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Registros recuperados: 25
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Agrobacterium-mediated transformation of sorghum: factors that affect transformation efficiency Genet. Mol. Biol.
Carvalho,Carlos Henrique S.; Zehr,Usha B.; Gunaratna,Nilupa; Anderson,Joseph; Kononowicz,Halina H.; Hodges,Thomas K.; Axtell,John D..
The results presented in this work support the hypothesis that Agrobacterium-mediated transformation of sorghum is feasible, analogous to what has been demonstrated for other cereals such as rice, maize, barley and wheat. The four factors that we found most influenced transformation were: the sensitivity of immature sorghum embryos to Agrobacterium infection, the growth conditions of the donor plant, type of explant and co-cultivation medium. A major problem during the development of our protocol was a necrotic response which developed in explants after co-cultivation. Immature sorghum embryos proved to be very sensitive to Agrobacterium infection and we found that the level of embryo death after co-cultivation was the limiting step in improving...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Agrobacterium tumefaciens; GUS; Transformation; Sorghum.
Ano: 2004 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572004000200022
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Antitumor and anti-angiogenic potentials of isolated crude saponins and various fractions of Rumexhastatus D. Don Biol. Res.
Ahmad,Sajjad; Ullah,Farhat; Ayaz,Muhammad; Zeb,Anwar; Ullah,Farman; Sadiq,Abdul.
BACKGROUND: Cancer, being the foremost challenge of the modern era and the focus of world-class investigators, gargantuan research is in progress worldwide to explore novel therapeutic for its management. The exploitation of natural sources has been proven to be an excellent approach to treat or minify the excessive angiogenesis and proliferation of cells. Similarly, based the ethnomedicinal uses and literature survey, the current study is designed to explore the anti-tumor and anti-angiogenic potentials of Rumex hastatus. Anti-tumor and anti-angiogenic activities were carried out using potato-disc model and chorioallantoic membrane (CAM) assay respectively. Moreover, R. hastatus was also assessed for antibacterial activity against Agrobacterium...
Tipo: Journal article Palavras-chave: Rumex hastatus; Agrobacterium tumefaciens; Saponins; Angiogenesis; Tumor.
Ano: 2016 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0716-97602016000100018
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Aumento da superfície radicular de tabaco mediada pelo gene Rootless Concerning Crown and Seminal Roots (RTCS) de milho. Infoteca-e
CAMPOLINO, M. L.; LANA, U. G. de P.; CARNEIRO, A. A.; TINOCO, S. M. de S..
A disponibilidade de fósforo (P) é um dos fatores mais limitantes para a produtividade agrícola em solos tropicais, uma vez que este nutriente apresenta menor eficiência de uso por plantas. Modificações na morfologia do sistema radicular são particularmente importantes para a eficiência na aquisição de P em plantas em razão da baixa mobilidade do fósforo no solo. O sistema radicular é complexo e formado por diferentes tipos de raízes. Durante a embriogênese, uma raiz primária é depositada no polo basal do embrião, enquanto um número variável de raízes seminais é formado no escutelo, que são relevantes somente nos estágios iniciais do desenvolvimento. Em estágios mais avançados de desenvolvimento, um extenso sistema radicular pós-embrionário forma a maior...
Tipo: Boletim de Pesquisa e Desenvolvimento (INFOTECA-E) Palavras-chave: Transgênico; Genética; Fumo; Fósforo; Sistema radicular; Raiz; Agrobacterium tumefaciens.
Ano: 2017 URL: http://www.infoteca.cnptia.embrapa.br/infoteca/handle/doc/1075961
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Development of pollen mediated activation tagging system for Phalaenopsis and Doritaenopsis Electron. J. Biotechnol.
Tsay,Hsin-Sheng; Ho,Hui-Min; Gupta,Sushim K; Wang,Chang-Sheng; Chen,Pei-Tsu; Chen,Emily Chin-Fun.
In the present study, a novel plant transformation system for Doritaenopsis and Phalaenopsis has been developed. The pollen-mediated activation tagging system was established by artificial pollination. The pollens, co-cultured with Agrobacterium tumefaciens strain EHA105 harbouring an activation tagging vector (pTAG-8), were used for pollination. In order to optimize the transformation efficiency, several factors (concentration of A. tumefaciens, concentration of acetosyringone during co-cultivation and the duration of co-cultivation) known to influence Agrobacterium-mediated DNA transfer were examined. A concentration of 0.5-1 x 10(8) CFU/ml for A. tumefaciens, 0.1 mM acetosyringone, and 6 hrs of co-culture period were found to be the optimal condition...
