Calcium and cell wall dynamics during microspore embryogenesis and double haploid production in rapeseed and eggplant

Calcium is widely known to have a role as a signaling molecule in many different processes, including stress response and activation of the embryogenic program. However, there are no direct clues about the role of calcium in microspore embryogenesis, an experimental process that combines a developmental switch towards embryogenesis and the simultaneous application of different stressing factors. In this work, we used FluoForte, a calcium-specific fluorescent vital dye, to track, by means of confocal microscopy, the changes in levels and subcellular distribution of calcium in living microspores and pollen grains during in vivo development, as well as during the first stages of in vitroinduced microspore embryogenesis. During in vivo development, a clear peak of cytosolic Ca 2+ was observed in vacuolate microspores and young pollen grains, the stages more suitable for embryogenesis induction. Just after in vitro induction, Ca 2+ levels increased specifically in embryogenic microspores at levels dramatically higher than during in vivo development. The increase was observed in the cytosol, but predominantly in the vacuoles. Non-embryogenic forms such as callus-like and pollen-like structures presented remarkably different calcium patterns. After the heat shock-based inductive treatment, Ca 2+ levels progressively decreased in all cases. Together, our results reveal a unique calcium dynamics in cells reprogrammed towards embryogenesis, establishing a link between changes in Ca 2+ level and subcellular distribution, and microspore embryogenesis.