Apoptosis during diploid parthenogenesis and early somatic embryogenesis of Norway spruce

In diploid parthenogenesis of Norway spruce, specific nuclei were ontogenetically eliminated in cells programmed for cell death or apoptosis. Nuclear elimination was defined morphologically and biochemically for the degenerating ventral canal nucleus of egg-equivalents, and in differentiating suspensors of the axial tier of early embryos. Nuclei became pycnotic and disintegrated with the release of nucleoli and nuclear fragments into the cytoplasm. Enucleation in tubular cells of the axial tier was preceded by a reduction in the number of interphase nucleoli. Apoptosis was biochemically characterized by the activation of endonuclease activity that contributed to massive morphological changes in nuclei and ultimately to cell death. Endonuclease activity in individual nuclei was determined by a terminal deoxynucleotidyl transferase (TdT). This enzyme labeled the 3'OH ends of DNA, generated by DNA nicking with biotin-conjugated dUTP, for visualization with a secondary detection system using a colorimetric substrate for peroxidase. The TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay stained apoptotic nuclei red for the expression of endogenous endonucleases. The salvage of nuclear degradation products was postulated to enhance embryo survival, growth, and multiplication by cleavage polyembryony. By contrast, the egg-equivalent nucleus, proembryos, and the large and rounded nonapoptotic nuclei of the embryonal group fluoresced strongly with DAPI (4'-6-diamidino-2-phenylindole dihydrochloride). Nuclei also reacted blue with Giemsa in cells probed by the TUNEL assay.