Partial synchronization of maize cells in liquid suspension culture

Mass isolation of somatic metaphase chromosomes might be possible from maize cells growing as a liquid suspension culture. Increasing the mitotic index of the culture should facilitate chromosome isolation procedures. Black Mexican Sweet Corn (BMS) suspension cells, kindly provided by C. E. Green, were grown for four days at 26 C in a modified Murashige and Skoog liquid medium (MS) prior to various treatments. Six general procedures for attaining partial synchronization were tested.

1. Temperature alternatives: Cell suspension cultures were shifted to 4 C for 10, 20, 36, or 42 hours or 35 C for 36 hours and then returned to 26 C.

2. Carbohydrate starvation: Cultures were transferred to a sucrose-free medium for 27, 35, 45 or 56 hours.

3. Double thymidine block: Thymidine (1 ug/ml medium) was added for 16 hours. Cultures were then washed with fresh media and grown for 8 hours. Thymidine was added again (1 ug/ml medium) for 8 hours. Cultures were then washed again.

4. 5-FudR synchronization: 5-Fluorodeoxyuridine (0.5-2 ug/ml medium) and uridine (1 ug/ml) were added to the medium for 20 or 24 hours. After washing with fresh media, thymidine (1 or 2 ug/ml) was added.

5. Hydroxyurea synchronization plus colchicine: Hydroxyurea (3-10 mM) was added to the medium for 24 or 36 hours. Cultures were then washed and subcultured into a conditioned medium (80% fresh, 20% used medium) with or without 0.02% colchicine.

6. Mitotic arrest with colchicine: Cells were cultured in the MS medium plus 0.02% colchicine.

After the treatments, cultures were maintained at 26 C on a shaker in the medium specified at the end of each procedure. Samples (1 ml) were taken every 1-2 hours, fixed in 3 parts 95% ethanol:1 part glacial-acetic acid, squashed and stained in propionic carmine.

The first four procedures did not significantly alter the mitotic index (M.I.) from the untreated control. The last two procedures, hydroxyurea plus colchicine and colchicine alone, resulted in a 2-2.5 fold increase in the M.I. (Fig. 1). We presume that the hydroxyurea blocks the active cells (probably a low percentage) at the end of G1. Upon release, the cells stay in G1 for several hours. This could be due to the transfer procedure or a hydroxyurea effect. The hydroxyurea without colchicine procedure required a longer time (50 hours) to reach the maximum effect compared with the hydroxyurea plus colchicine procedure. The reason for this is not clear. The "mitotic arrest with colchicine" procedure resulted in a mitotic index of 9% after 10 hours compared with less than 4% in the untreated control.

Although the BMS culture is variable in chromosome number, ploidy changes were expected with the colchicine procedure. The frequency of tetraploid cells (Table 1) increased significantly at around 48 hours of treatment and generally remained high. Octoploid cells also were observed.

Figure 1.

Table 1.

C. C. Mi,* A. S. Wang and R. L. Phillips

*Department of Agronomy, Beijing Agricultural Univ., Beijing, China


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