Use of Osmotic Inhibition in Studies of Shoot Formation in Tobacco Callus Cultures

Abstract

Tobacco (Nicotiana tabacum) callus was transferred from permissive shoot-forming (SF) to inhibitory conditions and vice versa at different days in culture. Inhibition occurred when high levels (10%, 12%, 15%, w/v) of sucrose were added to the medium. Reciprocal tissue transfers were made at key histological stages of the SF process, and known biochemical and biophysical correlates of the process were examined after the transfers. The earlier in culture the cells were exposed to the permissive SF conditions, the greater was the number of shoots formed. High sucrose levels inhibited all stages of the SF process. Transfer of the SF tissue to the inhibitory conditions caused a reduction of the preferential cell division zones, the disorganization of meristemoids into parenchymatous tissue, and the accumulation of starch. Osmotic adjustment took place in the reciprocal transfers occurring rapidly in the early stages of culture and more slowly in older tissue. The changes in the rate of respiration could not be correlated with the above events. This approach to the study of in vitro organogenesis appears to be useful for examining the mechanism of tissue and organ differentiation.