Abstract
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.
Highlights
The suspensor is a terminally differentiated embryonic structure, which connects the embryo to surrounding endosperms and seed coats in plants and is necessary for embryonic development by transporting nutrients and hormones from the mother tissues to the embryo [1,2,3]
[18], here, here, we describe a detailed protocol for the whole-mount detection of suspensor we describe a detailed protocol for the whole-mount detection of suspensor programmed cell death (PCD) using a TUNEL
A few studies reported the methods for the isolation of early embryos using either laser-capture microdissection (LCM) [19] or manual isolation [19,20]
Summary
The suspensor is a terminally differentiated embryonic structure, which connects the embryo to surrounding endosperms and seed coats in plants and is necessary for embryonic development by transporting nutrients and hormones from the mother tissues to the embryo [1,2,3]. A well-known characteristic of the suspensor is the timely initiation of programmed cell death (PCD) [4,5]. During this process, some classic markers of eukaryotic PCD have been observed in suspensor PCD, such as DNA fragmentation, nuclear degradation, and caspase-like activities [4,6,7,8,9,10,11,12]. Because embryos are deeply embedded in the maternal tissues, it is difficult to observe the spatial and temporal dynamics of suspensor PCD directly by conventional methods. The methods for the detection of suspensor PCD have been established for years in a few plants with a huge suspensor structure, such as Picea abies [6,7,8,9], Vicia faba [10], and Phaseolus coccineus [11,12]
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