The use of colchicine as an indicator of mitotic rate in broad bean root meristems

Abstract

1. Conditions of culture are described, under which the primary roots of broad bean seedlings show a constant growth rate and Mitotic Index for a limited period of time. It was found that the diurnal fluctuation in root growth and Mitotic Index is eliminated when the plumules of the seedlings are removed. 2. Experiments with colchicine, acenaphthene.p-diehlorobenzene, α-bromonaphthalene and 8-hydroxyquinoline showed that colchicine was the only spindle inhibitor which caused marked increases in metaphase counts. 3. Three concentrations of colchicine, 0.1, 0.05 and 0.025%, were found to have similar capacities for metaphase accumulation over treatments of from 1 to 6 hr. duration. With increased durations of treatment, concentration differences were expressed, the weaker concentrations being more efficient than the stronger ones. The depression of the rate of metaphase accumulation which was found in treatments of more than 6 hr. duration is attributed to an inhibitory effect of colchicine which results in slowing the rate of entry of cells into mitosis, i.e. increasing the time spent in interphase. 4. It was found that the time required for colchicine to break down metaphase spindles which were organized prior to the commencement of treatment, was directly dependent on the concentration of colchicine used. For this reason the rate of metaphase accumulation over the first hour of treatment cannot be used to determine mitotic rate. 5. The mean number of rnetaphases accumulated in a population of 1000 meristematic cells over a period of from 1 to 6 hr. colchicine treatment, was found to be 30.1 ± 1.3 cells per hour. Allowing for the escape of 2 ± 1 cells per hour from c-metaphase into interphase, the number of cells entering metaphase in 1 hr. was thus determined as being 32.1 ± 1.6. 6. The rate of entry of cells into metaphase was used to determine the time parameters of the mitotic cycle. The method of calculation and formulae for the kinetics of growth of the cell population are given. 7. When using the colchicine technique the mean mitotic time and total mitotic cycle time of the meristematic cells was found to be 3.9 ± 0.2 hr. and 24.6 ± 1.5 hr., respectively. It is shown that these values are in close agreement with values determined through the use of other methods.