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Live-cell Imaging by Super-resolution Confocal Live Imaging Microscopy (SCLIM): Simultaneous Three-color and Four-dimensional Live Cell Imaging with High Space and Time Resolution

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Efficient Agrobacterium-mediated Transformation of the Elite–Indica Rice Variety Komboka

Authors: Van T. Luu, Melissa Stiebner, Paula Emmerich Maldonado, Sandra Valdés, Didier Marín, Gerardo Delgado, Virginia Laluz, Lin-Bo Wu, Paul Chavarriaga, Joe Tohme, Inez H. Slamet-Loedin and Wolf B. Frommer, date: 09/05/2020, view: 1291, Q&A: 0

[Abstract] Genetic transformation is crucial for both investigating gene functions and for engineering of crops to introduce new traits. Rice (Oryza sativa L.) is an important model in plant research, since it is the staple food for more than half of the world’s population. As a result, numerous transformation methods have been developed for both ...

Visualization and Quantification of Cell-to-cell Movement of Proteins in Nicotiana benthamiana

Authors: Mila C Blekemolen, Mariliis Tark-Dame and Frank L.W Takken, date: 12/20/2018, view: 3276, Q&A: 0

[Abstract] Cell-to-cell movement of proteins through plasmodesmata is a widely-established mechanism for intercellular signaling in plants. Current techniques to study intercellular protein translocation rely on single-cell transformation using particle bombardment or transgenic lines expressing photo-inducible fluorophores. The method presented here allows ...

Generation and Selection of Transgenic Olive Plants

Authors: Elena Palomo-Ríos, Sergio Cerezo, Jose Ángel Mercado and Fernando Pliego-Alfaro, date: 11/20/2017, view: 5311, Q&A: 0

[Abstract] Olive (Olea europaea L.) is one of the most important oil crops in the Mediterranean basin. Biotechnological improvement of this species is hampered by the recalcitrant nature of olive tissue to regenerate in vitro. In previous investigations, our group has developed a reliable Agrobacterium-mediated transformation ...

Ensifer-mediated Arabidopsis thaliana Root Transformation (E-ART): A Protocol to Analyse the Factors that Support Ensifer-mediated Transformation (EMT) of Plant Cells

Authors: Dheeraj Singh Rathore, Fiona M. Doohan and Ewen Mullins, date: 10/05/2017, view: 6640, Q&A: 0

[Abstract] Ensifer adhaerens OV14, a soil borne alpha-proteobacteria of the Rhizobiaceae family, fortifies the novel plant transformation technology platform termed ‘Ensifer-mediated transformation’ (EMT). EMT can stably transform both monocot and dicot species, and the host range of EMT is continuously expanding across a diverse range of ...

DNA-free Genome Editing of Chlamydomonas reinhardtii Using CRISPR and Subsequent Mutant Analysis

Authors: Jihyeon Yu, Kwangryul Baek, EonSeon Jin and Sangsu Bae, date: 06/05/2017, view: 9343, Q&A: 0

[Abstract] We successfully introduced targeted knock-out of gene of interest in Chlamydomonas reinhardtii by using DNA-free CRISPR. In this protocol, the detailed procedures of an entire workflow cover from the initial target selection of CRISPR to the mutant analysis using next generation sequencing (NGS) technology. Furthermore, we introduce a ...

Use of Geminivirus for Delivery of CRISPR/Cas9 Components to Tobacco by Agro-infiltration

Authors: Kangquan Yin, Ting Han and Yule Liu, date: 04/05/2017, view: 9574, Q&A: 0

[Abstract] CRISPR/Cas9 system is a recently developed genome editing tool, and its power has been demonstrated in many organisms, including some plant species (Wang et al., 2016). In eukaryotes, the Cas9/gRNA complexes target genome sites specifically and cleave them to produce double-strand breaks (DSBs), which can be repaired by non-homologous end ...

Multiplexed GuideRNA-expression to Efficiently Mutagenize Multiple Loci in Arabidopsis by CRISPR-Cas9

Authors: Julia Schumacher, Kerstin Kaufmann and Wenhao Yan, date: 03/05/2017, view: 7948, Q&A: 0

[Abstract] Since the discovery of the CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein (Cas) as an efficient tool for genome editing in plants (Li et al., 2013; Shan et al., 2013; Nekrasov et al., 2013), a large variety of applications, such as gene knock-out, knock-in or transcriptional ...

Production of Guide RNAs in vitro and in vivo for CRISPR Using Ribozymes and RNA Polymerase II Promoters

Authors: Tao Zhang, Yangbin Gao, Rongchen Wang and Yunde Zhao, date: 02/20/2017, view: 11448, Q&A: 0

[Abstract] CRISPR/Cas9-mediated genome editing relies on a guide RNA (gRNA) molecule to generate sequence-specific DNA cleavage, which is a prerequisite for gene editing. Here we establish a method that enables production of gRNAs from any promoters, in any organisms, and in vitro (Gao and Zhao, 2014). This method also makes it feasible to conduct ...

Agrobacterium rhizogenes-Based Transformation of Soybean Roots to Form Composite Plants

Authors: Benjamin F. Matthews and Reham M. Youssef, date: 01/20/2016, view: 7720, Q&A: 0

[Abstract] Transgenic soybean roots of composite plants are a powerful tool to rapidly test the function of genes and activity of gene promoters. No tissue culture is needed, thus avoiding loss of valuable material due to contamination. This is a simple technique that requires less training and care than tissue culture techniques. Furthermore, it takes less ...

5’ Rapid Amplification of cDNA Ends (5’ RACE) of Agrobacterial T-DNA Genes within Transformed Plant Sample

Author: Yi Zhang, date: 09/20/2015, view: 9107, Q&A: 0

[Abstract] The T-DNA (transferred-DNA) region of virulent Agrobacterium tumefaciens (A. tumefaciens) strain is transferred and integrated into the plant genome, and thereby the T-DNA genes are expressed in transformed plant cells. This protocol was used to analyze the transcription start sites (TSSs) of agrobacterial T-DNA genes within ...

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