Abstract
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.
Keywords: Flow cytometry, Seed development, Endosperm, Flow cytometric seed screen, Apomixis
Materials and Reagents
Equipment
Procedure
Representative data
Figure 4. Sexual offspring (n + n) of diploid Boechera stricta. This histogram shows the fluorescence intensity on the x-axis and the number of counts for each intensity on the y-axis. The peaks are manually annotated and labeled according to ploidy inferred from the internal or external standard. Green represents the nuclei of the embryo. The 4x and 8x peaks are from endoreduplicated embryonic nuclei (autopolyploidization). Brown represents the endosperm nuclei. The ratio of the 3x:2x peaks is 275.99/187.4 = 1.47, indicating sexual development. The ratio of the 6x:4x peaks is 1.48. Using the autopolyploid peaks for ratio calculations provides an internal control. Figure 5. Apomictic offspring (2n+0) of triploid Boechera gunnisoniana. Green represents the nuclei of the embryo. The 3x, 6x, and 12x peaks represent autopolyploidization. As apomictic Boechera spp. are typically pseudogamous (Rushworth et al., 2011), we expect a fertilized endosperm (Table 1). In this case, the endosperm is 9x (9x:3x ratio is 2.86), indicating fertilization by an unreduced sperm cell. Figure 6. BIII hybrid offspring (2n+2n) of triploid Boechera gunnisoniana. This histogram is almost identical to that shown in Figure 6. However, the red peak (3x) is much smaller than the 6x peak, indicating that this is contamination from maternal sporophytic tissues, due to scraping the seeds from the silique. As B. gunnisoniana produces both unreduced female and male gametes, the 2n+2n embryo peak (6x) will be double the size of that from maternal nuclei (3x). These data were published by Schmidt and colleagues (2014).
Notes
Recipes
Acknowledgments
This protocol is based on previously published work (Matzk et al., 2000; Schmidt et al., 2014). Our research in this area was supported by the University of Zürich, the Marie Curie project IDEAGENA, a Syngenta Ph.D. Project of the Zurich-Basel Plant Science Center, and a grant from the “Staatssekretariat für Bildung und Forschung” in the framework of the COST Action FA0903. We thank Manuel Waller for help with flow cytometry, and Anja Herrmann and Margarida Sofia Nobre for careful reading of this protocol.
References
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