Detection of Embryonic Suspensor Cell Death by Whole-Mount TUNEL Assay in Tobacco

Embryonic suspensor in angiosperms is a short-lived structure that connects the embryo to surrounding maternal tissues, which is necessary for early embryogenesis. Timely degeneration via programed cell death is the most distinct feature of the suspensor during embryogenesis. Therefore, the molecular mechanism regulating suspensor cell death is worth in-depth study for embryonic development. However, this process can hardly be detected using conventional methods since early embryos are deeply embedded in the seed coats and inaccessible through traditional tissue section. Hence, it is necessary to develop a reliable protocol for terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) analysis using limited living early embryos. Here, we provide a detailed protocol for the whole-mount detection of suspensor cell death using a TUNEL system in tobacco. This method is especially useful for the direct and rapid detection of the spatial-temporal characters of programed cell death during embryogenesis, as well as for the diminishment of the artifacts during material treatment by traditional methods.

Direct evidence that suspensor cells have embryogenic potential that is suppressed by the embryo proper during normal embryogenesis

The suspensor is a temporary supporting structure of proembryos. It has been proposed that suspensor cells also possess embryogenic potential, which is suppressed by the embryo as an effect of the embryo–suspensor interaction. However, data to support this hypothesis are not yet available. In this report, using an in vivo living cell laser ablation technique, we show that Arabidopsis suspensor cells can develop into embryos after removing the embryo proper. The embryo proper plays a critical role in maintaining suspensor cell identity. However, this depends on the developmental stage; after the globular embryo stage, the suspensors no longer possess the potential to develop into embryos. We also reveal that hypophysis formation may be essential for embryo differentiation. Furthermore, we show that, after removing the embryo, auxin gradually accumulates in the top suspensor cell where cell division occurs to produce an embryo. Auxin redistribution likely reprograms the fate of the suspensor cell and triggers embryogenesis in suspensor cells. Thus, we provide direct evidence that the embryo suppresses the embryogenic potential of suspensor cells.

Rape embryogenesis I. The proembryo development

The development of the proembryo of rape <i>Brassica napus</i> L. from the zygote to the young embryo proper is described. A number of regularities were found in the direction, succession, and distribution of segmental and differentiating divisions of the proembryo. The direction of the divisions seems to foe determined by the direction of growth and the shape of the cells. The termyoung embryo proper is proposed to denote the globular embryo which already possesses separate plerome and periblem mother-cells and mother-cells of the iec layer and of clumella. The body of the embryo proper is derived from the apical cell ca which arose from the first division of the zygote and from the hypophysis - the only suspensor cell which closes the spheroid of the embryo. The development of the <i>Brassica napus</i> L. proembryo follows the sub-archetype <i>Capsella bursa-pastoris</i> in the IV megarchetype of Soueges.