Plant Science


    Protocols in Current Issue
    A Novel Method to Construct Binary CRISPR Vectors for Plant Transformation by Single Round of PCR Amplification
    Authors:  Kang Li, Yuhui Wang and Chuanying Fang, date: 04/05/2021, view: 411, Q&A: 0

    CRISPR/Cas9 is an established and flexible tool for genome editing. However, most methods used to generate expression clones for the CRISPR/Cas9 are time-consuming. Hence, we have developed a one-step protocol to introduce sgRNA expression cassette(s) directly into binary vectors (Liu et al., 2020). In this approach, we have optimized the

    Monitoring Real-time Temperature Dynamics of a Short RNA Hairpin Using Förster Resonance Energy Transfer and Circular Dichroism
    Authors:  Martin Balcerowicz, Marco Di Antonio and Betty Y. W. Chung, date: 03/20/2021, view: 1016, Q&A: 0

    RNA secondary structures are highly dynamic and subject to prompt changes in response to the environment. Temperature in particular has a strong impact on RNA structural conformation, and temperature-sensitive RNA hairpin structures have been exploited by multiple organisms to modify the rate of translation in response to temperature changes.

    Plant ARGONAUTE Protein Immunopurification for Pathogen Cross Kingdom Small RNA Analysis
    Authors:  Florian Dunker, Bernhard Lederer and Arne Weiberg, date: 02/05/2021, view: 683, Q&A: 0

    Over the last decade, it has been noticed that microbial pathogens and pests deliver small RNA (sRNA) effectors into their host plants to manipulate plant physiology and immunity for infection, known as cross kingdom RNA interference. In this process, fungal and oomycete parasite sRNAs hijack the plant ARGONAUTE (AGO)/RNA-induced silencing complex

    Cellulase and Macerozyme-PEG-mediated Transformation of Moss Protoplasts
    [Abstract] This protocol describes the generation of protoplasts from protonemal tissue of the moss Physcomitrium patens (syn. Physcomitrella patens), using Cellulase ONOZUKA R10 and Macerozyme R10, followed by polyethylene glycol (PEG) mediated transformation. The protonemal tissue grown in liquid suspension was harvested and treated with ...
    Efficient Transient Gene Knock-down in Tobacco Plants Using Carbon Nanocarriers
    Authors:  Gozde S. Demirer and Markita P. Landry, date: 01/05/2021, view: 1096, Q&A: 0
    [Abstract] Gene knock-down in plants is a useful approach to study genotype-phenotype relationships, render disease resistance to crops, and enable efficient biosynthesis of molecules in plants. Small interfering RNA (siRNA)-mediated gene silencing is one of the most common ways to achieve gene knock-down in plants. Traditionally, siRNA is delivered into ...
    Chromatin Immunoprecipitation (ChIP) to Assess Histone Marks in Auxin-treated Arabidopsis thaliana Inflorescence Tissue
    Authors:  André Kuhn and Lars Østergaard, date: 12/05/2020, view: 1116, Q&A: 0
    [Abstract] Chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR) or high-throughput sequencing (ChIP-seq) has become the gold standard for the identification of binding sites of DNA binding proteins and the localization of histone modification on a locus-specific or genome-wide scale, respectively. ChIP experiments can be divided into seven ...
    Dual sgRNA-based Targeted Deletion of Large Genomic Regions and Isolation of Heritable Cas9-free Mutants in Arabidopsis
    Authors:  Yu Jin and Sebastian Marquardt, date: 10/20/2020, view: 1721, Q&A: 0
    [Abstract] CRISPR/Cas9 system directed by a gene-specific single guide RNA (sgRNA) is an effective tool for genome editing such as deletions of few bases in coding genes. However, targeted deletion of larger regions generate loss-of-function alleles that offer a straightforward starting point for functional dissections of genomic loci. We present an ...
    Ratiometric Measurement of Protein Abundance after Transient Expression of a Transgene in Nicotiana benthamiana
    Authors:  Aashima Khosla and David C. Nelson, date: 09/05/2020, view: 2071, Q&A: 0
    [Abstract] Ratiometric reporters are tools to dynamically measure the relative abundance of a protein of interest. In these systems, a target protein fused to a fluorescent or bioluminescent reporter is expressed with fixed stoichiometry to a reference protein fused to a second reporter. Both fusion proteins are encoded on a single transcript but are ...
    RNA Stability Measurements Using RT-qPCR in Arabidopsis Seedlings
    Authors:  Tianran Jia and Brandon H. Le, date: 07/20/2020, view: 1353, Q&A: 0
    [Abstract] Steady-state mRNA levels are determined by both the rates of transcription and degradation. Regulation of mRNA stability and/or degradation are key factors that can significantly affect mRNA levels and its biological functions. mRNA stability can be measured indirectly after transcription inhibition. This protocol described a rapid and sensitive ...
    Methylation-sensitive Amplified Polymorphism as a Tool to Analyze Wild Potato Hybrids
    Authors:  Nicolás Cara, Carlos F. Marfil, María V. Bertoldi and Ricardo W. Masuelli, date: 07/05/2020, view: 1444, Q&A: 0
    [Abstract] Methylation-Sensitive Amplification Polymorphism (MSAP) is a versatile marker for analyzing DNA methylation patterns in non-model species. The implementation of this technique does not require a reference genome and makes it possible to determine the methylation status of hundreds of anonymous loci distributed throughout the genome. In addition, ...

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