Large quantities of purified chromosomes have been successfully isolated from animal cell cultures. The bulk isolation of plant chromosomes has been less successful probably due to difficulties in synchronizing cell division. Understanding the cell cycle parameters and other growth characteristics would facilitate achieving synchronization. We report here the doubling time and cell cycle parameters for Black Mexican Sweet (BMS) corn suspension cultures.
Doubling time: The BMS culture, kindly provided by C. E. Green, was grown in a modified Murashige and Skoog (MS) liquid medium for 4 days at 26 C on a rotary shaker. After standing for about 10 minutes, settled cells (0.5 ml) were transferred to fresh MS medium and grown for 2, 4, or 6 days at 26 C. An exponential growth pattern was apparent based on fresh weight:
Fresh weight was obtained by centrifuging the culture in a Beckman TJ-6 centrifuge at 1000 rpm for 5 minutes. Doubling time was estimated by using the following formulae: y = mx + b and y = ln (wt/wo) = kt, where m is the slope of ln (wt/wo) plotted against days, b the y-axis intercept, wo the initial fresh weight, wt the fresh weight at time t, and 1/k the doubling time. Based on ln (wt/wo) values in Table 1, y = 0.32x + 0.39. At x = 6 days, y = 0.32(6) + 0.39 = 2.31 and, thus, 2.31 = k(6) or 1/k = 2.6 days or 62 hours. Therefore, the doubling time is 62 hours.
Frequency of dividing cells: The BMS suspension cultures were grown in MS medium plus 3H-thymidine (3H-TdR) of specific activity 2 uCi/ml. A sample (1 ml) was collected every hour and fixed in 3 parts 95% ethanol:1 part glacial acetic acid overnight at room temperature. The fixed cells were washed twice with 0.1 M sodium acetate (pH 4.5) and digested for 2 hours with 0.5 ml cellulase (0.5%) plus pectinase (0.5%). The resulting protoplast preparation was rinsed twice with 45% acetic acid and stored in 45% acetic acid at 4 C. A drop of the protoplast preparation was placed on an acid-cleaned slide and a coverslip applied. The slide was then placed on dry ice for 5 minutes and the coverslip flipped off with a razor blade. The slide was then passed through 70% and 95% ethanol, air dried, coated with Kodak NTB-2 emulsion, exposed for 1 day at 4 C, and developed in Kodak D-19 developer.
Less than 30 minutes were required for 3H-TdR to be incorporated into certain nuclei (Fig. 1); 9% of the nuclei were labeled within 30 minutes of incubation. Labeling of interphase nuclei reached a maximum (28%) in 5 hours. Apparently, 72% of the cells were not actively dividing. The number of labeled prophase cells reached a plateau (80%) in 20 hours. The reason for 20% unlabeled prophase cells is not known but it might reflect a prolonged G2 period for cells in G2 at the time of initial labeling. The first labeled prophase cell was observed 7 hours after labeling and the first labeled telophase cell was observed after 11 hours. The mitotic period (M), therefore, is 4 hours.
Cell cycle parameters: The BMS culture was grown for 4 days, labeled with 3H-TdR for 1 hour, washed, and transferred to fresh MS medium. Samples (1 ml) were collected at various times as indicated in Figure 2 and prepared for autoradiography. Subculturing appeared to cause a growth lag of about 5 hours. The first labeled prophase cell was observed after 7 hours without subculturing after label introduction (Fig. 1) and after 12 hours with subculturing prior to labeling (Fig. 2). Based on the results displayed in Figure 2, G2 is 6 hours, S is 15 hours, M is 4 hours, G1 is 2 hours, and the total cycle time (T) is 27 hours. The 27-hour cycle time is in general agreement with the growth data presented in Table 1, assuming a doubling time of 62 hours and only 28% of the cells actively dividing.
A. S. Wang, R. L. Phillips, and C. C. Mi*
*Department of Agronomy, Beijing Agricultural University, Beijing, China
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