The apomixis-specific phenomenon: Appearance of aposporous embryo sac initial cell (AIC), AIC-derived embryo sac formation and embryogenesis without fertilization in apomictic guinea grass (Panicum maximum)

Apomixis is a reproductive mode which bypasses female meiosis and syngamy to produce embryos genetically identical to the maternal parent. Apomixis is usually classified into three modes, apospory, diplospory and adventitious embryogeny. Here, we describe the mechanism of aposporous initial cell appearance in guinea grass (Panicum maximum), which belongs to aposporous mode. Based on the observation of young buds by Nomarski differential interference-contrast microscopy (DIC), there was no cytological difference between sexual and apomicts up to megasporogenesis. After that, however, the megaspore usually degenerated and in final, disappeared in the apomicts in contradistinction to that in sexual to form 8-nucleate embryo sac. At the same time, aposporous embryo sac initial cell (AIC), derived from nucellar tissue around the megaspore, appeared, which divides into 4-nucleate embryo sac. And before anthesis, the AIC appears one by one and each AIC become mature AIC-derived embryo sac (AES), i.e. one egg, two synergids and one polar cell. After anthesis, the egg cell develops into embryo automatically without fertilization with sperm cell, but with the stimulation of the fertilization between the one polar and sperm cell. To further clarify process of AIC appearance, ultrastructural observation was studied by transmission electron microscopy (TEM). The first degeneration was observed in dyad stage. Accompanying the degeneration of sexual cell, the cell adjacent to the dyad in chalazal side, derived from nucellus began to increase its size of cell and change its organelles and shape, and thus, the cell become to the first AIC usually located in micropylar side. The AIC appeared in order continently adjacent to the first AIC in the chalazal side. The interdependent patterns of the nucellar cell and AIC, and their relationship are also discussed at subcellular level. At anthesis, the first AES located in micropylar end consists of egg apparatus with two synergids and one egg cell, and the central cell with one nucleus. The other AESs contain less than four cells. The egg cell contains high dense of cytoplasm, and which around the nucleus plastids and mitochondria are remarkable. There exist lipid bodies, few rough endoplasmic reticulum (rER), dictyosomes, and big vacuoles distributed in the cytoplasm. The polar cell is occupied almost by one big vacuole, the lower dense cytoplasm than that of egg cell wraps one or two nuclei and is distributed around egg apparatus. To clarify the cytological mechanism of seed-forming embryo development in polyembryonic ovules in facultative apomictic guinea grass, the samples staged after anthesis of seven facultative apomicts were collected every day up to 10th day after open pollination (DAP), and observed using DIC (Chen and Kozono 1994b). The following results were understood. 1) the first AIC is located dominantly in micropylar end; 2) the rates of ovules containing embryo at 4 DAP was 90% in micropylar end higher than 2% in the other end; 3) the embryo in micropylar end, in final, became a seed-forming embryo, and in contrast, the others were crowded out to chalazal end and degenerated at 10 or days. Based on cytological observation using ovary length as an index to sample the young buds, and differential screening method, AIC stage-specific genes were cloned. The gene analysis using in situ hybridization and transgenic plants carried out was also discussed.