II.1 DNA content of dormant barley leaf nuclei.
Zia Uddin Ahmed and Om P. Kamra, Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
The first leaf of the germinating barley seed is an interesting embryonic organ in which to study the metabolic events triggered by germination, such as the syntheses and relationship of protein, RNA and DNA. As a prelude to such a study it was considered important to characterize the cell population of the dormant leaf with respect to the replicative state of the genome, that is, whether the cells are in pre-DNA replicative (2C) or post-DNA replicative (4C) state. A 2C cell population will enter the post-dormancy cell cycle in G1 and proceed to accomplish genome replication in S while a 4C cell population will do so in G2 and proceed to mitosis. The early biochemical response of the embryo may be related to whether it is the genome replication or division that immediately follows the post-dormancy initiation of the cell cycle. The possibility of such a relationship has been discussed in a report we have submitted recently for publication elsewhere.
Dormant seeds of Hordeum vulgare L. var. Himalaya (1970 harvest) were fixed in ethanol-acetic acid, 3:1 for 24 hours and transferred to cold water overnight. The first leaf was dissected from the embryo, hydrolyzed for 8 min. in 1N HCl in a water bath at 60°C and stained with the Feulgen reagent for 2 hours. After three washes with SO2-water (5 min. each) the leaves were treated with 5% pectinase, pH 5.5, for 35 min. at 35°C, squashed on a drop of 45% acetic acid, made permanent by dry ice method and mounted in Euparal. 2C and 4C values were established from mitotic telophase nuclei and metaphase plates respectively from the leaf of a 31 h germinated embryo; these leaves were processed along with the dormant leaves. DNA content was determined by two-wavelength Feulgen cytophotometry following the procedure outlined by Pollister et al. (1966) using a Zeiss UMSP 1 Universal Microspectrophotometer. The two wavelengths were selected from the Feulgen absorption curve of a homogeneous interphase nucleus. Approximately 7 arbitrary units of DNA was found to correspond to 2C and 14 units to 4C. Field diameters used for telophase and metaphase measurements were 5 and 8 mu respectively. A sample of 100 nuclei from each of 8 different dormant leaves were measured (Table 1) and classified according to DNA value into 2C, 4C and intermediate.
The data indicate that approximately 98% of the dormant nuclei are in the pre-DNA replicative state and hence they will enter the post-dormancy cell cycle in G1. The cells with 4C DNA value may belong to the differentiated cell line and may not enter the cell cycle. Thus, the dormant cells of the first leaf of the barley embryo represent a fairly homogeneous cell population with respect to the replicative state of the genome.
Reference:
Pollister, A. W., Swift, and Rasch, E. Microphotometry with visible light. In "Physical techniques in biological research", A. W. Pollister ed., vol. 3 part C, P. 201, Academic Press, N. Y., 1966.
Acknowledgements:
Financial assistance of National Research Council grant A-4892 is gratefully acknowledged.
