Documenting the Sporangium Development of the Polypodiales Fern Pteris multifida.

Reconstructing the development of sporangia in seed-free vascular plants provides crucial information about key processes enabling the production of spores that are important in the life cycle of these plants. By applying fluorescence imaging in intact tissues using dyes and confocal microscopy, this study aimed to reconstruct the key steps during the development of sporangia. Special emphasis was taken on the cell wall structures of tapetum and spore mother cells that have been challenged by microscopical documentation in the past. After staining the cell wall and cytoplasm using calcofluor white and basic fuchsin, the sporangium development of Pteris multifida was observed using confocal microscopy. The clear cell lineages from the sporangial initial cell to stalk, epidermis, inner tapetum, outer tapetum, and sporogenous cells were revealed by confocal imaging. The sporangium development improved in this work will be useful for a general understanding of fern spore formation.

A dye combination for the staining of pollen coat and pollen wall.

The pollen coat, which forms on the pollen surface, consists of a lipid-protein matrix. It protects pollen from desiccation and is involved in adhesion, pollen-stigma recognition, and pollen hydration during interactions with the stigma. The classical methods used for pollen coat observation are scanning and transmission electron microscopy. In this work, we screened a collection of fluorescence dyes and identified two fluorescent brighteners FB-52 and FB-184. When they were used together with the exine-specific dye, Basic fuchsin, the pollen coat and the exine structures could be clearly visualized in the pollen of Brassica napus. This co-staining method was applied successfully in staining pollen from Fraxinus chinensis, Calystegia hederacea, and Petunia hybrida. Using this method, small pollen coat-containing cavities were detected in the outer pollen wall layer of Oryza sativa and Zea mays. We further showed these dyes are compatible with fluorescent protein markers. In the Arabidopsis thaliana transgenic line of GFP-tagged pollen coat protein GRP19, GRP19-GFP was observed to form particles at the periphery of pollen coat. This simple staining method is expected to be widely used for the studies of the palynology as well as the pollen-stigma interaction.

Development of the Middle Layer in the Anther of Arabidopsis.

The middle layer is an essential cell layer of the anther wall located between the endothecium and tapetum in Arabidopsis. Based on sectioning, the middle layer was found to be degraded at stage 7, which led to the separation of the tapetum from the anther wall. Here, we established techniques for live imaging of the anther. We created a marker line with fluorescent proteins expressed in all anther layers to study anther development. Several staining methods were used in the intact anthers to study anther cell morphology. We clarified the initiation, development, and degradation of the middle layer in Arabidopsis. This layer is initiated from both the inner and outer secondary parietal cells at stage 4, stopped cell division at stage 6, and finally degraded at stage 11. The neighboring cell layers, the epidermis, and endothecium continued cell division until stage 10, which led to a thin middle layer. The degradation of the tapetum cell wall at stage 7 lead to its isolation from the anther wall. This work presents fundamental information on the development of the middle layer, which facilitates the further investigation of anther development and plant fertility. These live imaging methods could be useful in future studies.