DURATION OF THE NUCLEAR CYCLE IN TRADESCANTIA ROOT TIPS AT THREE TEMPERATURES AS MEASURED WITH H3‐THYMIDINE

The duration of the subdivisions of the nuclear cycle in cells from the root tips of Tradescantia paludosa was determined by the labelled mitosis method. These times were found not to increase or decrease in direct proportion to each other at the three temperatures tested, 13, 21 and 30 C. The main difference between the nuclear cycles at 30 and 21 C was in the shortening of the pre-DNA synthetic period (G1) at the higher temperature. A comparison between the nuclear cycle at 21 and 13 C showed that at the lower temperature mitosis and the post-DNA synthetic period (G2) were approximately tripled in duration while DNA synthesis was doubled. Cell synchronization and radiosensitivity are discussed in relation to these findings. THE EFFECT of the environment on the girowth of cell populations has long been of interest and numerous studies have been performed in attempts to determine changes that may occur in populations grown at different environmental means. A factor that changes almost continuously under natural conditions is the temperature, and it is without doubt one of the major items that influences the rates at which cell populations grow (Brown, 1951; Evans and Savage, 1959; Hughes, 1952). This paper is devoted to a description of the growth of cells in root tips of Tradescantia paludosa at three different temperatures. The popuilation is analyzed by using the labelled-mitosis technique (Wimber, 1960) with tritiated thymidine and is subdivided with respect to the duration of the DNA synthetic period and mitosis. It is shown that the durations of the various subdivisions of the nuclear cycle are bDy no means uniformly lengthened or shortened by temperature changes, but some parts are altered much more profoundlv than others. MIATERIALS AND MIETHOI)S-Root tips of Tradescantia, paludosa were used as experimental material. Plantlets were removed from the clonal parent (B2-2, A. H. Sparrow's clone) and the bases placed in aerated Hoagland's solution. They were given constant light (ca. 500 ft-c) and constant temperature. Three temperatures were used, 13, 21 and 30 C. Well-developed roots were usually present within a week. Nuclei in the root meristems were labelled by growing the rooted plants in an aerated nutrient solution containing 0.5 ,c/ml H3-thymidine (New England Nuclear, 3.6 c/mmole) for 30 min .The roots were washed thoroughly in water following label' Received for publication February 15, 1965. Part of this study was carried out at Brookhaven National Laboratory under the auspices of the U. S. Atomic Energy Commission, and part was supported by Public Health Service Research Grant GM 11702-01 from the National Institute of General Medica.l Sciences. The technical assistance of Miss Mary Winnike is gratefully acknowledged. ling and placed in a nutrient solution for further growth. Root tips were collected at various intervals after labelling and fixed in acetic-alcohol (1:3); they were hydrolyzed for 10 min in N HCI at 60 C and stained by the Feulgen method. The root cap was removed and squashes of the intensely stained terminal portion of the root tip were prepared on slides; the cover glasses were removed by the method of Conger and Fairchild (1953). Autoradiographs were made using Kodak NTB liquid emulsion. Exposure was for approximately 3 months. The percentage of mitotic cells labelled was determined by using samples of 100 prophase cells from each slide. Eight roots were sampled at each time interval. Ratios of labelled cells to cells in mitosis were also determined in some cases, and with these a total of 1000 cells were scored on each slide. The labelled-mitosis technique is the method utilized here (Quastler and Sherman 1959; Wimber, 1960, 1963; Evans and Scott, 1964) for the determination of the nuclear cycle and its various subdivisions as they relate to DNA synthesis. The nuclear cycle (C) for this type of analysis may be divided into mitosis (M), DNA synthesis (S), pre-DNA synthesis (G1) and post-DNA synthesis (G2). A discussion of this method is given in the above references and need not be repeated here. Suffice it to say that the duration of the subdivisions of the cycle may be estimated from a labelled prophase (P) curve. An example is given in Fig. 1, and estimates of the durations of G2 + P/2, S and C may be read directly from the figure as distances on the abscissa, using as end points the positions at which the rising and descending curves cross the 50% mark. The duration of GI + mitosis P/2 may be calculated by subtraction of G2 + P/2 + S from C. The duration of mitotic stages may be estimated by observing the frequency of labelled cells and mitotic figures in root tips fixed immediately after a 30-min labelling period. In an asynchronous