Flow cytometry in plant breeding

Abstract

Since the first report on the flow cytometric study of plant material 35 years ago, analyzing the nuclear DNA content of field bean, an ever increasing number of applications of FCM has been developed and applied in plant science and industry, but a similar length of time elapsed before the appearance of the first complete volume devoted to FCM of plant cells. Most published information on the uses of FCM addresses various aspects of animal (including human) cell biology, thus failing to provide a pertinent substitute. FCM represents an ideal means for the analysis of both cells and subcellular particles, with a potentially large number of parameters analyzed both rapidly, simultaneously, and quantitatively, thereby furnishing statistically exploitable data and allowing for an accurate and facilitated detection of subpopulations. It is, indeed, the summation of these facts that has established FCM as an important, and sometimes essential, tool for the understanding of fundamental mechanisms and processes underlying plant growth, development, and function. In this review, special attention is paid to FCM as applied to plant cells in the context of plant breeding, and some new and less wellknown uses of it for plants will be discussed. ' 2008 International Society for Advancement of Cytometry

An efficient high‐throughput flow cytometric method for estimating DNA ploidy level in plants

“…FCM of plant cells is made difficult by structural irregularities (e.g., interlinked cells of irregular shape and rigid cell walls) typical of plant tissues. Furthermore, plant cells are frequently larger than the orifices in the flow chamber (50-100 lm diameter) of most flow cytometers (11)(12)(13). These problems associated with large and irregular cells may be overcome by isolating the nuclei that are smaller and more regularly shaped (13).…”

“…Furthermore, plant cells are frequently larger than the orifices in the flow chamber (50-100 lm diameter) of most flow cytometers (11)(12)(13). These problems associated with large and irregular cells may be overcome by isolating the nuclei that are smaller and more regularly shaped (13). In early protocols, nuclear suspensions were prepared from plant single-cell suspensions (protoplasts) using enzymes to digest cell wall materials.…”

Short Communication. A robust method for chromosome quantification and ploidy determination in woody species

“…Flow cytometry is commonly used to quantity nuclear DNA because of ease of sample preparation and high throughput (Galbraith et al 1983;Beck et al 2005;Loureiro et al 2007;Ochatt 2008). Living, dried or frozen plant material can be used for flow-cytometry analysis (Hopping 1993;Suda et al 2006;Roberts 2007;Ochatt 2008); however, disadvantages include contradictory results when analysing the same material in different laboratories, owing to variation in reference standards, sample preparation and staining protocols (Greilhuber and Obermayer 1998;Price et al 2000).…”

“…Living, dried or frozen plant material can be used for flow-cytometry analysis (Hopping 1993;Suda et al 2006;Roberts 2007;Ochatt 2008); however, disadvantages include contradictory results when analysing the same material in different laboratories, owing to variation in reference standards, sample preparation and staining protocols (Greilhuber and Obermayer 1998;Price et al 2000). Morgan and Westoby (2005) found that flow cytometry did not produce reliable results with taxa such as Myrtaceae and Rutaceae which contain high levels of secondary compounds, including phenolics, tannins and oils.…”

“…Chromosome counting in actively growing root-tip cells is the unambiguous method for ploidy assessment (De Lange et al 2004;Bachir and Abdellah 2006;Ochatt 2008) and mostly involves root-tip squashes and commonly used light-microscopy stains such as aceto-carmine. However, this technique can fail in species with many small chromosomes, such as Acacia mearnsii (Beck et al 2003a), and obtaining actively growing root tips from field-grown trees is difficult.…”