Kinetics of double fertilization in Torenia fournieri based on direct observations of the naked embryo sac

Abstract

Torenia fournieri Lind. has a naked embryo sac that protrudes from the micropyle. The precise time course of the entire process of double fertilization and the kinetics of fertilization events were determined in this species by the following methods: (i) without squashing, pollen tubes on the torn stylar canal were observed by fluorescence microscopy after staining with both 4′,6-diamidino-2-phenylindole (DAPI) and aniline blue; and (ii) large numbers of living embryo sacs were observed directly by differential interference microscopy before and after fertilization. The pollen began to germinate 5 min after pollination and extruded pollen tubes which elongated at a constant rate of 2.3 mm · h−1. At 4.0 h after pollination, the mitotic index of the generative cell within the pollen tube reached 88% and the two sperm cells were formed. Pollen tubes began to arrive at ovules 8.9 h after pollination and directly entered one of two synergids in the naked embryo sac. The time required for transport of sperm cells in the degenerated synergid was estimated statistically to be 1.9 ± 1.8 min for transport of the first cell and 7.4 ± 1.6 min for the second. In the nucleus of the fertilized egg cell, the male nucleolus began to emerge 10 h after pollination and the female nucleolus often decreased in size. The two nucleoli fused together prior to elongation of the zygote, which began 28 h after pollination. In the central cell, the secondary nucleus migrated to a region adjacent to the egg apparatus after pollination but prior to the arrival of the pollen tube. The primary endosperm nucleus rapidly returned to the inner region after fertilization. Prior to embryogenesis, the first division of the primary endosperm began about 15 h after pollination, at a defined site, to form the chalazal haustorium.