关键词 玉米 农杆菌介导法 抗草甘膦基因(CP4-EPSPS) 遗传转化
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反义RNA介导GmFAD2-1B基因沉默增强大豆种子中油酸的高效累积 被引量:9
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作者 杨静 邢国杰 +5 位作者 牛陆 贺红利 杜茜 郭东全 袁英 杨向东 《作物学报》 CAS CSCD 北大核心 2017年第11期1588-1595,共8页
油酸含量是评价大豆油食用品质和稳定性的重要指标。本研究采用农杆菌介导转化法,将反义GmFAD2-1B基因导入栽培大豆品种,获得油酸含量显著提高的转基因大豆新品系。Southern杂交检测表明,外源GmFAD2-1B基因片段已导入大豆基因组,其插入... 油酸含量是评价大豆油食用品质和稳定性的重要指标。本研究采用农杆菌介导转化法,将反义GmFAD2-1B基因导入栽培大豆品种,获得油酸含量显著提高的转基因大豆新品系。Southern杂交检测表明,外源GmFAD2-1B基因片段已导入大豆基因组,其插入拷贝数为1~5个。q RT-PCR检测表明,外源GmFAD2-1B主要在大豆种子中表达,并导致种子中内源GmFAD2-1 m RNA表达水平显著降低,而根、茎、叶、花组织中内源GmFAD2-1 m RNA表达水平无显著变化。脂肪酸组分分析表明,12份转基因大豆种子油酸含量为27.38%~80.42%,其中,L40和L72油酸含量分别为68.91%~80.42%和65.98%~80.22%,较对照品种Williams 82(17.8%~22.0%)提高2.65倍以上,亚油酸降低至4.84%~14.55%,饱和脂肪酸降低至10.34%~11.16%,但总脂肪和总蛋白含量与对照品种相比没有显著变化。农艺性状分析表明,转基因大豆在熟期、株高、叶形、花色、结荚高度、百粒重等方面与对照品种也没有显著差异。 展开更多
关键词 大豆 油酸 GmFAD2-1B 农杆菌介导转化法
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