The Experts below are selected from a list of 1671 Experts worldwide ranked by ideXlab platform
David S. Domozych - One of the best experts on this subject based on the ideXlab platform.
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Protoplast Isolation and manipulation in the unicellular model plant penium margaritaceum
Methods of Molecular Biology, 2020Co-Authors: David S. Domozych, Berke Tinaz, Anna Lietz, Eleanore Ritter, Sandra Cristina RaimundoAbstract:The unicellular freshwater green alga Penium margaritaceum has become a novel and valuable model organism for elucidating cell wall dynamics in plants. We describe a rapid and simple means for isolating Protoplasts using commercial enzymes in a mannitol-based buffer. Protoplasts can be cultured and cell wall recovery can be monitored in sequentially diluted mannitol-based medium. We also describe an optimized protocol to prepare highly pure, organelle-free nuclei fractions from Protoplasts using sucrose gradients. This technology provides a new and effective tool in Penium biology that can be used for analysis of cell wall polymer deposition, organelle Isolation and characterization, and molecular research including genetic transformation and somatic hybridization.
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Isolation and manipulation of Protoplasts from the unicellular green alga Penium margaritaceum
Plant Methods, 2018Co-Authors: Sandra Cristina Raimundo, Berke Tinaz, Iben Sørensen, Jocelyn K C Rose, Eleanore Ritter, David S. DomozychAbstract:Background The unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast Isolation and manipulation expedites a new range of experimental possibilities for Penium -based studies. These include enhanced means of Isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition. Results We present a methodology for enzyme-based digestion of the Penium cell wall and the Isolation of Protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from Protoplasts, which can be used for the Isolation of nuclear DNA. Conclusion Protoplast Isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium . The protocol described here offers a rapid and effective mechanism for fast and effective production of Protoplasts. Subsequently, the Protoplasts may be used for microscopy-based studies of specific subcellular components and the Isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.
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Isolation and manipulation of Protoplasts from the unicellular green alga penium margaritaceum
Plant Methods, 2018Co-Authors: Sandra Cristina Raimundo, Berke Tinaz, Iben Sørensen, Jocelyn K C Rose, Eleanore Ritter, David S. DomozychAbstract:The unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast Isolation and manipulation expedites a new range of experimental possibilities for Penium-based studies. These include enhanced means of Isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition. We present a methodology for enzyme-based digestion of the Penium cell wall and the Isolation of Protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from Protoplasts, which can be used for the Isolation of nuclear DNA. Protoplast Isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium. The protocol described here offers a rapid and effective mechanism for fast and effective production of Protoplasts. Subsequently, the Protoplasts may be used for microscopy-based studies of specific subcellular components and the Isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.
Miguel Lara - One of the best experts on this subject based on the ideXlab platform.
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Protoplast Isolation transient transformation of leaf mesophyll Protoplasts and improved agrobacterium mediated leaf disc infiltration of phaseolus vulgaris tools for rapid gene expression analysis
BMC Biotechnology, 2016Co-Authors: Kalpana Nanjareddy, Manoj Kumar Arthikala, Lourdes Blanco, Elizabeth S Arellano, Miguel LaraAbstract:Phaseolus vulgaris is one of the most extensively studied model legumes in the world. The P. vulgaris genome sequence is available; therefore, the need for an efficient and rapid transformation system is more imperative than ever. The functional characterization of P. vulgaris genes is impeded chiefly due to the non-amenable nature of Phaseolus sp. to stable genetic transformation. Transient transformation systems are convenient and versatile alternatives for rapid gene functional characterization studies. Hence, the present work focuses on standardizing methodologies for Protoplast Isolation from multiple tissues and transient transformation protocols for rapid gene expression analysis in the recalcitrant grain legume P. vulgaris. Herein, we provide methodologies for the high-throughput Isolation of leaf mesophyll-, flower petal-, hypocotyl-, root- and nodule-derived Protoplasts from P. vulgaris. The highly efficient polyethylene glycol-mannitol magnesium (PEG-MMG)-mediated transformation of leaf mesophyll Protoplasts was optimized using a GUS reporter gene. We used the P. vulgaris SNF1-related protein kinase 1 (PvSnRK1) gene as proof of concept to demonstrate rapid gene functional analysis. An RT-qPCR analysis of Protoplasts that had been transformed with PvSnRK1-RNAi and PvSnRK1-OE vectors showed the significant downregulation and ectopic constitutive expression (overexpression), respectively, of the PvSnRK1 transcript. We also demonstrated an improved transient transformation approach, sonication-assisted Agrobacterium-mediated transformation (SAAT), for the leaf disc infiltration of P. vulgaris. Interestingly, this method resulted in a 90 % transformation efficiency and transformed 60–85 % of the cells in a given area of the leaf surface. The constitutive expression of YFP further confirmed the amenability of the system to gene functional characterization studies. We present simple and efficient methodologies for Protoplast Isolation from multiple P. vulgaris tissues. We also provide a high-efficiency and amenable method for leaf mesophyll transformation for rapid gene functional characterization studies. Furthermore, a modified SAAT leaf disc infiltration approach aids in validating genes and their functions. Together, these methods help to rapidly unravel novel gene functions and are promising tools for P. vulgaris research.
