Chapter 1: Flow Cytometric Analysis of Transgene Expression in Higher Plants: Green-Fluorescent Protein

This chapter discusses the flow cytometric analysis of transgene expression in higher plants. The first consideration in flow cytometric analysis is the verification of proper alignment and instrument performance. This is usually done by checking the data obtained from running calibration beads or other standard cells. Once instrument performance has been verified, nontransfected protoplasts should be analyzed and photomultiplier voltages adjusted to obtain histograms with a clearly identifiable protoplast population. The growth and development of higher plants is governed to a large degree by the regulated expression of genes. The availability of robust techniques for the analysis of cell- and tissue-specific gene expression is therefore critical for progress in this area. One approach has been to develop transgenic markers, employing the coding regions of heterologous protein whose expression is placed under the control of various plant-derived regulatory DNA sequences. Biochemical and histological examination of patterns of expression of these markers can then provide information about the way in which gene expression patterns are regulated. Most heterologous protein markers are enzymes, because the amplification step inherent to biochemical assays provides great sensitivity.