Determination of the Developmental Origin of Seeds Containing Endosperm Using Flow Cytometric Analysis

Seeds derived from a diploid, sexual plant typically contain a 2n embryo (n+n) and 3n endosperm, a ratio characteristic for most flowering plants. However, this ratio is altered in apomictic species, which reproduce asexually through seeds (Koltunow and Grossniklaus, 2003). Apomixis is usually a facultative trait and encompasses several developmental steps: (1) apomeiosis (avoidance of meiosis), (2) parthenogenesis (embryo development without fertilization), and (3) functional endosperm formation
(autonomous without fertilization or pseudogamous requiring fertilization). If all three steps occur, this process results in maternal offspring (2n+0), which is genetically identical to the mother plant (clonal). Moreover, sexual and apomictic pathways can occur in the same plant and sometimes they cross over, producing polyhaploid offspring (n+0; resulting from meiosis and parthenogenesis) or BIII hybrids (2n+n; resulting from apomeiosis and fertilization) (Rutishauser, 1947). The different types of
offspring can be determined in a flow cytometric seed screen (FCSS), in which the relative chromatin content of stained nuclei is determined by measuring their fluorescence intensity. This allows a comparison of the ploidy of the endosperm to the ploidy of the embryo and, thus, an inference of the pathway by which a seed was
formed (Matzk et al., 2000). This method is particularly useful to characterize the developmental origin of seeds in apomictic plants or reproductive mutants of sexual species.
Here, we present the protocol for an FCSS in Brassicaceae that has specifically been adapted to plants of the genus Boechera. However, in principle this protocol can be applied to any species producing seeds that contain endosperm