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Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots
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作者 Huiyu Wang Yushan Zheng +3 位作者 Qian Zhou Ying Li Tongkun Liu Xilin Hou 《Horticultural Plant Journal》 SCIE CAS CSCD 2024年第2期450-460,共11页
Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation ... Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding. 展开更多
关键词 agrobacterium rhizogenes Non-heading Chinese cabbage Transgenic roots Composite plant transformation efficiency
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Establishment of a transformation system in close relatives of wheat under the assistance of TaWOX5
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作者 Yanan Chang Junxian Liu +7 位作者 Chang Liu Huiyun Liu Huali Tang Yuliang Qiu Zhishan Lin Ke Wang Yueming Yan Xingguo Ye 《Journal of Integrative Agriculture》 SCIE CAS CSCD 2024年第6期1839-1849,共11页
Species closely related to wheat are important genetic resources for agricultural production,functional genomics studies and wheat improvement.In this study,a wheat gene related to regeneration,TaWOX5,was applied to e... Species closely related to wheat are important genetic resources for agricultural production,functional genomics studies and wheat improvement.In this study,a wheat gene related to regeneration,TaWOX5,was applied to establish the Agrobacterium-mediated transformation systems of Triticum monococcum,hexaploid triticale,and rye(Secale cereale L.)using their immature embryos.Transgenic plants were efficiently generated.During the transformation process,the Agrobacterium infection efficiency was assessed by histochemical staining forβ-glucuronidase(GUS).Finally,the transgenic nature of regenerated plants was verified by polymerase chain reaction(PCR)-based genotyping for the presence of the GUS and bialaphos resistance(bar)genes,histochemical staining for GUS protein,and the QuickStix strip assay for bar protein.The transformation efficiency of T.monococcum genotype PI428182 was 94.4%;the efficiencies of four hexaploid triticale genotypes Lin456,ZS3297,ZS1257,and ZS3224 were 52.1,41.2,19.4,and 16.0%,respectively;and the transformation efficiency of rye cultivar Lanzhou Heimai was 7.8%.Fluorescence in situ hybridization(FISH)and genomic in situ hybridization(GISH)analyses indicated that the GUS transgenes were integrated into the distal or near centromere(proximal)regions of the chromosomes in transgenic T.monococcum and hexaploid triticale plants.In the transgenic hexaploid triticale plants,the foreign DNA fragment was randomly integrated into the AABB and RR genomes.Furthermore,the transgene was almost stably inherited in the next generation by Mendel’s law.The findings in this study will promote the genetic improvement of the three plant species for grain or forage production and the improvement of cereal species including wheat for functional genomics studies. 展开更多
关键词 Triticum monococcum hexaploid triticale RYE TaWOX5 agrobacterium transformation efficiency
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Development of a rapid and efficient system for CR genes identification based on hairy root transformation in Brassicaceae
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作者 Wenlin Yu Lu Yang +12 位作者 Yuanyuan Xiang Rongde Li Xueqing Zhou Longcai Gan Xianyu Xiang Yunyun Zhang Lei Yuan Yanqing Luo Genze Li Youning Wang Yinhua Chen Peng Chen Chunyu Zhang 《Horticultural Plant Journal》 SCIE CAS CSCD 2024年第4期1049-1060,共12页
Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection.Breeding of clubroot resistant cultivars bas... Many economically important crops and vegetables belonging to the cruciferous family are heavily endangered by clubroot disease caused by Plasmodiophora brassicae infection.Breeding of clubroot resistant cultivars based on mapping and cloning of resistant genes is commonly regarded as the most cost-effective and efficient way to fight against this disease.The traditional way of R gene functional validation requires stable transformation that is both time-and labor-consuming.In this study,a rapid and efficient hairy-root transgenic protocol mediated by Agrobacterium rhizogenes was developed.The transformation positive rate was over 80%in Brassica napus showed by GUS reporter gene and this transformation only took 1/6 of the time compared with stable transformation.The system was applicable to different B.napus varieties and other cruciferous crops including Brassica rapa and Brassica oleracea.In particular,two known CR genes,CRA3.7.1 and CRA8.2.4 were used respectively,as example to show that the system works well for CR gene study combined with subsequent P.brassicae infection in B.napus.Most importantly,it works both in over-expression that led to disease resistance,as well as in RNAi which led to disease susceptible phenotype.Therefore,this system can be used in batch-wise identification of CR genes,and also offered the possibility of manipulating key genes within the P.brassicae genome that could improve our knowledge on host-pathogen interaction. 展开更多
关键词 Brassicaceae agrobacterium rhizogenes Hairy root transformation CLUBROOT Gene function
