Physiological and biochemical characterization of a suspensionculture system for sustained exponential growth of Nicotiana silvestris

Abstract

A strong approach to understanding the regulation of enzymes in metabolic pathways, such as those responsible for amino acid biosynthesis, is to follow enzyme levels throughout the growth curve of higher plant cells in suspension culture. The rise and fall of enzyme levels can be traced as a function of physiological stage of growth Subculturing, as typically carried out by low‐factor dilution of stationary phase cells, yields a system suitable for the study of changes in enzyme and metabolite levels that accompany the transition from stationary‐phase physiology to exponential‐phase physiology. However, the short duration of exponential growth in such subculture protocols is inadequate to avoid carryover effects from previous stationary‐phase physiology. Suspension cultures of Nicotiana silvestris Speg, et Comes (2N = 24) were used to demonstrate substantial carryover levels of acid phosphatase, alkaline phosphatase and protease activities. A subculture routine is described for maintaining cell populations in exponential phase indefinitely. About 10 generations of sustained exponential growth is required to approach a true balanced state of exponential growth. Such exponential phase populations consist of cells termed EE cells. EE‐cell populations were similar to cells that have been in exponential phase for only a few generations (E cells), with respect to doubling time (about 40 h) and to minimal density of diluted populations able to resume growth (about 500 cells ml−1). EE cells possess a high content of soluble protein; they are smaller and more aggregated than are E cells. Upon dilution into fresh medium, EE cells resume exponential growth without a lag. In contrast to E cells, EE cells exhibit properties of balanced growth, since proportionate increases in cell number, dry weight, wet weight and packed‐cell volume were observed. E cells, sampled at different elapsed times of growth, are likely to differ in metabolite, enzyme and cell properties, whereas EE cells exhibit near‐constant properties.