Mitotic blocking agents for suspension cultures of Black Mexican Sweet cell lines

Colchicine has been used extensively since 1937 to block mitosis and induce polyploidy in a large number of plant species (A.F. Blakeslee, Compt. Rend. Acad. Sci. 205:476, 1937). The original studies of mitotic blockage were performed on tissues in planta, but, with the development of methods for culturing plant cells and the increasing use of these cells for research, attention has turned to the use of colchicine as a mitotic blocking agent in cell suspension cultures. Colchicine treatment gives satisfactory mitotic arrest in wheat and poppy suspension cultures. In the latter, the frequency of mitotic cells approaches 45% when the suspension is growing optimally (Hadlaczky et al., Planta 157:278, 1983).

Colchicine treatment, alone or in conjunction with hydroxyurea or physical methods, has recently been used to produce mitotic blocks in Black Mexican Sweet (BMS) suspension cultures (A.S. Wang et al., Plant Science 46:53, 1986). In those experiments the colchicine concentration used was 0.5mM (0.02%). This colchicine treatment alone doubled the frequency of mitotic figures (mitotic index: MD compared to the untreated control, but the maximum yield was 9%. Hydroxyurea pretreatment did not increase this frequency. However, a 4-day subculturing routine combined with the selection of large aggregations of cells (280µm or more) raised the mitotic index of the untreated control to 10%, and the further addition of 0.02% colchicine again doubled the frequency of mitotic cells so that a final yield of 23% was obtained. These results clearly illustrate the effect of culture conditions and health of the treated cells upon the success of a mitotic blockade, but, again, colchicine treatment only doubled the residual number of mitotic cells. The physical methods described for this combined procedure have proved difficult to replicate and are, therefore, not easily useful for routine preparations of large numbers of mitotic cells.

Because the optimal concentration of colchicine for mitotic arrest varies widely between different plant species (e.g., 1.25mM for poppy and 2.5mM for wheat, Hadlaczky et al., 1983), we have determined the effective concentration of colchicine for mitotic blockade in BMS suspension cultures. We also report excellent mitotic inhibition obtained with micromolar concentrations of amiprophos methyl (APM), a tubulin-specific phosphoric amide herbicide (Morejohn and Fosket, Science 224:874, 1984). Trifuralin, on the other hand, was unsuccessful as a mitotic blocking agent although the herbicide is known to have a colchicine-like effect on mitosis in tobacco callus cultures (Young and Camper, Pest. Biochem. Phys. 12:117, 1979) and to depolymerize microtubules in cotton root cells (Hess and Bayer, J. Cell Sci. 24:351, 1977).

Table 1. Mitotic indices after treatment of maize suspension cultures with colchicine, APM, and trifuralin.a
 
Colchicine
% Mitotic

Cells

APM

(µM)

% Mitotic

Cells

Trifuralin

(µM)

% Mitotic

Cells

(mM)
(%)
0
0
4
0
3.0
0
3.2
0.5
0.02
3-5
1
3.1
25
2.1
2.5
0.1
3-5
5
5.9
50
1.8
5.0
0.2
13
10
5.3
100
1.9
12.5
0.5
25
50
20.1
75
17.0
100
13.0
500
0b
aEach mitotic index is based upon 1,000 cells, and each experiment had two or three repetitions.
bComplete cell lysis.

Mitotic indices obtained after treatment with colchicine, APM, and trifuralin are shown in Table 1. Inhibition of mitosis by colchicine is concentration-dependent; 0.02% colchicine per se is insufficient for repeatable mitotic blockage, while treatment for 21 to 28h with 0.5% reliably arrests 25% of the cells in mitosis, almost all in metaphase. This mitotic arrest is also time-dependent, since in other experiments 0.5% colchicine applied for 10h yields only 6.8% mitotic cells. The effective blocking concentration of colchicine for maize cells (MI 25%) is 5 times that required for poppy cultures and 10 times that required for suspension-grown wheat cells. This result is not surprising in view of the previously mentioned species differences. This high-level colchicine requirement in mitotic blockade of maize cells is not understood. It could reflect either very low affinity of maize tubulin for colchicine or faulty entry of colchicine into the cell due to low affinity binding of the drug to transport molecules.

APM treatment produces a good mitotic blockage at low drug concentrations; 50 AM applied for 20 to 28h arrests 20% of the cells in metaphase. This result indicates that APM is effectively transported into the cell and that it is efficiently bound by maize tubulin. Trifuralin, however, does not induce any mitotic arrest although the concentration range tested was broad, 25 to 100µM.

Table 2. The effect of metaphase-blocking concentrations of APM on growth of BMS cell cultures.
APM
Initial growth lag
Population Td (d 4-7)
0
0
35h
25µM
4d
32h
50µM
4d
91h
100µM
No growth by seven days

Some experiments require reversible mitotic blockades, for example diploidization of haploid cultures or cell-cycle studies. For this work it is desirable to use drugs relatively nontoxic to the cells at blocking concentrations. Since APM is a newly reported mitotic inhibitor for maize cells, we have made some preliminary studies of the toxicity of this compound. Growth data are shown in Table 2. Suspension cultures were inoculated with cells (0.004 g/ml from populations in exponential growth, and several concentrations of APM were added at the time of subculture. Increments in fresh weight were determined in 2 to 3 replicate flasks at 2, 3, 4, 5, 6 and 7 d. Added 25, 50, and 75µM APM induces a strong lag in population growth for 4 days after cell transfer although the control (0 drug) doubled each 35h in this time period. This lag in population growth is only partially accounted for by mitotic arrest, since after 3 days the MI in cultures with 50µM APM was still only approximately 20%. Therefore, we must assume that other cells in the population were probably temporarily arrested by unknown toxic effects of the drug. After 4 days, populations treated with 25µM APM abruptly resumed growth and attained a population doubling time (Td) like controls. Cultures with 50µM APM had a Td of 91h and cultures with 75µM APM continued in arrest. When cells from 6-day populations were stained with fluorescein diacetate, those obtained from cultures with APM were at least 50% viable, as compared with the control. And, after 6 days of growth, the MI of cell populations treated with 50µM APM was only 1.5 percent.

These data show that APM is almost certainly cytotoxic, but not completely lethal, at the concentrations used for mitotic blockade applied to continuously growing cultures. Resumption of growth after a strong 4-day lag suggests metabolism or breakdown of APM. The drop in mitotic index by 6 days may indicate escape of metaphase cells from the block. Because this drug is such an effective mitotic inhibitor at low concentrations, we plan some further experiments which will investigate the toxicity of APM during 28h pulse treatments, which are sufficient for good metaphase blockade.

J. Stadler, J. Rugemer, R.L. Phillips and M. Leonard


Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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