Apoptosis-like cell death of Brassica napus leaf protoplasts

The cleavage of nuclear DNA into oligonucleosomal fragments that is the hallmark of apoptosis in animal cells occurs during the culture of Brassica napus leaf protoplasts. The changes in nuclei of cultured Brassica napus leaf protoplasts were studied by propidium iodide (PI) and 4', 6-diamino-2-phenylindole, dihydrochloride (DAPI) staining, transmission electron microscopy, flow cytometry analysis, and DNA laddering staining with ethidium bromide and Southern hybridization. Free 3'-OH termini of nuclear DNA fragments were labelled with DIG-dUTP, catalyzed by terminal deoxynucleotidyl transferase (TdT), and used as probes for Southern hybridization. This method (TUNEL on membrane) allowed visualization of DNA fragments with 3'-OH termini on a nylon membrane. These results suggest that loss of viability of protoplast with culture time is accompanied by apoptosis-like cell death. However, the forms or processes undergoing to apoptotic cell death in B. napus leaf protoplasts appears to be different in some details to those in animal cells.