Academic literature on the topic 'Agroinfiltratio'
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Journal articles on the topic "Agroinfiltratio"
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Liu, Pei-Feng, Yanhan Wang, Robert G. Ulrich, Christopher W. Simmons, Jean S. VanderGheynst, Richard L. Gallo, and Chun-Ming Huang. "Leaf-Encapsulated Vaccines: Agroinfiltration and Transient Expression of the AntigenStaphylococcal EndotoxinB in Radish Leaves." Journal of Immunology Research 2018 (2018): 1–9. http://dx.doi.org/10.1155/2018/3710961.
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Transgene introgression is a major concern associated with transgenic plant-based vaccines. Agroinfiltration can be used to selectively transform nonreproductive organs and avoid introgression. Here, we introduce a new vaccine modality in which Staphylococcal enterotoxin B (SEB) genes are agroinfiltrated into radishes (Raphanw sativusL.), resulting in transient expression and accumulation of SEBin planta. This approach can simultaneously express multiple antigens in a single leaf. Furthermore, the potential of high-throughput vaccine production was demonstrated by simultaneously agroinfiltrating multiple radish leaves using a multichannel pipette. The expression of SEB was detectable in two leaf cell types (epidermal and guard cells) in agroinfiltrated leaves. ICR mice intranasally immunized with homogenized leaves agroinfiltrated with SEB elicited detectable antibody to SEB and displayed protection against SEB-induced interferon-gamma (IFN-γ) production. The concept of encapsulating antigens in leaves rather than purifying them for immunization may facilitate rapid vaccine production during an epidemic disease.
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Ambrós, Silvia, Choaa El-Mohtar, Susana Ruiz-Ruiz, Leandro Peña, José Guerri, William O. Dawson, and Pedro Moreno. "Agroinoculation of Citrus tristeza virus Causes Systemic Infection and Symptoms in the Presumed Nonhost Nicotiana benthamiana." Molecular Plant-Microbe Interactions® 24, no. 10 (October 2011): 1119–31. http://dx.doi.org/10.1094/mpmi-05-11-0110.
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Citrus tristeza virus (CTV) naturally infects only some citrus species and relatives and within these it only invades phloem tissues. Failure to agroinfect citrus plants and the lack of an experimental herbaceous host hindered development of a workable genetic system. A full-genome cDNA of CTV isolate T36 was cloned in binary plasmids and was used to agroinfiltrate Nicotiana benthamiana leaves, with or without coinfiltration with plasmids expressing different silencing-suppressor proteins. A time course analysis in agroinfiltrated leaves indicated that CTV accumulates and moves cell-to-cell for at least three weeks postinoculation (wpi), and then, it moves systemically and infects the upper leaves with symptom expression. Silencing suppressors expedited systemic infection and often increased infectivity. In systemically infected Nicotiana benthamiana plants, CTV invaded first the phloem, but after 7 wpi, it was also found in other tissues and reached a high viral titer in upper leaves, thus allowing efficient transmission to citrus by stem-slash inoculation. Infected citrus plants showed the symptoms, virion morphology, and phloem restriction characteristic of the wild T36 isolate. Therefore, agroinfiltration of Nicotiana benthamiana provided the first experimental herbaceous host for CTV and an easy and efficient genetic system for this closterovirus.
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Wang, Zhiquan, Xiaoyang Xu, Longjie Ni, Jinbo Guo, and Chunsun Gu. "Efficient virus-induced gene silencing in Hibiscus hamabo Sieb. et Zucc. using tobacco rattle virus." PeerJ 7 (August 12, 2019): e7505. http://dx.doi.org/10.7717/peerj.7505.
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Background Hibiscus hamabo Sieb. et Zucc. is a semi-mangrove plant used for the ecological restoration of saline-alkali land, coastal afforestation and urban landscaping. The genetic transformation H. hamabo is currently inefficient and laborious, restricting gene functional studies on this species. In plants, virus-induced gene silencing provides a pathway to rapidly and effectively create targeted gene knockouts for gene functional studies. Methods In this study, we tested the efficiency of a tobacco rattle virus vector in silencing the cloroplastos alterados 1 (CLA1) gene through agroinfiltration. Results The leaves of H. hamabo showed white streaks typical of CLA1 gene silencing three weeks after agroinfiltration. In agroinfiltrated H. hamabo plants, the CLA1 expression levels in leaves with white streaks were all significantly lower than those in leaves from mock-infected and control plants. Conclusions The system presented here can efficiently silence genes in H. hamabo and may be a powerful tool for large-scale reverse-genetic analyses of gene functions in H. hamabo.
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Bridgeland, Aya, Sudip Biswas, Nikolaos Tsakirpaloglou, Michael J. Thomson, and Endang M. Septiningsih. "Optimization of gene editing in cowpea through protoplast transformation and agroinfiltration by targeting the phytoene desaturase gene." PLOS ONE 18, no. 4 (April 5, 2023): e0283837. http://dx.doi.org/10.1371/journal.pone.0283837.
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Cowpea (Vigna unguiculata) is a legume staple widely grown across Sub-Saharan Africa and other tropical and sub-tropical regions. Considering projected climate change and global population increases, cowpea’s adaptation to hot climates, resistance to drought, and nitrogen-fixing capabilities make it an especially attractive crop for facing future challenges. Despite these beneficial traits, efficient varietal improvement is challenging in cowpea due to its recalcitrance to transformation and long regeneration times. Transient gene expression assays can provide solutions to alleviate these issues as they allow researchers to test gene editing constructs before investing in the time and resource- intensive process of transformation. In this study, we developed an improved cowpea protoplast isolation protocol, a transient protoplast assay, and an agroinfiltration assay to be used for initial testing and validation of gene editing constructs and for gene expression studies. To test these protocols, we assessed the efficacy of a CRISPR-Cas9 construct containing four multiplexed single-guide RNA (sgRNA) sequences using polyethylene glycol (PEG)-mediated transformation and agroinfiltration with phytoene desaturase (PDS) as the target gene. Sanger sequencing of DNA from transformed protoplasts and agroinfiltrated cowpea leaves revealed several large deletions in the target sequences. The protoplast system and agroinfiltration protocol developed in this study provide versatile tools to test gene editing components before initiating plant transformation, thus improving the chance of using active sgRNAs and attaining the desired edits and target phenotype.