Tipo: Journal article Palavras-chave: Activation tagging; Agrobacterium tumefaciens; Doritaenopsis cultivars; Phalaenopsis amabilis; Transformation.
Ano: 2012 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000400009
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Efficient Agrobacterium tumefaciens-mediated transformation of Malus zumi (Matsumura) Rehd using leaf explant regeneration system Electron. J. Biotechnol.
Xu,Jin; Wang,Yu Zhen; Xia Yin,Heng; Jing Liu,Xiao.
Malus zumi is known as an excellent dwarfing apple rootstock occurring in natural or arid/semiarid soil or salina. Gene manipulation of M. zumi through transgenic technology can modify plant feature for further improvement fruit tree production by grafting the scion on a transgenic rootstock. Here, we report the establishment of an efficient, in vitro, shoot regeneration system and Agrobacterium tumefaciens- mediated transformation from the leaf explants for Malus zumi (Matsumura) Rehd. Leaf explants were infected with Agrobacterium strains containing nptII and gus gene. The highest frequency of shoot regeneration was obtained on MS medium containing 500 mg l-1 Lactalbumin hydrolysate, 30 g l-1 fructose, supplemented with 3.0 mg l-1 BA, 0.2 mg l-1...
Tipo: Journal article Palavras-chave: Agrobacterium tumefaciens; Apple rootstock; Genetic transformation.
Ano: 2009 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582009000100003
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Evaluation of the uniformity and stability of T-DNA integration and gene expression in transgenic apple plants Electron. J. Biotechnol.
Flachowsky,Henryk; Riedel,Marko; Reim,Stefanie; Hanke,Magda-Viola.
The generation of transgenic apple plants relies on the molecular analysis of transgene integration and expression based on polymerase chain reaction (PCR) analysis, blotting techniques and enzymatic assays on vitro leaves of putative transgenic regenerates. In order to assess the uniformity and the stability of transfer DNA (T-DNA) integration and gene expression, we studied 26 transgenic apple lines carrying the attacin E gene from Hyalophora cecropia, the β-glucuronidase gene, and the nptII gene. Plants were evaluated using standard molecular techniques, such as PCR, Southern blot, reverse transcription PCR (RT-PCR) and Enzyme Linked Immunosorbent Assay (ELISA), and propagated in vitro on non-selective antibiotic-free media for four years to...
Tipo: Journal article Palavras-chave: Agrobacterium tumefaciens; Apple; Chimeric tissue; Gene silencing; Malus domestica; T-DNA leakage.
Ano: 2008 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582008000100003
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EXPRESIÓN GUS EN EXPLANTES DE Solanum phureja (Juz. et. Buk) Var. Criolla Colombia , TRANSFORMADOS CON Agrobacterium tumefaciens Acta biol.Colomb.
BARRERO-FARFÁN,IVÁN DARÍO; CHAPARRO GIRALDO,ALEJANDRO.
La expresión transitoria y estable del gen gusA-intron en explantes internodales de papa criolla variedad Criolla Colombia cocultivados con Agrobacterium tumefaciens es reportada. Con el fin de determinar la susceptibilidad de esta variedad a la transformación mediada por A. tumefaciens, explantes internodales de Solanum phureja fueron infectados con la cepa LBA4404 de A. tumefaciens que contiene el plásmido pCAMBIA2301. Este plásmido contiene el gen ntpII que confiere resistencia a kanamicina y el gen reportero gusA-intron. La selección de los explantes potencialmente transgénicos fue realizada en medios con kanamicina. La eficiencia de transformación estable y transitoria fue calculada con base en la actividad GUS (ß-glucuronidasa), detectada por el...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Solanum phureja; Actividad Gus; Expresión estable y transitoria; Agrobacterium tumefaciens; Variedad Criolla Colombia.
Ano: 2008 URL: http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0120-548X2008000100008
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Expresión transitoria del gen GUS en caña de azúcar usando Agrobacterium tumefaciens Acta Agron. (Palmira)
Bonilla Betancourt,Martha Liliana; Muñoz Flórez,Jaime Eduardo; ángel Sánchez,Fernando.
En el estudio se desarrolló una metodología de transformación genética mediante Agrobacterium tumefaciens en cultivares colombianos de caña de azúcar. La transformación se evaluó mediante la expresión del gen GUS. Callos embriogénicos y explantes meristemáticos de los genotipos CC85-92, CC84-75 y CC87-505 se transformaron usando tres cepas (AGL-1, LBA4404 y EHA105) con el plásmido pCambia 1305.2 y dos (EHA105 y LBA4404) con pCambia 2301. Se usó el medio de infiltración (IM) con acetosiringona y se evaluó el tiempo de cocultivo y la densidad óptica de la bacteria al momento de la inducción. Los genotipos mostraron respuesta diferencial con las combinaciones cepa-plásmido: obtuvieron mayor expresión del gen GUS cuando el genotipo CC85-92 se transformó con la...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Agrobacterium tumefaciens; Saccharum spp; Caña de azúcar; Transformación genética.
