Apoptotic cell death induces temperature-sensitive lethality in hybrid seedlings and calli derived from the cross of Nicotiana suaveolens x N-tabacum

Hybrid lethality expressed in the interspecific hybrid of Nicotiana suaveol ens Lehm. x N. tabacum L. cv. Hicks-2 is one of the mechanisms for reproduc tive isolation and it is temperature-sensitive. Apoptotic changes were dete cted in the cells of hybrid seedlings and calli expressing lethality at 28 degreesC but not under high-temperature conditions (36 degreesC), when the lethality is suppressed. Condensation of chromatin, fragmentation of nuclei and cytoplasmic reduction are the cytological changes associated with apop tosis leading to hybrid lethality. Fragmentation of nuclei was correlated w ith the lethal symptoms in both hybrid seedlings and calli, as confirmed by fluorimetry of the nuclear DNA using laser scanning cytometry. Agarose gel analysis of DNA extracted from hybrid seedlings and calli showing lethal s ymptoms revealed a specific ladder pattern suggesting nucleosomal fragmenta tion which is one of the biochemical changes of apoptosis. In-situ detectio n using terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) showed that this process occurred in distinct st ages on each organ of hybrid seedlings and centripetally in hybrid calli. F rom these results, we confirmed that cell death inducing hybrid lethality w as indeed apoptosis.