Organization of Isolated Embryo Sacs and Eggs of Plumbago Zeylanica (Plumbaginaceae) Before and After Fertilization

Abstract

The organization of isolated embryo sacs and eggs of Plumbago zeylanica was described before and after fertilization using microscopic cytochemistry and scanning electron microscopy. Major developmental events of fertilization, including preferential fertilization and early embryogenesis, are described in isolated embryo sacs. The two sperms, one unassociated with vegetative nucleus (Sua) and the other physically associated with the vegetative nucleus (Svn), fuse with nuclei of egg and central cell, respectively. The zygote divides asymmetrically to form a two‐celled embryo, consisting of a massive suspensor occupying most of the micropylar portion of the embryo during early embryogenesis. Plastids are distributed in the perinuclear and micropylar regions of the egg cell and in cytoplasmic strands of the central cell before fertilization. Calcofluor white‐positive fibrillar material in the filiform apparatus (presumed β‐1,4 linked glucans) was investigated using scanning electron microscopy. The egg of P. zeylanica can easily be divided into three cytologically distinct regions: 1) perinuclear cytoplasm, 2) lateral cytoplasm, and 3) micropylar cytoplasm. Cytological differences are evident in the organization of the cell walls, general degree of vacuolization, and the distribution of heritable organelles, storage bodies, and microtubules. The present study supports the concept that the egg of P. zeylanica plays combined synergid and gamete functions.

Fertilization in Nicotiana tabacum: Cytoskeletal modifications in the embryo sac during synergid degeneration

“…Cortical Mts in this region may add rigidity to the egg cell and appear to be related to the area of the cell where fusion most frequently occurs. Similar patterns are seen in the chalazal region of the egg cell in Plumbago (Huang et al 1990(Huang et al , 1993a. Associations between Mts and organelles are less frequent in the egg cell of tobacco than in the synergids and central cell, perhaps reflecting the quiescent ultrastructural appearance of the egg cell reported in studies of tobacco (Mogensen and Suthar 1979).…”

“…Ovaries from these pollinations were collected 48 h later and used to provide both fertilized and unfertilized ovules (Huang and Russell 1992b). lmmunofluorescence and affinity labeling. Embryo sacs were processed as described previously (Huang et al 1990) with the following modifications. Ovaries were dissected and cut longitudinally into several pieces and incubated in PEMG buffer (50 mM Pipes [piperazine-N, N'-bis[2-ethanesulfonic acid]], 5 mM EGTA [ethylene glycol-bis[[3-aminoethyl ether]-N,N,N',N'-tetraacetic acid], 2 mM Mg-SO4 and 4% glycerol, pH 6.9) containing 5% dimethylsulfoxide, 0.1% Triton X-100 and 400 laM m-maleimidobenzoic acid N-hydroxysuccinimide ester (Sigma Chemical Company, St. Louis, Mo., USA) and mixed protease inhibitors (2 mM phenylmethylsulfonyl fluoride, 25 I~M pepstatin A and 25 gM leupeptin) for 10 min, and then fixed in 4% paraformaldehyde in the above buffer for 1 h. Ovules were then incubated for 30 min at 37~ in a solution of enzymes containing 2% cellulysin (Calbiochem Corporation, La Jolla, Calif., USA) and 2% pectinase (Sigma) in PEMG buffer containing protease inhibitors.…”

“…Although there are immunofluorescent microscopic studies on the localization of Mts during megasporogenesis (Bednara et al 1988;Willemse and van Lammeren 1988;Webb and Gunning 1990), in mature ESs (Huang et al 1990;Murgia et al 1993) and in zygotes (Webb and Gunning 1991), few details are available on fertilization (Huang et al 1993a) and on F-actin (Bednara et al 1990). …”

The Ovule and the Embryo Sac.

“…The arrangement of microtubules, as detected by immunofluorescence, indicates a role for the cytoskeleton in distribution and positioning of cytoplasmic contents during megasporogenesis and megagametogenesis (Huang et al, 1990;Webb and Gunning, 1990). Possibly, asymmetric distribution of morphogenetic determinants is mediated by the cytoskeleton, as is the case in other organisms (Hill and Strome, 1990;Yisraeli et al, 1990;Pokrywka and Stephenson, 1991).…”

Fertilization-induced changes in the microtubular architecture of the maize egg cell and zygote—an immunocytochemical approach adapted to single cells

“…Techniques for detailed three-dimensional observations of isolated embryo sacs and egg cells of Plumbago were developed (Huang et al 1990). Microtubules in the egg cell of the synergid-lacking embryo sac of Plumbago zeylanica are longitudinally aligned in the micropylar and mid-lateral areas, forming bundles near the filiform apparatus.…”