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Agrobacterium _mediated Transformation of Rice with Help of Bombardment 被引量:10
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作者 明小天 苑华毅 +1 位作者 王莉江 陈章良 《Acta Botanica Sinica》 CSCD 2001年第1期72-76,共5页
Oryza sativa L. ssp. japonica cv. Zhonghua 8, which is recalcitrant to infection of Agrobacterium tumefaciens (Smith et Townsend) Conn strain EHA105 with ordinary binary vector pCambia 1301, was transformed through Ag... Oryza sativa L. ssp. japonica cv. Zhonghua 8, which is recalcitrant to infection of Agrobacterium tumefaciens (Smith et Townsend) Conn strain EHA105 with ordinary binary vector pCambia 1301, was transformed through Agrobacterium-mediated transformation with help of bombardment. The transformation efficiency can be raised greatly. Single copy of gene insertion in the genome of transgenic rice plants was proved by Southern analysis and the expression of GUS gene was observed. GUS gene and hygromycin-resistant gene show 3:1 segregation in progenies of the transgenic rice plants. 展开更多
关键词 agrobacterium BOMBARDMENT transformation RICE
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Genetic Transformation of Aloe barbadensis Miller by Agrobacterium tumefaciens 被引量:1
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作者 何聪芬 张佳星 +4 位作者 陈杰 叶兴国 杜丽璞 董银卯 赵华 《Journal of Genetics and Genomics》 SCIE CAS CSCD 北大核心 2007年第12期1053-1060,共8页
Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe (Aloe barbadensis Miller) by genetic engineering was seldom reported previously. In this study, regeneration and transfor... Despite the importance of aloe in cosmetic and pharmaceutical industries, improvement of aloe (Aloe barbadensis Miller) by genetic engineering was seldom reported previously. In this study, regeneration and transformation conditions, including explant selection and surface sterilization, use of different Agrobacterium strains, and co-culture processing, are optimized. The use of 20.0% sodium hypochloride (25 rain) for sterilization was less detrimental to the health of explant than 0.1% mercuric chloride (10 min). Regeneration frequency from stems was much higher than that from leaves or sheaths. Explants were infected by Agrobacterium (30 rain) in liquid co-cultural medium, and this was followed by three days co-culture on sterile filter papers with light for 10 h per day at 24℃. Histochemical data demonstrated that the transient expression of GUS gene in the stem explants of aloe infected with Agrobacterium strains EHAI05 and C58CI was 80.0% and 30.0%, respectively, suggesting the higher sensitivity of the explants to EHAI05 than to C58C1. Infected tissues were selected using G418 (10.0-25.0 mg/L) to generate transformants. Sixty-seven G418 resistant plantlets were generated from the infected explants. Southern blotting, PCR, and ELISA analyses indicated that the alien gene were successfully transferred into aloe and was expressed in the transgenic plants. This newly established transformation system could be used for the genetic improvement of aloe. 展开更多
关键词 ALOE agrobacterium tumefaciens transformation molecular test ELISA analysis
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Optimization of Genetic Transformation System of Tobacco K326 Mediated by Agrobacterium 被引量:2
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作者 赵勤 《Agricultural Science & Technology》 CAS 2011年第1期62-64,共3页
[Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-... [Objective]The aim was to optimize genetic transformation system in tobacco K326 mediated by Agrobacterium.[Method]The leaf of tobacco aseptic seedling was taken as explants to study the optimization of Agrobacterium-mediated genetic transformation system.[Result] The highest transformation efficiency was obtained when the explants were pre-cultured in the medium of MS + 2 mg/L 6-BA + 0.2 mg/L IAA for 2 d,and then infected with Agrobacterium GV3101(OD600 =0.6) for 5 min.The PCR detection proved that npt II gene had been integrated into the regenerated tobacco plants.[Conclusion]A highly efficient genetic transformation system of tobacco leaf mediated by Agrobacterium was established. 展开更多
关键词 agrobacterium tumefaciens TOBACCO Genetic transformation
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Optimization of Agrobacterium Mediated Genetic Transformation System of Tomato Meifen No.1
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作者 李立芹 《Agricultural Science & Technology》 CAS 2010年第11期92-94,共3页
[Objective] The aim of this study was to carry out study on the optimization of Agrobacterium mediated genetic transformation system of tomato Meifen No.1.[Method] The cotyledon of tomato cultivar Meifen No.1 was used... [Objective] The aim of this study was to carry out study on the optimization of Agrobacterium mediated genetic transformation system of tomato Meifen No.1.[Method] The cotyledon of tomato cultivar Meifen No.1 was used as the explant,and the Agrobacterium mediation method was used to optimize its genetic transformation efficiency so as to establish the efficient Agrobacterium mediated genetic transformation system of tomato cotyledon.[Result] The highest transformation efficiency was obtained when the explants were cultivated for 2 d on MS + 2.0 mg/L 6-BA+ 0.5 mg/L IAA medium and then infected with Agrobacterium EHA105(OD = 0.4)for 5 min;it was proved by PCR analysis that the target nptII gene had been integrated into the genome of regenerated plants.[Conclusion] The result in this study had provided basis for the transfer of valuable genes into tomato Meifen No.1. 展开更多
