0000000000636937
AUTHOR
M. Rös
The First Cell Division Cycle in Nicotiana Mesophyll Protoplasts Cultured in Vitro. I. Methods to Determine Cycle Kinetics
Summary The progress of freshly isolated and in vitro cultured mesophyll protoplasts through the first mitotic cycle was studied with the aim of determining the frequency and timing of the G2-phase. Putative G1 and G2 phase cells were identified by measuring the relative DNA content through improved cytofluorimetry of DAPI-stained nuclei. S-phase nuclei were identified by labelling with the thymidine-analogue BrdU, which was subsequently immuno-localized with a BrdU-specific monoclonal antibody. The cells entered S-phase after 12–24h and a maximum of G2 cells was observed 24–36h after culture initiation. Most of the cells had divided after 48 to 72 h.
The First Cell Division Cycle in Nicotiana Mesophyll Protoplasts Cultured in vitro II. Microtubules and the Plane of Division
Summary Reorganization of the microtubular cytoskeleton prior to and during the first cell division cycle of Nicotiana mesophyll protoplasts was investigated using immunofluorescence microscopy. Bands of microtubules reminiscent of PPBs were found. However, their structure was comparatively diffuse. In addition, frequencies were low compared to those in meristem cells of organized tissues. Abnormalities during phragmoplast and cell plate formation were observed. The question is raised whether the formation of abnormal PPBs is related to strategic difficulties in co-ordinating the formation of a coherent plate of cytoplasm (phragmosome) traversing the vacuole to permit smooth bisecting of th…
Microtubules and the First Cell Cycle in Cultured Mesophyll Protoplasts of Nicotiana
When a protoplast is excised and cultured in vitro, an organized pattern of cell divisions must be reestablished to permit organogenesis, including plant regeneration. However, in a number of species protoplasts have proved to be very recalcitrant with regard to cell division and/or plant regeneration. In an attempt to find means to overcome the regeneration problem in recalcitrant species we are investigating the processes leading to cell division and morphogenesis in mesophyll protoplasts of the model system Nicotiana. Of particular interest are the initiation of the cell cycle and the coordination of progress through the cycles as a prerequisite for subsequent morphogenesis. Mesophyll pr…