Overexpression of RFP-tagged proteins in growing tobacco pollen tubes together with the genetically encoded Ca2+ sensor YC3.6 allows to analyze localization and dynamics of the protein of interest, as well as the impact of its overexpression on Ca2+ dynamics and pollen tube growth. Here, we describe a step-by-step instruction for transient transformation of N. tabacum pollen and subsequent in vitro germination and Ca2+ imaging.Live cell imaging at high resolution of pollen tubes growing in vitro requires an experimental setup that maintains the elongated cells in a single optical plane and allows for controlled exchange of growth medium. As a low-cost alternative to lithography-based microfluidics, we developed a silicone-based spacer system that allows introducing spatial features and flexible design. These growth chambers can be cleaned and reused repeatedly.Conspicuous intracellular gradients manifest and/or drive intracellular polarity in pollen tubes. However, quantifying these gradients raises multiple technical challenges. Here we present a sensible computational protocol to analyze gradients in growing pollen tubes and to filter nonrepresentative time points. As an example, we use imaging data from pollen tubes expressing a genetically encoded ratiometric Ca2+ probe, Yellow CaMeleon 3.6, from which a kymograph is extracted. The tip of the pollen tube is detected with CHUKNORRIS, our previously published methodology, allowing the reconstruction of the intracellular gradient through time. Statistically confounding time points, such as growth arrest where gradients are highly oscillatory, are filtered out and a mean spatial profile is estimated with a local polynomial regression method. Finally, we estimate the gradient slope by the linear portion of the decay in mean fluorescence, offering a quantitative method to detect phenotypes of gradient steepness, location, intensity, and variability. The data manipulation protocol proposed can be achieved in a simple and efficient manner using the statistical programming language R, opening paths to perform high-throughput spatiotemporal phenotyping of intracellular gradients in apically growing cells.Successful fertilization and seed set require the pollen tube to grow through several tissues, to change its growth orientation by responding to directional cues, and to ultimately reach the embryo sac and deliver the paternal genetic material. buy LUNA18 The ability to respond to external directional cues is, therefore, a pivotal feature of pollen tube behavior. In order to study the regulatory mechanisms controlling and mediating pollen tube tropic growth, a robust and reproducible method for the induction of growth reorientation in vitro is required. Here we describe a galvanotropic chamber designed to expose growing pollen tubes to precisely calibrated directional cues triggering reorientation while simultaneously tracking subcellular processes using live cell imaging and confocal laser scanning microscopy.Mutant phenotype observation is the most useful and important method to study which biological process a gene-of-interest is involved in. In flowering plants, excessive pollen grains land and germinate on the stigma, then pollen tubes grow through the transmitting tract to reach the ovules, eventually enter the micropyle to complete double fertilization. First, for mutants whose homozygotes could not be obtained due to pollen tube defects, it is difficult to observe the defect phenotype since the pollen grains of different genotypes are mixed together. Here, we provide a detailed protocol to pick out mutant pollen grains from the heterozygous mutant plants in Arabidopsis thaliana. By using this method, we could obtain sufficient mutant pollen grains for phenotypic analysis. Second, it is difficult to compare the pollen/pollen tube behavior of two different genotypes/species in vivo in a same pistil. Here, we develop a new dual staining method which combines GUS staining with aniline blue staining. By using this method, we can analyze the competence of the two different pollen tubes in the same pistil.Determining pollen viability and other physiological parameters is of critical importance for evaluating the reproductive capacity of plants, both for fundamental and applied sciences. Flow cytometry is a powerful high-performance high-throughput tool for analyzing large populations of cells that has been in restricted use in plant cell research and in pollen-related studies, it has been minimized mostly for determination of DNA content. Recently, we developed a flow cytometry-based approach for robust and rapid evaluation of pollen viability that utilizes the reactive oxygen species (ROS) fluorescent reporter dye H2DCFDA (Luria et al., Plant J 98(5)942-952, 2019). This new approach revealed that pollen from Arabidopsis thaliana and Solanum lycopersicum naturally distribute into two subpopulations with different ROS levels. This method can be employed for a myriad of pollen-related studies, primarily in response to stimuli such as biotic or abiotic stress. In this chapter, we describe the protocol for H2DCFDA staining coupled with flow cytometry analysis providing specific guidelines. These guidelines are broadly applicable to many other types of cellular reporters to further develop this novel approach in the field of pollen biology.Pollen tubes have been key models to study plant cell wall elongation. Arabidopsis thaliana, although small, is a nice model, easy to grow and with a large set of studies to simplify result integration and interpretation. Pollen tubes may be used for gene expression essays, but also for biochemical characterization of the cell wall composition. However, pollen tube culture methods though seemingly straightforward have often a multitude of small technical details crucial for success, quickly deterring the more inexperienced and setting back experiments for months at the time. Here we propose a detailed method to set up easily a pollen tube culture routine in any lab, with a minimal set of equipment, to isolate and process pollen tubes for gene expression and/or cell wall biochemistry studies.