Ano: 2008 URL: http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0120-28122008000300002
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Expressão eficiente do gene reporter beta-glucuronidase nos tecidos vasculares de batata (Solanum tuberosum L.) utilizando de um promotor específico (BRA3) de Agrobacterium rhizogenes Horticultura Brasileira
Torres,Antonio Carlos; Ferreira,Adriana T.; Widholzer,Carlos F. N.; Romano,Eduardo; Peters,José A..
Promotores tecido-específico controlam a transcrição de genes em diferentes tecidos vegetais bem como em diferentes estádios de desenvolvimento da planta, levando à indução de distintos níveis de atividade transiente e/ou estável do gene. Tais promotores podem ser empregados para a expressão seletiva de genes de interesse. O promotor rol A de Agrobacterium rhizogenes, por exemplo, é floema-específico, sugerindo que possa ser empregado em estratégias de defesa de plantas que são infectadas por vírus com replicação restrita ao floema. A expressão do gene marcador da ß-glucuronidase (gus) dirigido pelo promotor rol A (pBRA3) foi observada em plantas transgênicas de batata (cvs. Macaca e Baronesa). Entrenós e secções de folhas foram submetidos ao cocultivo com...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Agrobacterium tumefaciens; Solanum tuberosum; Transformação genética; Expressão do gene gus.
Ano: 2003 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0102-05362003000200011
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Expression of Hemagglutinin-Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco Electron. J. Biotechnol.
Ghaffar Shahriari,Amir; Bagheri,Abdolreza; Bassami,Mohammad Reza; Malekzadeh-Shafaroudi,Saeid; Afsharifar,Alireza; Niazi,Ali.
Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin-Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco ( Nicotiana tabacum) plant by Agrobacterium-mediated transformation. Results: Putative transgenic plants were screened in a selection medium containing 50 mg/L...
Tipo: Journal article Palavras-chave: Agrobacterium tumefaciens; ELISA; Recombinant vaccine; Transgenic plants.
Ano: 2016 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582016000400006
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Histological study of shoot organogenesis in melon (Cucumis melo L.) after genetic transformation National Institute of Agronomic Research
Chovelon, V.; Restier, V.; Dogimont, C.; Aarouf, J..
Leave and cotyledon explants from two melon genotypes ‘Vedrantais’ and ‘Paul’ were cultured on different media and their regeneration was followed during 60 days. High frequency of plant regeneration was observed with ‘Vedrantais’ cotyledons cultured on MS medium added with BAP and 2-iP. Using this protocol, Agrobacterium mediated transformation was developed and a significant reduction of the plant regeneration rate was observed. Explants histological analysis revealed the presence of transformed meristematic areas but a rapid and premature cell vacuolization was observed. During plant regeneration, meristematic cells were affected by important cytological damages: cell dislocation accompanied by cell death and final collapsus. This disruption of...
Tipo: Poster Palavras-chave: Cucumis melo; Agrobacterium tumefaciens; GUS uidA; Genetic transformation; Regeneration.
Ano: 2008 URL: http://hdl.handle.net/2174/285
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Influence of antibiotics on embryogenic tissue and Agrobacterium tumefaciens suppression in soybean genetic transformation Bragantia
Wiebke,Beatriz; Ferreira,Fabricio; Pasquali,Giancarlo; Bodanese-Zanettini,Maria Helena; Droste,Annette.
The influence of different antibiotic treatments in soybean genetic transformation was evaluated. First, an assay was performed to verify how different antibiotic treatments affect soybean embryogenic tissues. The effect of carbenicillin at 500 mg L-1 was genotype-dependent. This antibiotic did not affect embryo survival of cv. IAS5, but a three-fold increase of embryo proliferation was observed for cv. Bragg, when compared to the control. On the other hand, cefotaxime at 350 and 500 mg L-1 caused death of embryogenic tissues of both cultivars. Finally, the association of cefotaxime (250 mg L-1) + vancomycin (250 mg L-1) did not affect negatively the somatic embryos of tested cultivars until 63 days of treatment. Thereafter, a second experiment was carried...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Agrobacterium tumefaciens; Carbenicillin; Cefotaxime; Genetic transformation; Glycine max; Vancomycin.
