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This volume explores a collection of experimental techniques used to investigate different aspects of pollen development and function, including its role in reaching the ovule and delivering the two sperm cells. The techniques discussed range from basic methodology to cultivate pollen in vitro to the sophisticated experiments involving micromanipulation, Lab-on-Chip technology, or high-end imaging. The chapters in this book cover topics such as pollen grain counting using a cell counter; restricted pollination for tracing individual pollen tubes in a pistil; obtaining mutant pollen for phenotypic analysis and pollen tube dual-staining; analyzing intracellular gradients in pollen tubes; and measuring exocytosis rate in Arabidopsis pollen tube using corrected fluorescence recovery after photoconversion (cFRAPc) technique. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessarymaterials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Cutting-edge and thorough, Pollen and Pollen Tube Biology: Methods and Protocols is a valuable resource for novice and expert scientists interested in learning more about the field of plant reproduction.
List of contents
Pollen Grain Counting Using a Cell Counter.- A Toolkit for teasing Apart the Early Stages of Pollen-Stigma Interactions in Arabidopsis thaliana.- Decapitation Crosses to Test Pollen Fertility Mutations for Defects in Stigma-Style Penetration.- Isolation of the Pistil-Stimulated Pollen Tube Secretome.- Restricted Pollination for Tracing Individual Pollen Tubes in a Pistil.- Semi-In Vivo Assay for Pollen Tube Attraction.- Isolation and Cloning of Suppressor Mutants with Improved Pollen Fertility.- Workflow to Characterize Mutants with Reproductive Defects.- Internally-Controlled Methods to Quantify Pollen Tube Growth and Penetration Defects in Arabidopsis thaliana.- Arabidopsis thaliana Pollen Tube Culture for Multi-Omics Studies.- Large Scale Analysis of Pollen Viability and Oxidative Level using H2DCFDA-Staining Coupled with Flow Cytometry.- Obtaining Mutant Pollen for Phenotypic Analysis and Pollen Tube Dual-Staining.- Galvanotropic Chamber for Controlled Reorientation of Pollen Tube Growth and Simultaneous Confocal Imaging of Intracellular Dynamics.- Analyzing Intracellular Gradients in Pollen Tubes.- Silicone Chambers for Pollen Tube Imaging in Microstructures In Vitro Environments.- Analyzing the Impact of Protein Overexpression on Ca2+ Dynamics and Development in Tobacco Pollen Tubes.- Imaging and Analysis of the Content of Callose, Pectin, and Cellulose in the Cell Wall of Arabidopsis Pollen Tubes Grown In Vitro.- Characterization of Growth Behavior and the Resulting Forces Applied by Pollen Tubes in a 3D Matrix.- Flow Chamber Assay to Image the Response of FRET-Based Nanosensors in Pollen Tubes to Changes in Medium Composition.- Quantification of Mechanical Forces and Physiological Processes Involved in Pollen Tube Growth Using Microfluidics and Microrobotics.- Measuring Exocytosis Rate in Arabidopsis Pollen Tube Using Corrected Fluorescence Recovery afterPhotoconversion (cFRAPc) Technique.- Testing Pollen Tube Proteins for In Vivo Binding to Phosphatidic Acid by n0Butanol Treatment and Confocal Microscopy.
