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

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.

Morphological, biochemical and molecular hallmarks of programmed cell death in stigmatic papillae of Brassica oleracea L.

“…Besides, nucleus and chromatin deformations in anther wall cells of Lathyrus undulatus (Vardar and Ünal 2012) and, chromatin condensation in stamen primordia of Cucumis sativus (Pawełkowicz et al 2019) were indicated as doped hallmarks by various researchers. Also, Shi et al (2020) have been reported chromatin fragmentation in suspensor cells undergoing PCD of Nicotiana tabacum. In parallel to these literatures, we detected the various invaginations and fragmentation in papillae nuclei at the post-anthesis stage which we detected the dPCD.…”

Assessment of antimitotic and programmed cell death stimulation potentials of Galium sinaicum (Delile ex Decne) Boiss. at cellular level

“…DNA fragments is one of the hallmarks for apoptosis Matilla 2020, subsequently, Detection of DNA fragmentation is currently considered as one of the most frequently used techniques in the study of programmed cell death (Hanna et al 2013;Shi et al 2020). Different concentrations (0.1%,0.3% and 0.5%) of GsEE DNA was degraded into multimers of 180 bp.…”

Programmed Cell Death (PCD) in Plant: Molecular Mechanism, Regulation, and Cellular Dysfunction in Response to Development and Stress

“…Pollen ROS mediate [14] Osatg7-1 mediate [15] ROS mediate [16] PERSISTENT TAPETAL CELL2 mediate [17] AtLSD1 deathosome mediate [18] Female floral buds AMC9, MeGI, BAG5, AifA, and HSP70 mediate [19] Female gametophyte Auxin efflux [20] Fruit Ca 2+ -dependent nuclease [21] Double Fertilization ROS mediate [22] Vegetative cells PrpS-PrsS modulate PCD vegetative cells [23] Zygotic Embryogenesis - [24] Somatic Embryogenesis K + ATP channel activity [25] Phenol-storing cells [26] Embryonic Suspensor Cell - [27] Table 1.…”