A case study demonstrating dynamic range and measurement linearity
Plant Flow Cytometry
Flow cytometers were originally designed to operate with suspensions of cells, prototypically blood cells, and most applications still involve samples of this type. Despite the fact that plant cells and their protoplasts are generally larger than mammalian blood cells, these cells can still be analyzed by flow cytometry. At most growth stages, higher plants are comprised of tissues and organs, complex three-dimensional assemblies of cells held together by cell walls. Consequently, prior to analysis, plants must be first processed to produce cellular suspensions that are compatible with flow cytometry. One approach is to dissolve the cell walls, using enzymes that hydrolyze their carbohydrate components, to release protoplasts, which then can be readily analyzed on the cytometer.
The second approach, detailed here with the CytoFLEX, involves analysis not of protoplasts, but of suspensions of nuclei released from plant tissues and organs via a simple chopping procedure (2). The procedure involves cutting open the cells within the tissues using a standard double-edged razor blade. The homogenate is clarified using a nylon mesh filter through which the released nuclei are passed. The nuclei are then stained with DNA-specific fluorochromes,
which allows flow cytometric determination of the DNA content of the individual nuclei. Interest in measurement of plant genome sizes derives from the observation that higher plants, when analyzed across species, display an extraordinarily large range of genome sizes: a current lower limit of around 0.13pg per 2C nucleus for Genlisea margaretae to an upper limit of 304.40pg for Paris japonica (3). The latter genome is roughly 50 times larger than the human genome, and the DNA from a single cell of Paris japonica, when stretched out, would extend approximately 91 meters.
Flow cytometry coupled with the chopping procedure provides a simple means of determining genome sizes that is generally applicable across species and the methodology has been widely adopted across the globe to address numerous problems in basic and applied biology, ecology, and agriculture. This includes applications in biotechnology, such as monitoring genetic stability following transformation and tissue culture, prioritizing species for whole genome sequencing, validating the completeness of whole genome assemblies, and providing appropriate planning for genetic engineering procedures such as DNA library production. Applications in agriculture that benefit from flow cytometry include production of haploid and dihaploid lines using anther/ovary culture, followed by colchicine doubling, production of seed-sterile triploid lines, production of plants having tetraploid and higher ploidy levels, associated with desired traits such as fruit size, quality control of euploidy status for commercial seed lots, identification of individuals having novel ploidy levels and displaying novel modes of reproduction (including apomixis), characterization of interspecific hybridization based on intermediate nuclear DNA contents, classification of ploidy distributions within germplasm collections, sex determination in dioecious plants, and identification of hybrids formed between wild species, and between wild and cultivated species.
Applications in plant anatomy, cell biology, physiology, and development that benefit from flow cytometry include the study of the regulation of the cell division cycle and of endoreduplication in plant development, and the effects of abiotic and biotic stress on these processes. Flow cytometry is also useful for detection of mixoploidy and chimerism.
Analysis of gene expression as a function of cell type, using flow sorted
nuclei, is increasingly recognized as an important field of research.
Objectives
• Learn about the background of plant genome analysis via flow cytometry.
• Find out how to prepare plant samples for analysis of nuclear DNA content.
• Obtain step-by-step instructions for setting up the CytoFLEX for multi-parametric DNA content analysis.
