W. Yanpaisan et al., ANALYSIS OF CELL-CYCLE ACTIVITY AND POPULATION-DYNAMICS IN HETEROGENEOUS PLANT-CELL SUSPENSIONS USING FLOW-CYTOMETRY, Biotechnology and bioengineering, 58(5), 1998, pp. 515-528
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.