关键词 agrobacterium tumefaciens TOMATO Transgenic transformation
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Factors Affecting Agrobacterium-Mediated Transformation Efficiency in Rice 被引量:7
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作者 CHENEn-hui ZHANGPing +4 位作者 ZUOShi-min LIAi-hong ZHANGYa-fang CHENZong-xiang PANXue-biao 《Rice science》 SCIE 2004年第4期181-185,共5页
Several important factors affecting the efficiency of Agrobacterium-mediated rice transformation were studied with several predominant commercial indica and japonica rice cultivars. As far as indica rice callus was co... Several important factors affecting the efficiency of Agrobacterium-mediated rice transformation were studied with several predominant commercial indica and japonica rice cultivars. As far as indica rice callus was concerned, CC medium was the best and the quality of callus was improved with the addition of 1.0 to 2.0 mg/L ABA. It decreased the percentage of browning calli and improved the callus growing state by addition of a certain amount of sorbitol to the subculture medium. NB medium was the best for callus initiation of japonica rice, but the improvement in the quality of callus of japonica subspecies was not obvious by adding ABA. During the period of subculture, to a certain degree, increasing the sucrose concentration could improve the proportion of hygromycin resistant calli. Furthermore, the transformation efficiency would be higher by applying selection pressure in the selection stage, removing selection pressure during the plantlet differentiation period and applying selection pressure again during seedling hardening period. Besides, suitable combination of plant hormones was beneficial for callus differentiation. An efficient /Agrobacterium-mediated rice transformation system had been established for several rice cultivars and a lot of transgenic rice plants had been obtained. 展开更多
关键词 RICE agrobacterium CALLUS HORMONES transformation
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Meropenem as an Alternative Antibiotic Agent for Suppression of Agrobacterium in Genetic Transformation of Orchid 被引量:9
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作者 CAO Ying Niimi Yoshiyuki HU Shang-lian 《Agricultural Sciences in China》 CAS CSCD 2006年第11期839-846,共8页
A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies (PLBs) has been reported in this ar... A case of Meropenem as a novel antibacterial agent to suppress and eliminate Agrobacterium tumefaciens in the Agrobacterium-mediated transformation of orchid protocorm-like bodies (PLBs) has been reported in this article. The in vitro activities of meropenem and four comparator antibacterial agents against three Agrobacterium tumefaciens strains, LBA4404, EHA101, and GV3101, were assessed. In addition, the effect of meropenem on the growth of Dendrobium phalaenopsis PLBs was determined. Compared with other commonly used antibiotics (including ampicillin, carbenicillin, cefotaxime, and cefoperazone), meropenem showed the highest activity in suppressing all tested A. tumefaciens strains (minimum inhibitory concentration [MIC] 〈 0.5 mg L^-1, which is equal to minimum bactericidal concentration [MBC]). Meropenem, at all tested concentrations, except for 10 mg L^-1 concentration, had little negative effect on the growth of orchid tissues. The A. tumefaciens strain EHA101 in genetic transformation with vector plG121Hm in infected PLBs of the orchid was visually undetectable after a two-month subculture in 1/2 MS medium with 50 mg L^-1 meropenem and 25 mg L^-1 hygromacin. The expression and incorporation of the transgenes were confirmed by GUS histochemical assay and PCR analysis. Meropenem may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation. 展开更多
关键词 ANTIBIOTICS MEROPENEM agrobacterium tumefaciens antibacterial activity genetic transformation protocormlike bodies (PLBs)
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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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Soybean hairy roots produced in vitro by Agrobacterium rhizogenes-mediated transformation 被引量:4
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作者 Li Chen Yupeng Cai +6 位作者 Xiujie Liu Chen Guo Shi Sun Cunxiang Wu Bingjun Jiang Tianfu Han Wensheng Hou 《The Crop Journal》 SCIE CAS CSCD 2018年第2期162-171,共10页
Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the Agroba... Soybean is one of the world's most important oil and protein crops. Efficient transformation is a key factor for the improvement of soybean by genetic modification. We describe an optimized protocol for the Agrobacterium rhizogenes-mediated transformation of soybean and the induction of hairy root development in vitro. Cotyledons with 0.5-cm hypocotyls were cut from 5-day-old seedlings and used as explants. After infection and co-cultivation,hairy roots were produced in induction culture medium after 10–12 days. Using this method, 90%–99% of the infected explants of five different cultivars produced hairy roots within one month. Observations using reporter constructs showed that 30%–60% of the hairy roots induced were transformed. Based on high transformation efficiency and short transformation period, this method represents an efficient and rapid platform for study of soybean gene function. 展开更多
关键词 agrobacterium RHIZOGENES HAIRY ROOT Protocol SOYBEAN transformation
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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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The Establishment of an Agrobacterium-Mediated Transformation Platform for the Non-Embryogenic Calli of Vitis vinifera L. 被引量:3
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作者 ZHAO Feng-xia CHEN Shang-wu +3 位作者 Perl Avihai DAI Ru XU Hai-ying MA Hui-qin 《Agricultural Sciences in China》 CAS CSCD 2011年第5期686-694,共9页
Non-embryogenic calli (NEC) was inevitably and heavily produced when grape embryogenic calli (EC) was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform ... Non-embryogenic calli (NEC) was inevitably and heavily produced when grape embryogenic calli (EC) was induced from explants or during the subculture of EC.A stable and highly efficient NEC transformation platform is required to further sort out and verify key genes which determine/switch the identity of NEC and EC.In this research,a vector pA5 containing a chitinase signal sequence fused to gfp (green fluorescent protein) and an HDEL motive was used to target and immobilize into Agrobacterium strain EHA105 to establish a transformation platform for Vitis vinifera L.cv.Chardonnay NEC.It was determined that NEC 10 d after subculture was the best target tissue;30 min for inoculation followed by 3 d co-cultivation with the addition of 200 μmol L-1 acetosyringone (AS) was optimized as protocol.The use of bacterial densities as 1.0 at OD600 did not result in serious tissue hypersensitive reaction and it had higher efficiency.Kanamycin at 200 mg L-1 was picked for positive expression selection.The stable transformation of NEC was proved by reverse transcription-polymerase chain reaction techniques (RT-PCR) and fluorescent microscopy after three sub-cultures of the selected cell line.Highly efficient genetic transformation protocol of grape NEC was achieved and some of the optimized parameters were different from that reported for EC.This transformation platform could facilitate the verification of candidate somatic embryogenesis (SE) decisive genes,and the successfully transformed NEC with certain genes can also be used as bioreactors for the production of functional products,as NEC not only proliferates fast,but also keeps in a rather stable condition. 展开更多
关键词 agrobacterium tumefaciens non-embryogenic calli transformation Vitis vinifera L. RT-PCR
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Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum 被引量:10
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作者 Ji-ye WANG Hong-ye LI 《Journal of Zhejiang University-Science B(Biomedicine & Biotechnology)》 SCIE CAS CSCD 2008年第10期823-828,共6页
Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformat... Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alter-natively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics. 展开更多
关键词 Penicillium digitatum agrobacterium tumefaciens-mediated transformation (ATMT) Hygromycin B resistance gene lnsertional mutagenesis
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Transformation of Chinese Cabbage(Brassica rapa L. ssp. pekinensis)by Agrobacterium Micro-Injection into Flower Bud 被引量:3
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作者 YANJi-yong HEYu-ke CAOJia-shu 《Agricultural Sciences in China》 CAS CSCD 2003年第8期906-911,共6页
We obtained two lines of Chinese head cabbage(Brassica rapa L. ssp. pekinensis)selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their prima... We obtained two lines of Chinese head cabbage(Brassica rapa L. ssp. pekinensis)selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their primary transformants(T0)with Agrobacterium tumefaciens strain LBA4404. 31 positive plants resistant to kanamycien were recovered. Southern blot analysis confirmed the presence of T-DNA in two transgenic plants. One(DHZ-13-1)exhibits the characteristics of out-toward rosette and cauline leaves, and nested flower model in which secondary complete flower developed from the base of the primary ovary and the third flower from the ovary in the secondary flower, and so on, while another(DHZ-6-1)has no phenotype change. ABA and IAA affected the root growth of progeny of DHZ-13-1, but 6-BA was insensitive to hypocotyl growth during its seedling development. 展开更多
关键词 Brassica rapa agrobacterium tumefaciens Anti-sense gene of BcpLH in planta transformation bud injection
