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Full Description
This detailed volume explores common and numerous specialized methods to study various aspects of plant germline development and targeted manipulation, including imaging and hybridization techniques to study cell-type specification, cell lineage, signaling and hormones, cell cycle, and the cytoskeleton. In addition, cell-type specific methods for targeted ablation or isolation are provided, protocols to apply "omics" technologies and to perform bioinformatics data analysis, as well as methods relevant for aspects of biotechnology or plant breeding. This includes protocols that are relevant for the targeted manipulation of pathways, for crop plant transformation, or for conditional induction of phenotypes. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Plant Germline Development: Methods and Protocols serves as a comprehensive guide not only to studying basic questions related to different aspects of plant reproductive development but also for state of the art methods, in addition to being a source of inspiration for new approaches and research questions in many laboratories.
Contents
An Introduction to Male Germline Development.- Apomixis: Engineering the Ability to Harness Hybrid Vigor in Crop Plants.- 3D Imaging of Whole-Mount Ovules at Cellular Resolution to Study Female Germline Development in Rice.- Live-Cell Imaging of F-Actin Dynamics During Fertilization in Arabidopsis thaliana.- Development and Observation of Mature Megagametophyte Cell-Specific Fluorescent Markers.- Analysis of Fluorescent Reporter Activity in the Male Germline During Pollen Development by Confocal Microscopy.- In Vivo Ploidy Determination of Arabidopsis thaliana Male and Female Gametophytes.- Staining and Clearing of Arabidopsis Reproductive Tissue for Imaging of Fluorescent Proteins.- Live Cell Imaging of Auxin and Cytokinin Signaling in Maize Female Gametophytes.- Imaging Ca2+ Dynamics in Wild-Type and NADPH Oxidase-Deficient Mutant Pollen Tubes with Yellow Cameleon and Confocal Laser Scanning Microscopy.- Immunolocalization of AGPs and Pectins in Quercus suber Gametophytic Structures.- Optimization of Cell Spreading and Image Quality for the Study of Chromosomes in Plant Tissues.- Whole Mount RNA-FISH on Ovules and Developing Seeds.- Analysis of Peroxisome Biogenesis in Pollen by Confocal Microscopy and Transmission Electron Microscopy.- Transmission Electron Microscopy (TEM) to Study Histology of Pollen and Pollen Tubes.- Isolation of Arabidopsis Pollen, Sperm Cells, and Vegetative Nuclei by Fluorescence-Activated Cell Sorting (FACS).- Isolation of Rice Sperm Cells for Transcriptional Profiling.- Manual Isolation of Living Cells from the Arabidopsis thaliana Female Gametophyte by Micromanipulation.- Isolation and Detection of Small RNAs from Pollen.- Analysis of Proteins Enriched in Rice Gamete.- Phosphoprotein Enrichment from Tobacco Mature Pollen Crude Protein Extract.- Identification of Cis-Regulatory Modules that Function in the Male Germline of Flowering Plants.- RNA-Seq Data Analysis: From Raw Data Quality Control to Differential Expression Analysis.- RNA-Seq Data Analysis Protocol: Combining In-House and Publicly Available Data.- Impedance Flow Cytometry as a Tool to Analyze Microspore and Pollen Quality.- Agrobacterium-Mediated Sorghum Transformation.- Use of the Cas9 Orthologues from Streptococcus thermophilus and Staphylococcus aureus for Non-Homologous End-Joining Mediated Site-Specific Mutagenesis in Arabidopsis thaliana.- Transgenic Reproductive Cell Ablation.- In Vivo Reporters for Protein Half-Life.- Conditional Modulation of Biological Processes by Low-Temperature Degrons.
