Microtubules and the First Cell Cycle in Cultured Mesophyll Protoplasts of Nicotiana

Abstract

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 protoplasts are highly vacuolated. In vacuolated cells a plate of cytoplasm (phragmosome) must be formed prior to mitosis in order to permit positioning of the nucleus and bisecting of the cell through the vacuole during cytokinesis (1). Thus, the phragmosome determines the orientation of the plane of cell division. Another structure which is thought to be involved in determining the plane of cell division, is a cortical band of microtubules commonly termed the preprophase band of microtubules (PPB). The term refers to the time when the bands first appear and not to the life span, which includes prophase (2). There seems to be a close temporal and spatial correlation between the establishment of phragmosomes and PPBs (2,3,4).