Flow cytometric characterization and sorting of cultured Brassica napus microspores

Abstract

A flow cytometric procedure was used to characterize and sort embryogenic Brassica napus microspore cultures. Embryogenic microspores continued to increase in size throughout the culture period and fluoresced when stained with fluoresecein diacetate (FDA). However, most of the cells in culture (greater than 95%) lost their viability over the culture period and the correlation between the percentage viable cells in a culture and the productivity of the culture was not significant until day 3. By sorting large fluorescent cells on day 1 and day 3 of the culture period populations of cells that were 7-18-fold more embryogenic than sorted mixed cells were obtained. The flow cytometric procedure would be useful for rapidly assessing the effects of a large variety of culture and media conditions on embryogenesis is rapeseed microspore cultures. Furthermore, the populations of potentially embryogenic microspores isolated by flow cytometry might be useful for studies of the early cellular and molecular changes that occur during androgenesis in rapeseed.