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Debler, Johannes W., Bernadette M. Henares, and Robert C. Lee. "Agroinfiltration for transient gene expression and characterisation of fungal pathogen effectors in cool-season grain legume hosts." Plant Cell Reports 40, no. 5 (April 3, 2021): 805–18. http://dx.doi.org/10.1007/s00299-021-02671-y.
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Abstract Key message Modified pEAQ-HT-DEST1 vectors were used for agroinfiltration in legumes. We demonstrate protein expression and export in pea, lentil, and faba bean; however, the method for chickpea was not successful. Abstract Agroinfiltration is a valuable research method for investigating virulence and avirulence effector proteins from pathogens and pests, where heterologous effector proteins are transiently expressed in plant leaves and hypersensitive necrosis responses and other effector functions can be assessed. Nicotiana benthamiana is widely used for agroinfiltration and the characterisation of broad-spectrum effectors. The method has also been used in other plant species including field pea, but not yet developed for chickpea, lentil, or faba bean. Here, we have modified the pEAQ-HT-DEST1 vector for expression of 6 × histidine-tagged green-fluorescent protein (GFP) and the known necrosis-inducing broad-spectrum effector necrosis and ethylene-inducing peptide (Nep1)-like protein (NLP). Modified pEAQ-based vectors were adapted to encode signal peptide sequences for apoplast targeting of expressed proteins. We used confocal microscopy to assess the level of GFP expression in agroinfiltrated leaves. While at 3 days after infiltration in N. benthamiana, GFP was expressed at a relatively high level, expression in field pea and faba bean at the same time point was relatively low. In lentil, an expression level of GFP similar to field pea and faba bean at 3 days was only observed after 5 days. Chickpea leaf cells were transformed at low frequency and agroinfiltration was concluded to not be successful for chickpea. We concluded that the pEAQ vector is suitable for testing host-specific effectors in field pea, lentil, and faba bean, but low transformation efficiency limits the utility of the method for chickpea.
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Dickinson, Christopher C., Alexandra J. Weisberg, and John G. Jelesko. "Transient Heterologous Gene Expression Methods for Poison Ivy Leaf and Cotyledon Tissues." HortScience 53, no. 2 (February 2018): 242–46. http://dx.doi.org/10.21273/hortsci12421-17.
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Poison ivy [Toxicodendron radicans (L.) Kuntz] is a widely recognized native plant species because of its production of urushiol, which is responsible for delayed contact dermatitis symptoms in humans. Poison ivy is predicted to become both more prevalent and more noxious in response to projected patterns of climate change. Future studies on poison ivy chemical ecology will require reverse genetics to investigate urushiol metabolism. A prerequisite for reverse genetic procedures is the introduction and expression of recombinant DNA into poison ivy tissues. Poison ivy leaves and cotyledons were marginally susceptible to vacuum- and syringe-agroinfiltration and expression of two firefly luciferase (LUC)–based reporter genes. The efficacy of agroinfiltration and transient LUC expression was dependent on leaf age and plant growth environmental conditions, with young leaves grown in magenta boxes showing highest transient LUC expression levels. Agroinfiltrated leaves showed an Agrobacterium-dependent accumulation of brown–colored pigments. Biolistic transformation of a LUC reporter gene did not show brown pigment accumulation and readily displayed transient LUC bioluminescence in both leaves and cotyledon tissues. These studies establish best practices for introducing and transiently expressing recombinant DNA into poison ivy leaf and cotyledon tissues, on which future reverse genetic procedures can be developed.
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Chong, Xinran, Yue Wang, Xiaoyang Xu, Fan Zhang, Chuanyong Wang, Yanwei Zhou, Ting Zhou, Yunlong Li, Xiaoqing Lu, and Hong Chen. "Efficient Virus-Induced Gene Silencing in Ilex dabieshanensis Using Tobacco Rattle Virus." Forests 14, no. 3 (February 28, 2023): 488. http://dx.doi.org/10.3390/f14030488.
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Ilex dabieshanensis is not only an important ornamental plant, but can also be used to produce Kuding tea, owing to its lipid-lowering and anti-inflammatory medicinal properties. The genetic transformation of I. dabieshanensis is currently difficult, which restricts functional gene studies and molecular breeding research on this species. Virus-induced gene silencing (VIGS) is a powerful tool for determining gene functions in plants. The present study reports the first application of VIGS mediated by a tobacco rattle virus (TRV) vector in I. dabieshanensis. We tested the efficiency of the VIGS system to silence Mg-chelatase H subunit (ChlH) gene through agroinfiltration. The agroinfiltrated leaves of I. dabieshanensis exhibited a typical yellow-leaf phenotype of ChlH gene silencing at 21 days post infiltration. Endogenous ChlH expression levels in the leaves of yellow-leaf phenotype plants were all significantly lower than that in the leaves of mock-infected and control plants. Overall, our results indicated that the TRV-based VIGS system can efficiently silence genes in I. dabieshanensis, and this system will contribute to efficient functional genomics research in I. dabieshanensis.
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Chiba, Sotaro, Kamal Hleibieh, Alice Delbianco, Elodie Klein, Claudio Ratti, Véronique Ziegler-Graff, Salah Bouzoubaa, and David Gilmer. "The Benyvirus RNA Silencing Suppressor Is Essential for Long-Distance Movement, Requires Both Zinc-Finger and NoLS Basic Residues but Not a Nucleolar Localization for Its Silencing-Suppression Activity." Molecular Plant-Microbe Interactions® 26, no. 2 (February 2013): 168–81. http://dx.doi.org/10.1094/mpmi-06-12-0142-r.
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The RNA silencing-suppression properties of Beet necrotic yellow vein virus (BNYVV) and Beet soil-borne mosaic virus (BSBMV) cysteine-rich p14 proteins have been investigated. Suppression of RNA silencing activities were made evident using viral infection of silenced Nicotiana benthamiana 16C, N. benthamiana agroinfiltrated with green fluorescent protein (GFP), and GF-FG hairpin triggers supplemented with viral suppressor of RNA silencing (VSR) constructs or using complementation of a silencing-suppressor-defective BNYVV virus in Chenopodium quinoa. Northern blot analyses of small-interfering RNAs (siRNAs) in agroinfiltration tests revealed reduced amounts of siRNA, especially secondary siRNA, suggesting that benyvirus VSR act downstream of the siRNA production. Using confocal laser-scanning microscopy imaging of infected protoplasts expressing functional p14 protein fused to an enhanced GFP reporter, we showed that benyvirus p14 accumulated in the nucleolus and the cytoplasm independently of other viral factors. Site-directed mutagenesis showed the importance of the nucleolar localization signal embedded in a C4 zinc-finger domain in the VSR function and intrinsic stability of the p14 protein. Conversely, RNA silencing suppression appeared independent of the nucleolar localization of the protein, and a correlation between BNYVV VSR expression and long-distance movement was established.
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Tu, Liqin, Shuhua Wu, Danna Gao, Yong Liu, Yuelin Zhu, and Yinghua Ji. "Synthesis and Characterization of a Full-Length Infectious cDNA Clone of Tomato Mottle Mosaic Virus." Viruses 13, no. 6 (June 1, 2021): 1050. http://dx.doi.org/10.3390/v13061050.
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Tomato mottle mosaic virus (ToMMV) is a noteworthy virus which belongs to the Virgaviridae family and causes serious economic losses in tomato. Here, we isolated and cloned the full-length genome of a ToMMV Chinese isolate (ToMMV-LN) from a naturally infected tomato (Solanum lycopersicum L.). Sequence analysis showed that ToMMV-LN contains 6399 nucleotides (nts) and is most closely related to a ToMMV Mexican isolate with a sequence identity of 99.48%. Next, an infectious cDNA clone of ToMMV was constructed by a homologous recombination approach. Both the model host N. benthamiana and the natural hosts tomato and pepper developed severe symptoms upon agroinfiltration with pToMMV, which had a strong infectivity. Electron micrographs indicated that a large number of rigid rod-shaped ToMMV virions were observed from the agroinfiltrated N. benthamiana leaves. Finally, our results also confirmed that tomato plants inoculated with pToMMV led to a high infection rate of 100% in 4–5 weeks post-infiltration (wpi), while pepper plants inoculated with pToMMV led to an infection rate of 40–47% in 4–5 wpi. This is the first report of the development of a full-length infectious cDNA clone of ToMMV. We believe that this infectious clone will enable further studies of ToMMV genes function, pathogenicity and virus–host interaction.
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Sindarovska, Yana, and Mykola Kuchuk. "Long-Term Potato Virus X (PVX)-Based Transient Expression of Recombinant GFP Protein in Nicotiana benthamiana Culture In Vitro." Plants 10, no. 10 (October 15, 2021): 2187. http://dx.doi.org/10.3390/plants10102187.
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Plant molecular farming has a great potential to produce valuable proteins. Transient expression technology provides high yields of recombinant proteins in greenhouse-grown plants, but every plant must be artificially agroinfiltrated, and open greenhouse systems are less controlled. Here, we propose to propagate agrobacteria-free plants with high-efficient long-term self-replicated transient gene expression in a well-controlled closed in vitro system. Nicotiana benthamiana plant tissue culture in vitro, with transient expression of recombinant GFP, was obtained through shoot induction from leaf explants infected by a PVX-based vector. The transient expression occurs in new tissues and regenerants due to the natural systemic distribution of viral RNA carrying the target gene. Gene silencing was delayed in plants grown in vitro, and GFP was detected in plants for five to six months. Agrobacteria-free, GFP-expressing plants can be micropropagated in vitro (avoiding an agroinfiltration step), “rejuvenated” through regeneration (maintaining culture for years), or transferred in soil. The mean GFP in the regenerants was 18% of the total soluble proteins (TSP) (0.52 mg/g of fresh leaf weight (FW). The highest value reached 47% TSP (2 mg/g FW). This study proposes a new method for recombinant protein production combining the advantages of transient expression technology and closed cultural systems.
Dissertations / Theses on the topic "Agroinfiltratio"
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Abe, Valeria Yukari. "Expressão transiente de genes de Phakopsora pachyrhizi em genótipos resistentes de soja visando a identificação de genes de avirulência." Universidade Federal de Viçosa, 2012. http://locus.ufv.br/handle/123456789/4404.
