Flow cytometric characterization of embryogenic and gametophytic development in Brassica napus microspore cultures

Abstract

Microspore cultures are ideal systems for studying plant embryogenesis because the resulting embryos are very similar to zygotic embryos, all the stages of development are readily accessible and the process can be induced by a simple heat treatment. However, not all microspores are embryogenic and the mixture of cells that develops in the cultures complicates the use of this system. Brassica napus microspore cultures cultured at 30°C (induced) and at 25°C (non-induced) were compared by flow cytometry to obtain structure and function information for several types of cells in the culture. Clear differences in light scatter and fluorescence were found between induced and noninduced cultures that are related to early stages of embryo development. Viable, round cells that were unique to induced cultures were sorted into culture media and developed into embryos confirming that they were embryogenic. The present study provided flow cytometric identifiers for embryogenic and gametophytic cells, demonstrated how flow sorting can be used to isolate specific cell types and defined bench-marks for assessing the embryogenic potential of microspore cultures.