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Additional file 1: of Protoplast Isolation, transient transformation of leaf mesophyll Protoplasts and improved Agrobacterium-mediated leaf disc infiltration of Phaseolus vulgaris: tools for rapid gene expression analysis
2016Co-Authors: Kalpana Nanjareddy, Manoj Kumar Arthikala, Lourdes Blanco, Elizabeth Arellano, Miguel LaraAbstract:Selection of appropriate Phaseolus vulgaris tissue material for Protoplast Isolation. (A) Wing and keel petals were excised, retaining the pink-colored portions, for Protoplast Isolation. (B) The roots of 3-day-old germinated seeds were cut 3 mm from tip and were used for Protoplast Isolation. (C) Three-day-old germinating seeds were decotyledoned, and ~10 mm hypocotyls were used to isolate Protoplasts. (D) Sliced 18-dpi nodule that was inoculated with R. tropici harboring the pSN30-GFP plasmid expressing GFP fluorescence protein as seen under a laser scanning confocal microscope. Hc, hypocotyl; dpi, days post inoculation; dashed line, site of excision. (DOC 576 kb
Neal C Stewart - One of the best experts on this subject based on the ideXlab platform.
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switchgrass panicum virgatum l cell suspension cultures establishment characterization and application
Plant Science, 2011Co-Authors: Hani Alahmad, Mary R Rudis, Neal C Stewart, Mitra Mazarei, Blake L JoyceAbstract:a b s t r a c t Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable atten- tion as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell sus- pension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using Protoplast Isolation. A high Protoplast Isolation rate of up to 10 6 Protoplasts/1.0 g of cells was achieved for the fine milky culture, whereas only a few Protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of Protoplast Isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research. © 2011 Elsevier Ireland Ltd. All rights reserved.
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Protoplast Isolation and transient gene expression in switchgrass panicum virgatum l
Biotechnology Journal, 2008Co-Authors: Mitra Mazarei, Hani Alahmad, Mary R Rudis, Neal C StewartAbstract:Transient assay systems using Protoplasts have been utilized in several plant species and are a powerful tool for rapid functional gene analysis and biochemical manipulations. A Protoplast system has not been used in switchgrass (Panicum virgatum L.), even though it is a bioenergy crop that has received considerable attention. Here we report the first protocol to isolate large numbers of viable Protoplasts from both leaves and roots of two switchgrass genotypes. Furthermore, we demonstrate transient expression of PEG-mediated DNA uptake in the isolated Protoplasts by measuring the activity of β-glucuronidase (GUS) reporter gene driven by either the Cauliflower mosaic virus (CaMV) 35S promoter or the maize ubiquitin 1 promoter. Protoplast transformation with either the 35S or the ubiquitin promoter resulted in an increase in GUS activity compared to the untransformed controls; however, the extent of GUS activity was considerably higher for the ubiquitin promoter than for the 35S promoter. Collectively, our results indicate an efficient Protoplast Isolation and transient assay system that can be used to facilitate gene expression studies in switchgrass.
Sandra Cristina Raimundo - One of the best experts on this subject based on the ideXlab platform.
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Protoplast Isolation and manipulation in the unicellular model plant penium margaritaceum
Methods of Molecular Biology, 2020Co-Authors: David S. Domozych, Berke Tinaz, Anna Lietz, Eleanore Ritter, Sandra Cristina RaimundoAbstract:The unicellular freshwater green alga Penium margaritaceum has become a novel and valuable model organism for elucidating cell wall dynamics in plants. We describe a rapid and simple means for isolating Protoplasts using commercial enzymes in a mannitol-based buffer. Protoplasts can be cultured and cell wall recovery can be monitored in sequentially diluted mannitol-based medium. We also describe an optimized protocol to prepare highly pure, organelle-free nuclei fractions from Protoplasts using sucrose gradients. This technology provides a new and effective tool in Penium biology that can be used for analysis of cell wall polymer deposition, organelle Isolation and characterization, and molecular research including genetic transformation and somatic hybridization.
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Isolation and manipulation of Protoplasts from the unicellular green alga Penium margaritaceum
Plant Methods, 2018Co-Authors: Sandra Cristina Raimundo, Berke Tinaz, Iben Sørensen, Jocelyn K C Rose, Eleanore Ritter, David S. DomozychAbstract:Background The unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast Isolation and manipulation expedites a new range of experimental possibilities for Penium -based studies. These include enhanced means of Isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition. Results We present a methodology for enzyme-based digestion of the Penium cell wall and the Isolation of Protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from Protoplasts, which can be used for the Isolation of nuclear DNA. Conclusion Protoplast Isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium . The protocol described here offers a rapid and effective mechanism for fast and effective production of Protoplasts. Subsequently, the Protoplasts may be used for microscopy-based studies of specific subcellular components and the Isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.
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Isolation and manipulation of Protoplasts from the unicellular green alga penium margaritaceum
Plant Methods, 2018Co-Authors: Sandra Cristina Raimundo, Berke Tinaz, Iben Sørensen, Jocelyn K C Rose, Eleanore Ritter, David S. DomozychAbstract:The unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast Isolation and manipulation expedites a new range of experimental possibilities for Penium-based studies. These include enhanced means of Isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition. We present a methodology for enzyme-based digestion of the Penium cell wall and the Isolation of Protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from Protoplasts, which can be used for the Isolation of nuclear DNA. Protoplast Isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium. The protocol described here offers a rapid and effective mechanism for fast and effective production of Protoplasts. Subsequently, the Protoplasts may be used for microscopy-based studies of specific subcellular components and the Isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.
Berke Tinaz - One of the best experts on this subject based on the ideXlab platform.
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Protoplast Isolation and manipulation in the unicellular model plant penium margaritaceum
Methods of Molecular Biology, 2020Co-Authors: David S. Domozych, Berke Tinaz, Anna Lietz, Eleanore Ritter, Sandra Cristina RaimundoAbstract:The unicellular freshwater green alga Penium margaritaceum has become a novel and valuable model organism for elucidating cell wall dynamics in plants. We describe a rapid and simple means for isolating Protoplasts using commercial enzymes in a mannitol-based buffer. Protoplasts can be cultured and cell wall recovery can be monitored in sequentially diluted mannitol-based medium. We also describe an optimized protocol to prepare highly pure, organelle-free nuclei fractions from Protoplasts using sucrose gradients. This technology provides a new and effective tool in Penium biology that can be used for analysis of cell wall polymer deposition, organelle Isolation and characterization, and molecular research including genetic transformation and somatic hybridization.
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Isolation and manipulation of Protoplasts from the unicellular green alga Penium margaritaceum
Plant Methods, 2018Co-Authors: Sandra Cristina Raimundo, Berke Tinaz, Iben Sørensen, Jocelyn K C Rose, Eleanore Ritter, David S. DomozychAbstract:Background The unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast Isolation and manipulation expedites a new range of experimental possibilities for Penium -based studies. These include enhanced means of Isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition. Results We present a methodology for enzyme-based digestion of the Penium cell wall and the Isolation of Protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from Protoplasts, which can be used for the Isolation of nuclear DNA. Conclusion Protoplast Isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium . The protocol described here offers a rapid and effective mechanism for fast and effective production of Protoplasts. Subsequently, the Protoplasts may be used for microscopy-based studies of specific subcellular components and the Isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.
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Isolation and manipulation of Protoplasts from the unicellular green alga penium margaritaceum
Plant Methods, 2018Co-Authors: Sandra Cristina Raimundo, Berke Tinaz, Iben Sørensen, Jocelyn K C Rose, Eleanore Ritter, David S. DomozychAbstract:The unicellular charophycean green alga Penium margaritaceum has emerged as an appealing experimental organism in plant cell wall and cell biology research. Innovative practical approaches in the manipulation and maintenance of this unicellular model alga are needed in order to probe the complexities of its subcellular and molecular machinery. Protoplast Isolation and manipulation expedites a new range of experimental possibilities for Penium-based studies. These include enhanced means of Isolation of subcellular components and macromolecules, application of intracellular probes for high resolution microscopy of live cells, transformation studies and analysis of the fundamental mechanisms of plant cell expansion and wall polymer deposition. We present a methodology for enzyme-based digestion of the Penium cell wall and the Isolation of Protoplasts. The subcellular events associated with this technology are presented using multiple microscopy-based techniques. We also provide protocols for applying an array of intracellular dyes that can be used as markers for specific organelles and membrane microdomains in live cells. Finally, we present a protocol for the purification of a nuclei-rich fraction from Protoplasts, which can be used for the Isolation of nuclear DNA. Protoplast Isolation, culturing and manipulation provide valuable means for molecular and cellular studies of Penium. The protocol described here offers a rapid and effective mechanism for fast and effective production of Protoplasts. Subsequently, the Protoplasts may be used for microscopy-based studies of specific subcellular components and the Isolation of organelles and nuclear DNA. These methods offer a new practical methodology for future studies of this model organism in cell and molecular biology.