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Previous issue date: 2012-02-17Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
Brazil is the second world soybean producer. Currently, the main limiting factor in this crop production is the Asian soybean rust (ASR) whose etiologic agent is the fungus Phakopsora pachyrhizi. The rust fungi are obligate parasites that during their interaction with the plant, they secrete effector proteins that manipulate host metabolism and interfere with their defense responses. Some of these effector proteins, called Avr proteins, are recognized by encoded proteins by resistant R genes, which usually trigger a hypersensitivity response (HR) and resistance phenotype. At least, there are five described R genes (Rpp1 to Rpp5) that confer resistance to ASR and several genes that encode secreted proteins by this fungus were recently identified. However, the effector proteins (Avr) recognized by encoded proteins by Rpp genes were not identified yet. Since there is not a transformation assay protocol for P. pachyrhizi, a strategy to identify this fungus Avr proteins is the transient expression of R proteins in resistant varieties cytoplasm and the observation of a possible HR response. Thus, the specific objectives of this work were: to try to establish a methodology for transient expression in soybean by agroinfiltration using the gene GUSPlus as reporter gene; to establish a protocol for translocation of effector proteins by the type III secretion system (SST3) of Pseudomonas syringae pv. glycinea race 4 (Psg4), and also to evaluate the effector activity of candidate genes in soybean resistant genotypes to the isolate PPUFV02 of P. pachyrhizi. There was no expression of the gene GUSPlus in Agrobacterium tumefaciens strain EHA105 infiltrated soybean leaves, while using the same inoculum preparation and concentration of bacterial cells, there was a consistent expression of the gene GUSPlus in tobacco. This result derailed the use of agroinfiltration in the functional study of candidate genes in soybean effectors. All soybean genotypes evaluated were susceptible to Psg4, demonstrating that the viability to use this bacterial on functional analysis of candidate effector proteins mediated by SST3. Better symptoms reproducibility was observed with inoculation by vacuum infiltration of Psg4 in a bacterial concentration of OD600 = 0,01, for allowing a gradually symptoms analysis. The encoded protein by avrB gene is recognized by the RPG1 gene product, which is present in some genotypes of soybean. The construction pVSP61-avrB was able to induce HR in the genotype Williams 82, that contains the corresponding gene RPG1 and to induce it in the genotypes Conquista and PI 459025. This result allowed the use of this construction as a positive control for functional analysis of P. pachyrhizi putative effector proteins. Because of this, 12 sequences were cloned into vector pEDV6. This vector allows the expression of proteins of interest fused to secretion signal sequences by SST3, aiming its subsequent translocation into the cytosol. Nine from the constructions with pEDV6-PHPA_RSP transformed into Psg4 were submitted to functional analysis. The inoculated plants varied in severity of observed symptoms and no HR phenotype was observed. Instead, it was observed reduction, increase or absence of a significant change in the symptoms evolution of genotype-dependent manner in treated plants. These studies allowed a first screening of P. pachyrhizi effector candidates, selecting the candidates PHPA_RSP_71 and PHPA_RSP_78 as the most promising candidates for further detailed analysis.
O Brasil é o segundo maior produtor mundial de soja. Atualmente, o principal fator limitante na produção desta cultura é a ferrugem asiática da soja (FAS), cujo agente etiológico é o fungo Phakopsora pachyrhizi. Os fungos causadores das ferrugens são parasitas obrigatórios que durante sua interação com a planta secretam proteínas efetoras que manipulam o metabolismo do hospedeiro e interferem com suas respostas de defesa. Algumas dessas proteínas efetoras, denominadas proteínas Avr, são reconhecidas pelas proteínas codificadas por genes de resistência R, o que desencadeia a resposta de hipersensibilidade (HR) e fenótipo de resistência. Já foram descritos pelo menos cinco genes R (Rpp1 a Rpp5) que conferem resistência a FAS e vários genes que codificam proteínas secretadas por esse fungo foram recentemente identificados. Todavia, ainda não foram identificadas as proteínas efetoras (Avr) reconhecidas pelas proteínas codificadas pelos genes Rpp. Como não existe ainda um sistema de transformação para P. pachyrhizi, uma estratégia para identificar as proteínas Avr desse fungo é a expressão transiente das proteínas efetoras candidatas no citoplasma de variedades resistentes e a observação do possível desencadeamento de resposta de HR. Desta forma, os objetivos específicos deste trabalho foram tentar estabelecer uma metodologia de expressão transiente em soja via agroinfiltração utilizando como gene repórter o gene GUSPlus; estabelecer um protocolo de translocação de proteínas efetoras via sistema de secreção tipo III (SST3) de Pseudomonas syringae pv. glycinea raça 4 (Psg4), e também avaliar a atividade efetora de genes candidatos em genótipos de soja resistentes ao isolado monopustular PPUFV02 de P. pachyrhizi. Não se observou a expressão do gene GUSPlus em folhas de soja agroinfiltradas com Agrobacterium tumefaciens estirpe EHA105, enquanto que utilizando do mesmo preparo de inóculo e concentração de células bacterianas, observou-se a expressão consistente do gene GUSPlus em tabaco. Este resultado invibializou o uso de agroinfiltração no estudo funcional de genes efetores candidatos na soja. Todos os genótipos de soja avaliados foram suscetíveis a Psg4, demonstrando a vialibilidade do uso desta bactéria na análise funcional de proteínas candidatas a efetores mediada por SST3. Melhor reprodutibilidade de sintomas foi observada com a inoculação por infiltração a vácuo de Psg4 numa concentração bacteriana de OD600=0,01, por permitir uma análise dos sintomas de forma gradual. O produto do gene avrB é reconhecido pela produto do gene RPG1, presente em alguns genótipos de soja. A construção pVSP61-avrB, foi capaz de induzir HR no genótipo Williams 82, que contêm o gene RPG1 correspondente, e nos genótipos Conquista e PI 459025. Este resultado permitiu o uso desta construção como controle positivo para a análise funcional de proteínas efetoras putativas de P. pachyrhizi. Assim, 12 sequências foram clonadas no vetor pEDV6. Este vetor permite a expressão de proteínas de interesse fusionadas a sequências-sinais de secreção via SST3, visando a sua posterior translocação para o citosol. Nove das construções com pEDV6-PHPA_RSP transformadas em Psg4 foram submetidas à análise funcional. As plantas inoculadas variaram quanto à severidade dos sintomas observados e não foi constatado fenótipo de HR. Ao invés disso, nos tratamentos foi observado redução, aumento ou ausência de alteração significativa na evolução dos sintomas, de maneira genótipo-dependente. Esses estudos permitiram uma primeira triagem de candidatos a efetores de P. pachyrhizi, selecionando os candidatos PHPA_RSP_71 e PHPA_RSP_78 como os mais promissores para estudos futuros mais detalhados.
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Tah, Tapashree Schoelz James E. "Chloroplast GFP expression in tobacco plants agroinfiltrated with tobacco mosaic virus based vectors." Diss., Columbia, Mo. : University of Missouri--Columbia, 2009. http://hdl.handle.net/10355/6604.