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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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Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage(Brassica rapa L.ssp.pekinensis) 被引量:6
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作者 Xiaonan Li Haiyan Li +3 位作者 Yuzhu Zhao Peixuan Zong Zongxiang Zhan Zhongyun Piao 《Horticultural Plant Journal》 SCIE CSCD 2021年第2期117-128,共12页
Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is genera... Chinese cabbage,belonging to Brassica rapa species,is an important vegetable in Eastern Asia.It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low.The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies.In this study,we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors,with 93.50%regeneration rate on average.Based on this,a simple and efficient Agrobacteriummediated genetic transformation methodwas established,without pre-culture procedure and concentration adjustment of hormone and AgNO_(3) in co-cultivation and selection media.Using this system,transformants could be obtained within 3.5–4.0 months.Average transformation frequency is up to 10.83%.The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage. 展开更多
关键词 Chinese cabbage agrobacterium tumefaciens genetic transformation
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Production of Transgenic Tall Fescue Plants with Enhanced Stress Tolerances by Agrobacterium tumefaciens-Mediated Transformation 被引量:3
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作者 WU Guan-ting CHEN Jin-qing +5 位作者 HU Zhang-hua LANG Chun-xiu CHEN Xiao-yun WANG Fu-lin JIN Wei XIA Ying-wu 《Agricultural Sciences in China》 CAS CSCD 2006年第5期330-338,共9页
In order to improve stress tolerances of turf-type tall fescue (Festuca arundinacea Schreb.), Agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidop... In order to improve stress tolerances of turf-type tall fescue (Festuca arundinacea Schreb.), Agrobacterium tumefaciens strain EHA105 carrying plasmid pCMD containing stress tolerance-related CBF1 gene from Arabidopsis thaliana was used to transform mature seeds-derived embryogenic calli of four cultivars. A total of 112 transgenic plants were regenerated from 32 independent lines and verified by histochemical detection of GUS activity, PCR assay and Southern hybridization analysis. The transformation frequency ranged from 0.92 to 2.87% with apparent differences among the cultivars. Stress tolerances of transgenic plants were enhanced, which was shown by the facts that transgenic plants had distinct growth superiority and significantly higher survival rate than non-transformed ones under high salinity and high osmosis stresses, and that relative electronic conductivity of in vitro leaves treated with low and high temoeratures, dehvdration and high salinity stresses was 25-30% lower in transgenic plants than in control plants.In addition,it was observed that growth of transgenic plants was inhibited due to constitutive overexpression of CBF1 gene under normal environmental conditions. 展开更多
关键词 tall fescue (Festuca arundinacea Schreb.) agrobacterium tumefaciens genetic transformation CBF1 gene stress tolerance
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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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Mutation in the Agrobacterium his I gene enhances transient expression in pepper 被引量:1
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作者 Dan Liu Shengnan Zhao +3 位作者 Jubin Wang Xi Zhang Yingtian Deng Feng Li 《Horticultural Plant Journal》 SCIE CAS CSCD 2024年第3期809-822,共14页
Agrobacterium-mediated plant transformation is widely used in plant genetic engineering.However,its efficiency is limited by plant immunity against Agrobacterium.Chili pepper(Capsicum annuum L.)is an important vegetab... Agrobacterium-mediated plant transformation is widely used in plant genetic engineering.However,its efficiency is limited by plant immunity against Agrobacterium.Chili pepper(Capsicum annuum L.)is an important vegetable that is recalcitrant to Agrobacterium-mediated transformation.In this work,Agrobacterium was found to induce a strong immune response in pepper,which might be the reason for T-DNA being difficult to express in pepper.An Agrobacterium mutant screen was conducted and a point mutation in the hisI gene was identified due to a weak immune response and enhanced transient expression mediated by this Agrobacterium mutant in pepper leaves.Further genetic analysis revealed that histidine biosynthesis deficiency caused by mutations in many genes of this pathway led to reduced pepper cell death,presumably due to reduced bacterial growth.However,mutation analysis of threonine and tryptophan biosynthesis genes showed that the biosynthesis of different amino acids may play different roles in Agrobacterium growth and stimulating the pepper immune response.The possible application of Agrobacterium amino acid biosynthesis mutations in plant biology was discussed. 展开更多
关键词 Capsicum annuum L. agrobacterium Transient transformation efficiency MUTATION Amino acid Immune response
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