An ultrastructural study of cell wall regeneration by isolated mesophyll protoplasts of Zea mays L. and the effect of the antioxidant, ascorbic acid, on wall regeneration

Includes bibliographical references.Regeneration of a cell wall around enzymatically isolated mesophyll protoplasts of corn, Zea mays (inbred line W64A) was studied by scanning and transmission electron microscopy. The effect of the antioxidant ascorbic acid on the developmental process of cell wall regeneration around the protoplasts was also analyzed. At time zero (immediately upon removal from the protoplast isolation medium, Earle et al. , 1978) the protoplasts were completely free of new or residual wall material. Regenerated wall material was clearly evident following six hours of culturing in a modified culture medium of Murashige and Skoog, and displayed a discontinuous distribution over the plasmalemma surface. These "plaques" were devoid of discernible fibrillar components when viewed with the SEM, however, short fibrillar segments were seen with the TEM in negatively stained preparations of mildly sonicated protoplast surfaces and thin sections. Plaques were more continuous with increased culture time up to about 18 hr. Further changes were not evident through a 120 hr culture period. Ascorbic acid showed no apparent qualitatively effects on wall regeneration over the entire range of concentrations (0-60mM) tested. However, at the higher concentrations (30 and 60mM) the surface morphology of the protoplasts was quite different from that of control samples or the lower ascorbic acid concentrations. This difference was expressed in the appearance of projections from the cell surface accompanying cell wall regeneration. Such projections became visible as early as 18 hr from the time of isolation. They were seen to originate from amorphous centers within the chloroplasts. Our results suggest that the pattern of wall regeneration by mesophyll protoplasts of Zea mays is different from the pattern observed for tobacco, tomato and other dicotyledonous species by other investigators. The major differences appear to be the plaque-like deposition of a new wall and the absence of an abundant fibrillar component within the regenerated wall during the period of incubation studied. The latter may indicate a difference in the sequence of deposition of matrix and framework components of the primary wall between monocots and dicots. The formation of the ascorbic acid-induced projections at higher concentrations resulted in a marked deformation of the plasmalemma surface. Their protrusion to the surface appeared to be related to areas where little cell wall deposition occurred.M.S. (Master of Science