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Title from PDF of title page (University of Missouri--Columbia, viewed on Feb 19, 2010). The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file. Thesis advisor: Dr. James E. Schoelz. Includes bibliographical references.
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Joh, Lawrence Day. "High-level transient expression, extraction, and purification of recombinant [beta]-glucuronidase from agroinfiltrated lettuce /." For electronic version search Digital dissertations database. Restricted to UC campuses. Access is free to UC campus dissertations, 2005. http://uclibs.org/PID/11984.
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Thesis (Ph. D.)--University of California, Davis, 2005.Degree granted in Biological Systems Engineering. On t.p. "[beta]" appears as Greek letter. Also available via the World Wide Web. (Restricted to UC campuses)
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Bunwaree, Heemee Devi. "Implementation of a genetic screen for the identification of resistances to beet virus yellows." Electronic Thesis or Diss., Strasbourg, 2024. http://www.theses.fr/2024STRAJ042.
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Depuis l'interdiction des néonicotinoïdes dans l'Union Européenne, la production de betteraves sucrières est fortement menacée par des épidémies de jaunisses virales. Ces maladies sont causées par un complexe de plusieurs virus transmis par les pucerons. Parmi eux, les polérovirus, responsables de jaunisses modérées, tels que le beet mild yellowing virus (BMYV) et le beet chlorosis virus (BChV), sont particulièrement répandus. Afin d’améliorer le criblage des variétés de betteraves sucrières résistantes ou tolérantes aux jaunisses virales, nous avons mis au point un virus recombinant provoquant des symptômes visibles permettant de distinguer facilement et sans autre technologie les plantes infectées des plantes saines. Il s’agit d’un clone de BMYV capable d'induire l'extinction d'un gène endogène via le phénomène de virus induced gene silencing (VIGS) dont l’infection se manifeste par l'apparition accélérée de jaunisses au niveau des nervures des feuilles des betteraves, dès dix jours après l'agroinoculation. Les analyses moléculaires ont révélé que le virus recombinant présente un pouvoir infectieux comparable à celui du virus sauvage, et que l'insertion génétique est stable dans la descendance virale pendant au moins cinq mois après infiltration. Nos résultats ont également montré que le pourcentage de plantes présentant des symptômes de VIGS est représentatif du taux d'infection pour chaque lignée de betteraves testée. L'utilisation de cet outil nous a permis d'identifier visuellement au sein de quarante-deux lignées de betteraves sucrières, une lignée potentiellement résistante au BMYV ainsi que trois lignées partiellement résistantes. De telles lignées représentent des candidats potentiels intéressants pour les programmes de sélection. Ainsi, ce travail valide l'utilisation d'un polérovirus comme vecteur de VIGS, adapté à la betterave sucrière, permettant des criblages visuels et robustes à grande échelle pour l'identification de gènes de résistance ou pour des études fonctionnellesSince the ban on neonicotinoids in the European Union, sugar beet production has been severely threatened by virus yellows (VY) epidemics. VY are caused by a complex of several aphid-transmitted viruses, among which the poleroviruses beet mild yellowing virus (BMYV) and beet chlorosis virus (BChV) are highly represented. In order to improve the screening of sugar beet varieties resistant or tolerant to viral yellows, we produced a recombinant virus, allowing easy and rapid visual discrimination between infected and healthy plants, without the need of additional equipment. It is a clone of BMYV capable of inducing the silencing of an endogenous gene via the phenomenon of virus induced gene silencing (VIGS), with infection manifesting as accelerated vein clearing of leaves, starting as early as ten days after agroinoculation. Molecular analyses revealed that the recombinant virus displays the same infectivity as the wild-type virus and that the insert is stable within the viral progeny, till at least five months post-infiltration. Our results also indicated that the percentage of VIGS-symptomatic plants is representative of the infection rate for each evaluated line. The use of this tool allowed us to visually identify one BMYV resistant and three partial resistant lines from forty-two sugar beet lines. Such lines represent interesting potential candidates for breeding programs. Thus, this work validates the use of a polerovirus as a VIGS vector, adapted to sugar beet, allowing large-scale, robust visual screenings for the identification of resistance genes or for functional studies
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Bandyopadhyay, Amrita. "Analysis of the Arabidopsis Polyadenylation Factors PAP1, CstF64 and CstF77 and their characteristic inter-relationship." UKnowledge, 2009. http://uknowledge.uky.edu/gradschool_theses/601.
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3’-end modification by polyadenylation is a ubiquitous feature of almost all eukaryotic mRNA species and is catalyzed by a consortium of enzymes, the polyadenylation factors. Poly(A) polymerase (PAP), the enzyme catalyzing the addition of adenosine residues during the polyadenylation stage, exists in four isoforms within Arabidopsis. In silico and yeast two-hybrid studies showed that PAP1 has unique expression and interaction pattern in Arabidopsis, suggesting non-canonical functions of PAP1. Its exclusive interaction with PAP4 has not been reported in other living systems until now and hints at a difference in polyadenylation in plants with respect to mammals and yeast. Cleavage Stimulation Factor (CstF), a heterotrimeric complex of the polyadenylation factors CstF50, CstF64 and CstF77, plays a role largely in cleavage of pre-mRNA. This study highlights some aspects of the Arabidopsis homologs of CstF64 and CstF77, central to various cellular processes other than nuclear polyadenylation. In silico studies showed an elevated expression of CstF64 in the pollen while that of CstF77 remained fairly low. Yeast two-hybrid assays indicated a novel kind of interaction of CstF64 with Fip1(V). It is also speculated from sub-cellular localization techniques by agroinfiltration in tobacco leaves that CstF64 localizes in the cytoplasm and CstF77 in the nucleus, as found for the orthologs of CstF77 in other systems.
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Lin, Yi-Jyun, and 林怡君. "Examination Effect of Accessory Protein in T-DNA transferring by Transient Agroinfiltration of Tobacco." Thesis, 2011. http://ndltd.ncl.edu.tw/handle/82128429955580164605.