Ano: 2006 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0006-87052006000400002
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Morfogênese in vitro de variedades brasileiras de cana-de-açúcar PAB
Cidade,Daniela Anhel de Paula; Garcia,Renata de Oliveira; Duarte,Aline Castellar; Sachetto-Martins,Gilberto; Mansur,Elisabeth.
O objetivo deste trabalho foi estabelecer sistemas de multiplicação de plantas de cana-de-açúcar in vitro e avaliar sua utilização, como material inicial, para a indução de regeneração a partir de ápices caulinares. Três métodos de cultivo foram avaliados: cultura em meio semi-sólido, cultura líquida estacionária e cultura líquida sob agitação. A taxa de multiplicação mais elevada foi alcançada por meio da cultura líquida sob agitação. Ápices caulinares, excisados dessas plantas, apresentaram taxas de regeneração in vitro compatíveis com sua utilização em protocolos de transformação. Calos resistentes a PPT e GUS-positivos foram obtidos de explantes da variedade Chunnee com inoculação de Agrobacterium tumefaciens C58C1 (pMP90) (pDUBarA9). O protocolo...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Saccharum; Micropropagação; Transformação genética; Agrobacterium tumefaciens.
Ano: 2006 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0100-204X2006000300003
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Organogenesis and transient genetic transformation of the hybrid Eucalyptus grandis × Eucalyptus urophylla Scientia Agricola
Alcantara,Giovana Bomfim de; Bespalhok Filho,João Carlos; Quoirin,Marguerite.
The hybrid Eucalyptus grandis x Eucalyptus urophylla presents high levels of productivity and potential for use in paper, cellulose and fiber industries. The bud organogenesis from leaf explants of two clones of E. grandis × E. urophylla was studied in order to verify the effect of several factors: subculture duration on multiplication medium, type of explants, entire and half leaves: basal and apical portions, and duration of the culture on a regeneration medium. Differences in organogenic capacity of the two clones tested were observed. The explant most recommended for organogenesis is the basal section of the leaf collected from shoot clusters subcultured every 17 days. Moreover, the leaf explants must be transferred to a fresh bud induction medium...
Tipo: Info:eu-repo/semantics/report Palavras-chave: Agrobacterium tumefaciens; Micropropagation; Tissue culture; Transgenic plant; Woody species.
Ano: 2011 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0103-90162011000200016
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Plant regeneration and Agrobacterium-mediated transformation of Achyranthes bidentata using cotton EREBP gene BABT
Duan,Hong Ying; Ding,Xiao Sheng; Song,Jian Ying; He,Yun Long; Zhou,Yan Qing.
The aim of this work was to study the plant regeneration and Agrobacterium-mediated transformation of Achyranthes bidentata using cotton EREBP gene. Results showed that the callus induction rate of stems from A. bidentata was the highest (100%) and bud was in approximately 70% of calli from stems. However bud differentiation rate of the callus from leaves and petioles was very low. Compared with ceftriaxone, 200mg/L cefotaxime could completely control Agrobacterium tumefaciens and had relatively less toxic action on the stems of A. bidentata. In addition, the induction rate of callus resistant to hygromycin was the highest when infected for 3 min and co-cultivated for 3 d. Six positive transgenic plants transformed with pCAMBIA1304-GhEREB2 expression...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Achyranthes bidentata; EREBP gene; Agrobacterium tumefaciens; Genetic transformation.
Ano: 2013 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132013000300001
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REGENERACION Y SENSIBILIDAD A MANOSA EN ENTRENUDOS DE PAPA (Solanum tuberosum spp. andígena Var Diacol Capiro). Acta biol.Colomb.
YAYA-LANCHEROS,MARY LUZ; CHAPARRO- GIRALDO,ALEJANDRO.
Se estableció un sistema de organogénesis indirecta para la obtención de brotes múltiples a partir de segmentos internodales de la variedad Diacol Capiro. La ubicación de explantes en medio Murashige y Skoog (MS) suplementado con 2 mg/l de zeatina ribosido (ZR), 0,02 mg/l de ácido naftalenácetico (ANA) y 0,02 mg/l de ácido giberélico (AG3), permite la obtención de plántulas entre la séptima y novena semana con una efectividad del 80-100%. Mediante ubicación de explantes previamente cocultivados con la cepa LBA4404 de Agrobacterium tumefaciens que contiene el plásmido recombinante pNOV022, se verificó la utilidad del medio para procesos de transformación, obteniéndose tasas hasta del 100% de regeneración. Finalmente, con el objetivo de determinar el uso...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Organogénesis indirecta; Selección positiva; Plantas transgénicas; Agrobacterium tumefaciens.
