Tracking gene and protein expression during microspore embryogenesis by Confocal Laser Scanning Microscopy

Abstract

Confocal Laser Scanning Microscopy (CLSM) technology and bioimaging are powerful tools for three-dimensional (3D) and colocalization molecular analysis of the microspore embryogenesis. Strategies with fluorescent-labelled probes for in situ hybridization and immunofluorescence have provided unique images of the spatial and temporal pattern of the expression of genes and proteins, and of the sub-cellular rearrangements that accompany the microspore embryogenesis. Various signalling and stress proteins were differentially expressed in reprogrammed microspores and young embryos, and specific endosperm and embryo genes were expressed at different stages, supporting the existence of an endosperm-like domain, in cereals. Specific features such as changes in cell wall components and pectin esterification, presence of callose in special walls, and different behaviour of Cajal nuclear bodies were found in embryogenic microspores and young embryos, constituting early embryogenic markers. The 3D analysis of the nuclear dynamics at early stages of microspore embryogenesis has proved that the nuclear fusion was the mechanism of the spontaneous diploidization.