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碩士國立中興大學
生物科技學研究所
99
For the Agrobacterium-mediated plant transformation, the long journey for T-DNA transferring from bacteria till the final destination not only requires bacterial proteins, such as VirE2, VirE3, etc., but also is aided by various plant factors, including BTI1/BTI2/BTI3/Rab8, VIP1, VIP2, Ku70/Ku80, H2A, etc. Many reports had demonstrated that T1 progenies of transgenic Arabidopsis overproducing various accessory proteins exhibited higher transformation efficiency. Gene orthologs encoding the accessory proteins were isolated from rice and designated as “ET genes” for purpose of “enhance transformation”. Co-transformation strategy, infiltrate one Agrobacterium carries an ET gene and the other contains the mGFP5 reporter gene, was employed to evaluate transient transformation efficiency in tobacco. Several ET genes were already identified to be effective. In this study, ET gene expressed by 2X35S promoter mostly exerted better results than the previous constructs. Synergistic effects were observed when several ET genes were combined, with the best effect observed for mixture of VirE2, VIP1 and H2A genes. Moreover, to demonstrate that enhancement of transformation is caused by expression of ET proteins, truncated ET genes were generated. As a result, disrupt open reading frame of ET gene concomitantly diminish its ET effect. A preliminary test using floral dip of Arabidopsis revealed that BTI3 enhance permanent transformation frequency to be about ~2.2 fold. Besides, Agrobacterium EHA105 was transformed with plasmids to overproduce VirG proteins in its wildtype or constitutively active (N54D) version. VirG gene from C58, but not from LBA4404, was found to increase the “virulence” of EHA105.
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Lin, Jia-Ying, and 林佳螢. "Development of an in Planta System to Monitor Phosphorus Status by Agroinfiltration and Agroinjection." Thesis, 2014. http://ndltd.ncl.edu.tw/handle/21081124561212623300.
Full textAbstract:
碩士國立臺灣大學
園藝暨景觀學系
102
Phosphorous (P), one of the essential mineral nutrients for plant growth and development, is involved in the regulation of several physiological and biochemical processes in plants. In our study, it was found that, in the tobacco and tomato plants at vegetative growth stage under phosphorus deficiency treatment, the phosphate (Pi) concentration in shoot and root decreased rapidly, the chlorophyll fluorescence (Fv/Fm), chlorophyll content and shoot fresh weight decreased relatively slowly, while the root/shoot fresh weight ratio increased gradually. Furthermore, in tomato plants at reproductive growth stage under Pi starvation, the Pi concentration in fruit decreased rapidly, and then the fruit yield and total soluble solids decreased. Accordingly, it is shown that the vegetative growth of tobacco and tomato plants as well as the final yield and quality of tomato depend on whether phosphorus fertilizer is supplied sufficiently. It was further found that, while temporary phosphorus deficiency may immediately lead to the decrease of the Pi concentration in shoot or fruit, the shoot fresh weight or the fruit yield and quality may not be adversely impacted thereby so long as phosphorus fertilizer is timely supplemented to rapidly increase the Pi concentration in shoot or fruit. This indicates that the yield and quality of crops can be ensured by monitoring the nutritional status of the crops and supplying fertilizer as appropriate. By Agroinfiltration and Agroinjection, this study demonstrated that the expression of GUS reporter gene driven by tomato TPSI1 promoter can rapidly and truly reflect the phosphorus deficiency stress in tobacco leaf and tomato fruit. This indicates that the employed Agroinfiltration/Agroinjection transient expression system is useful in monitoring the Pi status in plants. Further, the aforesaid expression of GUS reporter gene is independent of the deficiency of the mineral nutrients other than phosphorus, indicating the specificity of the employed Agroinfiltration transient expression system. Because the application of the aforesaid system is convenient and leads to rapid and accurate reflection of phosphorus status in plants, the aforesaid system should be applicable to other crops or to monitor other mineral nutrient status if the tomato TPSI1 promoter is replaced by other appropriate promoters.
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Ribeiro, Diana Margarida da Costa. "Optimization of transient expression procedures in Catharanthus roseus and Arabidopsis thaliana for subcellular localization studies." Master's thesis, 2010. http://hdl.handle.net/1822/15963.
Full textAbstract:
Dissertação de mestrado em Biotecnologia e Bio-Empreendedorismo em Plantas Aromáticas e MedicinaisNowadays, the availability of much gene sequence information demands the development of tools for their fast characterization at the protein level, where function actually resides. Here, the interest in the characterization of certain of the known Arabidopsis class III peroxidase (Prx) genes, as well as the interest in the characterization of candidate genes implicated in the metabolism of the anticancer terpenoid indole alkaloids of Catharanthus roseus, has led to the need of establishing transient expression procedures for these two species. Therefore, the main goal of this work was the development and optimization of simple/fast, efficient and reproducible transient expression protocols for subsequent subcellular localization studies of proteins coded by Prx genes, and for characterization of candidate genes provided from omic approaches, namely implicated in the regulation, biosynthesis or transport of the valuable alkaloids from C. roseus. A complementary goal was to investigate the subcellular localization and sorting determinants of Prxs, namely the vacuolar sorting capacity of a C-terminal amino acid sequence extension (CTE) present in vacuolar Prxs, using as examples the well characterized and most abundant vacuolar Prx from C. roseus leaves, CrPrx1, and the most abundant Prx in the leaves of Arabidopsis, AtPrx34. For this, already available CrPrx1-GFP fusions and newly generated AtPrx34-GFP fusions were used in the transient expression assays. The successful establishment of protocols for PEG-mediated transformation of both Arabidopsis and C. roseus mesophyll protoplasts was achieved and validated as excellent transient expression systems. Transient expression by Agrobacterium infiltration of Arabidopsis and C. roseus leaves was also attempted, but it was only successful with in vitro C. roseus plants. However, promising insights were made into the development of this technique. Expression of CrPrx1-GFP fusions in Arabidopsis and C. roseus protoplasts using the established protocols confirmed the vacuolar localization of this Prx. Additionally the CrPrx1 signal peptide (SP) and CTE were confirmed as sorting determinants that target GFP to the ER and vacuole, respectively. The characterization of the subcellular localization and sorting determinants of AtPrx34 was not elucidated, possibly due to malfunctioning of the vector plasmid used for protoplast infiltration. In fact, upon agroinfiltration of in vitro C. roseus plants, it was possible to observe sorting to the ER of an SPAtPrx34-GFP fusion coded by a construct harboured in a binary vector plasmid, different from the one used for protoplast transformation. Thus, a resolution of the subcellular sorting of AtPrx34 should be possible in the near future. The transient expression assays described in the present study were highly reproducible, resulted in very satisfactory transformation efficiencies, and constitute a reliable and inexpensive methods that can be performed in most labs, and that are suitable test-systems to characterize genes of unknown function. This is also the the first time a transient expression system for C. roseus protoplasts is reported, using a PEGmediated transformation protocol.
