3D Imaging, Segmentation, and Cell Annotation of the Ovule During Megaspore Mother Cell Differentiation in Paspalum spp.

Reproductive development in apomictic plants diverges from the sexual pathway at different key steps. The early steps take place in the ovule, the female organ hosting female sporogenesis and gametogenesis. Cell identities are notably more plastic in the ovule of facultative aposporous plants, where somatic cells can shift to germinal fate. This plasticity likely starts during the early morphogenesis of the ovule, concomitant with gradual differentiation of the sexual megaspore mother cell (MMC). In sexual species, 3D morphogenetic analyses have shown that ovule shape conditions MMC plasticity. However, in aposporous grasses, the morphogenetic events shaping ovule primordia are currently undescribed in 3D and at the cellular level, largely due to the inaccessibility of this organ. To fill this gap, we propose here a comprehensive workflow from ovule sampling to the extraction of 3D cellular quantitative parameters, established for the tropical apomictic grass Paspalum rufum. First, this protocol describes 3D imaging of whole-mount ovules at successive developmental stages, covering MMC differentiation, using ClearSee clearing procedure and double cell walls/nuclei staining. Second, it provides a detailed image analysis workflow in the open-source platform, MorphoGraphX. The workflow enables semiautomatic 3D cell segmentation, cell location, and annotation according to tissue layers or adjacency networks, leading to the final extraction of cellular parameters that describe geometry and topology dynamics along with ovule primordia development. This protocol applies to various species of the Paspalum genus and is potentially useful for 3D studies of large, curved, and hidden organs in multiple plant species.