Flow cytometric analysis of Brassica junceacell and pollen cultures treated with destruxin B, a toxin produced by Alternaria brassicae

Abstract

A rapid and reliable flow cytometric method of determining plant cell and pollen grain viability upon treatment with Alternaria brassicaepathotoxin (destruxin B) has been standardized on the basis of light scattering properties of cells of Brassica junceacv. Kranti stained with fluorescein diacetate. Data on relative fluorescence of cells treated with 60 and 120μg destruxin Bml -1were collected. The frequency distribution of fluorescence and histogram statistics showed a gradual loss of fluorescence in destruxin B-treated cells. The cell viability was 81% in untreated cells and 39% in 120μg destruxin B ml -1treated cells after 96h of culture in the Murashige and Skoog medium. These observations were similar to those obtained by manual counting with a light microscope using Evans blue. Optical sectioning with a confocal laser scanning microscope revealed plasmolysis of destruxin B-treated cells. Viability of pollen grains of B.junceacv. Kranti treated with destruxin B was also determined on the basis of their relative fluorescence.