Actualmente, a disponibilidade de inúmeras sequências genómicas exige o desenvolvimento de ferramentas para uma rápida caracterização ao nível protéico, onde de facto reside a função. Neste trabalho a caracterização de determinados genes de Peroxidases de Classe III (Prx) de Arabidopsis, assim como o interesse na caracterização de possíveis genes envolvidos no metabolismo de alcalóides indólicos terpenóides anticancerígenos de Catharanthus roseus, impulsionou a necessidade de estabelecer procedimentos de expressão transiente para estas duas espécies. Consequentemente, o objectivo principal deste trabalho foi o desenvolvimento e optimização de protocolos de expressão transiente simples/rápidos, eficientes e reproduzíveis para estudos de localização subcelular de proteínas codificados por genes Prx, e para a caracterização de possíveis genes obtidos de abordagens omicas, nomeadamente implicados na regulação, biossíntese ou transporte de alcalóides relevantes de C. roseus. Como objectivo complementar investigar a localização subcelular e sinais de direccionamento de Prxs, designadamente a capacidade de direccionamento vacuolar da extensão C-terminal aminoacídica (CTE) presente em Prxs vacuolares, utilizando como exemplos a Prx vacuolar mais abundante e estudada presente nas folhas de C. roseus, Crprx1, e a Prx mais abundante nas folhas de Arabidopsis, AtPrx34. Para tal, fusões CrPr1-GFP já disponíveis e fusões AtPrx34-GFP recém geradas foram utilizadas em procedimentos de expressão transiente. O estabelecimento com sucesso de protocolos de transformação mediada por PEG para protoplastos de mesófilo de Arabidopsis e C. roseus foi alcançado e validado como um excelente sistema de transformação transiente. Transformação transiente por infiltração com Agrobacterium de folhas de Arabidopsis e C. roseus foi abordado, mas apenas foram obtidos resultados positivos com plantas de C. roseus in vitro. Todavia progressos promissores foram realizados para o desenvolvimento desta técnica. Expressão de fusões CrPrx1-GFP em protoplastos de Arabidopsis e C. roseus utilizando os protocolos estabelecidos confirmaram a localização vacuolar desta Prx. Adicionalmente o péptido sinal (SP) e a extensão C-terminal (CTE) de CrPrx1 foram confirmados com sinais determinantes que direccionam a GFP para o RE e o vacúolo, respectivamente. A caracterização da localização subcelular e sinais de direccionamento de AtPrx34 não foram elucidados, possivelmente devido a uma irregularidade funcional do vector plasmídeal utilizado na transformação de protoplastos. De facto, após agroinfiltraçao de plantas C. roseus in vitro, foi possível observar o direccionamento para o RE da fusão SPAtPrx34-GFP codificada por um constructo incluído em vector binário, diferente do vector utilizado na transformação de protoplastos. Portanto, a caracterização do direccionamento subcelular da AtPrx34 poderá ser possível num futuro próximo. Os procedimentos de expressão transiente descritos no presente estudo manifestaram-se bastante reproduzíveis, resultando em níveis satisfatórios de eficiência de transformação, e constituem métodos fidedignos e de baixo custo que podem ser realizados na maioria dos laboratórios, e são sistemas-teste convenientes para caracterizar genes de função desconhecida. Foi também reportado pela primeira vez um sistema de transformação transiente em protoplastos de C. roseus, utilizando um protocolo de transformação mediada por PEG. Palavras-chave: Catharanthus roseus, Arabidopsis, transformação mediada por PEG, Agroinfiltração, fusões-sGFP, localização subcelular, via secretora, sinais de direccionamento, vacúolo, microscopia confocal.
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Atnaseo, Chuthamat. "Transgenic Plant and Fungal Expression to Assay in vitro and in planta Activity of Sus scrofa beta-Defensin 1 and Nicotiana tabacum Defensin 1." Thesis, 2011. http://hdl.handle.net/10214/3193.
Full textAbstract:
To explore the use of defensins for transgenic plant disease resistance, expression by agroinfiltration of plants, stable transformation of plants and stable transformation of yeast were tested for porcine β-defensin 1 (pbd-1) and Nicotiana tabacum defensin 1 (Ntdef1). Attempts to screen constructs by agroinfiltration of Nicotiana benthamiana leaves revealed that agroinfiltration alone induced localized resistance against Colletotrichum destructivum. A comparison of Agrobacterium tumefaciens strains showed that the induced resistance required the transfer of type IV effectors into plant cells and was independent of salicylic acid or ethylene signaling. Stable expression of pbd-1 in N. tabacum and Pichia pastoris showed that PBD-1 purified from P. pastoris had varying degrees of antimicrobial activity against a broad range of microbes, including P. syringae pv. tabaci, C. destructivum and C. orbiculare, but in transgenic N. tabacum, the protein could not be detected and resistance increased only slightly to P. syringae pv. tabaci but not to C. destructivum or C. orbiculare. Stable expression of Ntdef1 in P. pastoris yielded a protein with no or little antimicrobial activity, and stable expression in N. tabacum did not result in detectable Ntdef1 or increased resistance to those pathogens. Although PBD-1 had strong antimicrobial activity against plant pathogens, plant disease resistance likely did not increase because of the low level of the protein in the plants, whereas resistance did not increase with Ntdef1 likely because of low antimicrobial activity and low levels of the protein in the plant. This research demonstrates that agroinfiltration is not appropriate for testing genes for antimicrobial activity in planta, while the P. pastoris expression system is useful for producing protein for in vitro tests of a gene prior to its transfer to plants.
