Analysis of Gametophytic Apomixis Using Confocal Microscopy

Apomixis is an asexual reproductive mechanism that takes place deeply inside the female reproductive organs of the plant, in ovules and seeds. In gametophytic apomixis, an unreduced female gametophyte is produced by a modified meiosis of the megaspore mother cell (dipolspory) or from a somatic initial cell (apospory). The unreduced, nonrecombined egg cell develops subsequently into an embryo by parthenogenesis. The cyto-embryological study of apomixis is challenging because of the inaccessibility of these structures. Consequently, images of apomeiosis and parthenogenesis with high definition are limited to a few species. In this chapter, we show the application of a Feulgen staining protocol combined with confocal microscopy for the study of nonreductional megasporogenesis and autonomous embryo formation in diplosporous apomictic Taraxacum officinale and aposporous apomictic Pilosella piloselloides var. praealta. Using a rapid and technically simple method, performed on whole-mount ovaries, we have obtained high-resolution images of the female reproductive cells. Furthermore, we highlight the application of this protocol for the study of loss-of-diplospory and loss-of-parthenogenesis mutants in the same species.