Characterization of polarity development through 2- and 3-D imaging during the initial phase of microspore embryogenesis in Brassica napus L

Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32 °C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECM-like structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical–basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis.