ANALYSIS OF CELL-CYCLE ACTIVITY AND POPULATION-DYNAMICS IN HETEROGENEOUS PLANT-CELL SUSPENSIONS USING FLOW-CYTOMETRY

Flow cytometry was used to measure cell cycle parameters in Solanum av iculare plant cell suspensions. Methods for bromodeoxyuridine (BrdU) l abeling of plant nuclei were developed so that cell cycle times and th e proportion of cells participating in growth could be determined as a function of culture time and conditions. The percentage of cells acti ve in the cell cycle at 25 degrees C decreased from 52% to 19% within 7.6 d of culture; presence of a relatively large proportion of non-act ive cells was reflected in the results for culture growth. While the m aximum specific growth rate of the suspensions at 25 degrees C was 0.3 4 d(-1) (doubling time: 2.0 d), the specific growth rate of active cel ls was significantly greater at 0.67 d(-1), corresponding to a cell cy cle time of 1.0 d. A simple model of culture growth based on exponenti al and linear growth kinetics and the assumption of constant cell cycl e time was found to predict with reasonable accuracy the proportion of active cells in the population as a function of time. Reducing the te mperature to 17 degrees C lowered the culture growth rate but prolonge d the exponential growth phase compared with 25 degrees C; the percent age of cells participating in the cell cycle was also higher. Exposure of plant cells to different agitation intensities in shake flasks had a pronounced effect on the distribution of cells within the cell cycl e. The proportion of cells in S phase was 1.8 times higher at a shaker speed of 160 rpm than at 100 rpm, while the frequency of G(0) + G(1) cells decreased by up to 27%, Because of the significant levels of int raculture heterogeneity in suspended plant cell systems, flow cytometr y is of particular value in characterizing culture properties and beha vior. (C) 1998 John Wiley & Sons, Inc.