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Peng, Hsien-Chieh, and 彭宣傑. "Study on the relatedness of LsGRP1 to the salicylic acid-induced resistance of Lilium cv. Star Gazer by agroinfiltration." Thesis, 2006. http://ndltd.ncl.edu.tw/handle/77579617912117147541.
Full textAbstract:
碩士國立臺灣大學
植物病理與微生物學研究所
94
Lilium is a bulbous crop, severely affected by a major fungal disease, gray mold, caused by Botrytis elliptica. As know, development of necrotic lesions in the leaves of Oriental lily cultivar Star Gazer could be suppressed by the application of salicylic acid (SA). A cDNA sequence, named LsGRP1, coding for a 138-amino acid protein has been identified via suppression subtractive hybridization and differential screening. When the lily plants were treated with SA solution, the amount of LsGRP1 transcript analyzed by Northern blot hybridization increased. In the study of the relatedness between LsGRP1 and salicylic acid-induced disease resistance, agroinfiltration was applied for analysis of LsGRP1 transient expression in the leaves of Lilium cv. Star Gazer. In Northern blot hybridization, LsGRP1 transcript increased 72 hours after agroinfiltration of sense strand LsGRP1–expressing Agrobacterium tumefaciens EHA101 strain and more obviously 96 hours after agroinfiltraion. When the lily plants were treated with SA solution, the amount of LsGRP1 mRNA decreased in ‘Star Gazer’ leaves infiltrated with A. tumefaciens EHA101 strains expressing sense, anti-sense and hairpin LsGRP1, but the lesion numbers increased as compared to that in the control. These results showed that a gene silencing reaction was induced by introducing sense, anti-sense or hairpin LsGRP1, that abated the resistance of ‘Star Gazer’ leaves to gray mold disease. Thus, LsGRP1 playing an important role in salicylic acid-induced disease resistance of lily wasconcluded.
Book chapters on the topic "Agroinfiltratio"
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Spiegel, Holger, Stefan Schillberg, and Greta Nölke. "Production of Recombinant Proteins by Agrobacterium-Mediated Transient Expression." In Recombinant Proteins in Plants, 89–102. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2241-4_6.
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AbstractThe agroinfiltration of plant tissue is a robust method that allows the rapid and transient expression of recombinant proteins. Using wild-type plants as biomass, agroinfiltration exploits the ability of plants to synthesize even complex multimeric proteins that require oxidative folding and/or post-translational modifications, while avoiding the expensive and time-consuming creation of stably transformed plant lines. Here we describe a generic method for the transient expression of recombinant proteins in Nicotiana benthamiana at the small to medium laboratory scale, including appropriate binary vectors, the design and cloning of expression constructs, the transformation, selection, and cultivation of recombinant Agrobacterium tumefaciens, the infiltration of plants using a syringe or vacuum device, and finally the extraction of recombinant proteins from plant tissues.
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Helm, Jutta Maria, Elena Dadami, and Kriton Kalantidis. "Local RNA Silencing Mediated by Agroinfiltration." In Methods in Molecular Biology, 97–108. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-123-9_7.
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Liu, Lijing, Qingzhen Zhao, and Qi Xie. "In Vivo Ubiquitination Assay by Agroinfiltration." In Methods in Molecular Biology, 153–62. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-809-2_12.
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Chen, Qiang, Matthew Dent, Jonathan Hurtado, Jake Stahnke, Alyssa McNulty, Kahlin Leuzinger, and Huafang Lai. "Transient Protein Expression by Agroinfiltration in Lettuce." In Methods in Molecular Biology, 55–67. New York, NY: Springer New York, 2016. http://dx.doi.org/10.1007/978-1-4939-3289-4_4.
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D'Aoust, Marc-André, Pierre-Olivier Lavoie, Julie Belles-Isles, Nicole Bechtold, Michèle Martel, and Louis-P. Vézina. "Transient Expression of Antibodies in Plants Using Syringe Agroinfiltration." In Recombinant Proteins From Plants, 41–50. Totowa, NJ: Humana Press, 2009. http://dx.doi.org/10.1007/978-1-59745-407-0_3.
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Von Lanken, Carol, and Arthur G. Hunt. "Transient Expression Using Agroinfiltration to Study Polyadenylation in Plants." In Methods in Molecular Biology, 127–33. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-2175-1_11.
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Picard, Kelsey, Robyn Lee, Roger Hellens, and Richard Macknight. "Transient Gene Expression in Medicago truncatula Leaves via Agroinfiltration." In Legume Genomics, 215–26. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-613-9_15.
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Menassa, Rima, Adil Ahmad, and Jussi J. Joensuu. "Transient Expression Using Agroinfiltration and Its Applications in Molecular Farming." In Molecular Farming in Plants: Recent Advances and Future Prospects, 183–98. Dordrecht: Springer Netherlands, 2011. http://dx.doi.org/10.1007/978-94-007-2217-0_9.
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Cournoyer, Patrick, and S. P. Dinesh-Kumar. "Studying NB-LRR Immune Receptor Localization by Agroinfiltration Transient Expression." In Methods in Molecular Biology, 1–8. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61737-998-7_1.
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Vaghchhipawala, Zarir, Clemencia M. Rojas, Muthappa Senthil-Kumar, and Kirankumar S. Mysore. "Agroinoculation and Agroinfiltration: Simple Tools for Complex Gene Function Analyses." In Methods in Molecular Biology, 65–76. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60761-682-5_6.
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