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Fluorescence Lifetime Imaging (FLIM) Measurements in Salinity Research

Plant response to salt stress involves changes in intracellular ion concentrations that can be estimated by ion-selective fluorescent dyes. Conventional confocal/fluorescent imaging does not always accurately reflect the process, since it is dependent not only on the ion concen ...

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Quantitative Cryo-Analytical Scanning Electron Microscopy (CEDX): An Important Technique Useful for Cell-Specific Localization of Salt

Advances in the techniques required for the X-ray microanalysis of cryo-fixed, naturally hydrated plant tissues in the cryo-scanning electron microscope have reached the stage that accurate, cell-specific localization and quantification of the nutrient and toxic elements can be ...

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Determination of Reactive Oxygen Species in Salt-Stressed Plant Tissues

Reactive oxygen species (ROS) participate in signaling events that regulate ion channel activity and gene expression. However, excess ROS exert adverse effects that stem from their interaction with macromolecules. Thus, the assessment of the effects of salinity on ROS changes are cen ...

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Quantification of the Antioxidant Activity in Salt-Stressed Tissues

Biochemical methods available for the measurement of antioxidant activity in salt-stressed tissues are reviewed, outlining the most important advantages and shortcomings of the methods. Here we consider commonly used methods for measuring total antioxidant capacity and phe ...

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Quantification of Abscisic Acid, Cytokinin, and Auxin Content in Salt-Stressed Plant Tissues

Plant hormones cytokinins, auxin (indole-3-acetic acid), and abscisic acid are central to regulation of plant growth and defence to abiotic stresses such as salinity. Quantification of the hormone levels and determination of their ratios can reveal different plant strategies to cope w ...

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Fluorescence-Activated Cell Sorting for Analysis of Cell Type-Specific Responses to Salinity Stress in Arabidopsis and Rice

Fluorescence-activated cell sorting (FACS) provides a rapid means of isolating large numbers of fluorescently tagged cells from a heterogeneous mixture of cells. Collections of transgenic plants with cell type-specific expression of fluorescent marker genes such as green fluo ...

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Dienes Staining and Light Microscopy for Phytoplasma Visualization

The chapter describes the main light microscopy techniques used for the diagnosis of phytoplasmas. Because the described techniques are generally only effective in cases where the concentration of the pathogen inside the host phloem tissue is high, they are useful as preliminary metho ...

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Molecular Identification of Phytoplasma Vector Species

The correct identification of the insect species involved in phytoplasma transmission is an essential condition for managing phytoplasma diseases and employing control strategies. The taxonomy of leafhoppers, planthoppers, and psyllids traditionally relies on morphol ...

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Insect Vector Transmission Assays

Phytoplasmas are transmitted in a persistent propagative manner by phloem-feeding vectors belonging to the order Hemiptera, suborder Homoptera. Following acquisition from the infected source plant, there is a latent period before the vector can transmit, so transmission assays ...

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Insect Maintenance and Transmission

Phytoplasmas are plant pathogens of huge economic importance due to responsibility for crop yield losses worldwide. Institutions around the world are trying to understand and control this yield loss at a time when food security is high on government agendas. In order to fully understand the m ...

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Capturing Insect Vectors of Phytoplasmas

Insect vectors of phytoplasmas are limited to leafhoppers, planthoppers, and psyllids. While populations can be monitored by a number of passive techniques in the field, the capture of live insects is necessary for manipulation and study. A number of physical methods for capturing these in ...

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Dodder Transmission of Phytoplasmas

Transmission of phytoplasmas from naturally infected plant host species using the parasitic plant Cuscuta spp. (dodder) to Catharanthus roseus (Madagascar periwinkle) is an effective way to maintain a wide range of phytoplasmas for further research. Here, we describe transmissi ...

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Micropropagation and Maintenance of Phytoplasmas in Tissue Culture

Maintenance of phytoplasma strains in tissue culture is achievable for all strains transmitted to periwinkle (Catharanthus roseus), and also for other naturally infected plant host species. Shoots of 1–3 cm length are grown in a solid medium containing Murashige and Skoog (MS) micro- and ma ...

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Mapping the Phytoplasma Chromosome

Physical and genetic mapping of the phytoplasma chromosome can be a useful tool in a genome sequencing project in order to assemble the in silico-predicted contigs robustly. Mapping consists of four distinct steps: preparation of phytoplasma chromosomes from infected plants, single- ...

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Pulsed-Field Gel Electrophoresis for Isolation of Full-Length Phytoplasma Chromosomes from Plants

Pulsed-field gel electrophoresis (PFGE) is a powerful technique for genomic studies of unculturable plant-pathogenic phytoplasmas, which enables separation of full-length phytoplasma chromosomes from contaminating host plant nucleic acids. The PFGE method described h ...

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Cesium Chloride-Bisbenzimide Gradients for Separation of Phytoplasma and Plant DNA

CsCl–bisbezimide gradient centrifugation is a method to separate phytoplasma from host plant DNA. Bisbenzimide forms a complex with A + T-rich DNA thereby lowering its relative density. During centrifugation the A + T-rich phytoplasma DNA is spatially separated from the less A + T-rich host p ...

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Phytoplasma Plasmid DNA Extraction

Phytoplasma plasmids have generally been detected from DNA extracted from plants and insects using methods designed for the purification of total phytoplasma DNA. Methods include extraction from tissues that are high in phytoplasma titre, such as the phloem of plants, with the use of CsCl– ...

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Preparation of Phytoplasma Membrane Recombinant Proteins

The study of phytoplasma membrane protein interactions with host cell proteins is crucial to understand the life cycle of these unculturable microorganisms within their hosts. A step-by-step protocol for the heterologous expression of phytoplasma membrane proteins in Escheri ...

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Techniques for the Maintenance and Propagation of Phytoplasmas in Glasshouse Collections of Catharanthus roseus

Phytoplasma collections are a vital resource for researchers and diagnosticians studying phytoplasma diseases. They provide material as a point of reference and a research tool to increase our understanding of phytoplasmas and the diseases they cause. This chapter describes the te ...

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Phytoplasma Proteomic Analysis

Proteome analysis is becoming a powerful tool in the functional characterization of organisms, and takes a broad, comprehensive, systematic approach to understanding biology. Following the sequencing of the phytoplasma genomes, the next step is to characterize the expressed pro ...

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