Ano: 2009 URL: http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0120-548X2009000100009
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Relative susceptibility of peach rootstocks to crown gall and Phytophthora root and crown rot in Chile Ciencia e Investigación Agraria
Guzman,Gonzalo; Latorre,Bernardo A; Torres,Rene; Wilcox,Wayne F.
Crown gall (Agrobacterium tumefaciens) and Phytophthora root and crown rot (PRCR), caused by P. cryptogea, are two major limiting factors affecting peach (Prunus pérsica) production in Chile. At present, Nemaguard is the most important rootstock used in peach orchards, and although several clonal rootstocks are under development worldwide, there is little information regarding their susceptibility to crown gall and PRCR. Therefore this investigation was undertaken to study the susceptibility to A. tumefaciens and P. cryptogea of the clonally propagated rootstocks Cadman®-Avimag (P. pérsica x P. davidiana), Mr.S 2/5 (P. cerasifera x P. spinosa), Viking ([P. pérsica x P. davidiana] x [P. dulcís 'Jordanolo'x P. blireiana]), and Nemaguard (P. pérsica x P....
Tipo: Journal article Palavras-chave: Agrobacterium tumefaciens; Disease control; Nectarines; Rootstock resistance.
Ano: 2007 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0718-16202007000100004
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Selective agent and A. tumefaciens overgrowth-control antibiotics in Eucalyptus camaldulensis Cotiledonary culture BABT
Quisen,Regina; Oliveira,Yohana de; Pileggi,Marcos; Cuquel,Francine; Quoirin,Marguerite.
The objectives of the present work were to establish the minimal lethal dose of the selective agent to determine the type and concentration of appropriate antibiotics for the elimination of Agrobacterium tumefaciens inoculated explants, without interfering with the regenerative potential of the E. camaldulensis cotyledonary explants. Non-transformed explants were cultivated in medium supplemented with kanamycin. The results showed that the antibiotic was suitable for the selection of transformed cells in the concentration of 9 mg L-1 as it inhibited the growth of non-transformed cells. Cotyledons infected with A. tumefaciens were cultivated in MS N/2 medium supplemented with BAP, ANA, Km and cefotaxime or AugmentinÒ . The highest average of regenerated...
Tipo: Info:eu-repo/semantics/article Palavras-chave: Morphogenesis; Kanamycin; Cefotaxime; Augmentin; Agrobacterium tumefaciens; Eucalypt.
Ano: 2009 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132009000600020
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Synthetic and minimalist vectors for Agrobacterium tumefaciens-mediated transformation of fungi Genet. Mol. Biol.
Nora,Luísa Czamanski; Gonçales,Relber Aguiar; Martins-Santana,Leonardo; Ferreira,Beatriz Henriques; Rodrigues,Fernando; Silva-Rocha,Rafael.
Abstract We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluorescent proteins, mCherry or eGFP, flanked by a multiple cloning site and a transcription terminator site. They also present a synthetic gene allowing resistance to Hygromicin B flanked by alternate promoters, one for yeast and another for filamentous fungi. Left and right borders were added for Agrobacterium tumefaciens recognition, and a minimal broad-host range RK2 replication origin. Transformation was validated in the pathogenic fungus Paracoccidioides lutzii. Hence, we developed an efficient and reliable molecular tool for fungal transformation:...
Tipo: Info:eu-repo/semantics/other Palavras-chave: Synthetic biology; Fungi; Agrobacterium tumefaciens; Transformation; Vectors..
Ano: 2019 URL: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1415-47572019000300395
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Towards genetic transformation of local onion varieties Electron. J. Biotechnol.
Marinangeli,Pablo; Deluchi,Bernardo; Ríos,Raúl; Franzone,Pascual; Galmarini,Claudio; Rosselló,Fernando; Curvetto,Néstor.
The aim of this work was to explore the possibility of obtaining transgenic plants of onion varieties cultivated in Argentina, starting from calli induced from mature zygotic embryos, using two strains of Agrobacterium tumefaciens as transfection vectors. Mature embryos from three varieties of 'Valenciana' onion, Torrentina, Cobriza INTA and Grano de oro were in vitro cultivated for callus induction. After three to four months an average of 57.4% success for the three varieties was reached. Transformation was carried out with Agl1 or LBA 4404 A. tumefaciens strains, both carrying a binary vector containing the marker gene gus a and the selection gene nptII. Selection was done in callus induction medium containing 10 mgL-1 geneticin during three...
Tipo: Journal article Palavras-chave: Agrobacterium tumefaciens; Allium cepa L.; Callus; Zygotic embryos.
Ano: 2006 URL: http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582006000300030
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