Techniques to study the cell cycle in the shoot apex of conifers
Date
1990
Authors
Grob, James Arnold
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Abstract
Techniques were developed to measure mitotic index (MI) and identify nuclei in G 1, S, and G2 stages of the cell cycle in squash preparations of conifer shoot apices. Feulgen staining and sampling by horizontal scanning at fixed vertical intervals were used. These procedures allowed indices for the entire apex to be accurately determined with a minimum of 10 apices. Using this technique, the resumption of mitosis from a G2 population of cells was observed after 10-14 hours in loblolly pine (Pinus taeda L.) shoot apices which were removed from cold storage and placed in growth promoting conditions. The potential for using this procedure to determine the growth potential of dormant shoot apices is discussed.
A method was developed to identify nuclei in the interphase stages of G 1, S, and G2. This involved labelling of shoot apices with tritiated thymidine (203.3 kBq/ml) in 1 % DMSO for two hours to identify nuclei in S stage. Nuclei in G 1 and G2 were distinguished visually. In loblolly pine, abnormal mitotic figures and a 26% reduction in MI resulted from this treatment, possibly due to the presence of DMSO. Labelling of western redcedar (Thuja plicata D.) shoot apices was significantly higher than loblolly pine apices and may have been due to exposure of vascular tissue to the treatment solution. Procedural modifications which may improve incorporation, decrease disruption to cells, and improve criteria for determining labelled nuclei are discussed.
The morphology of nuclei in G 1, S, and G2 was examined using microdensiometry. This allowed G 1 and G2 nuclei to be identified by their staining intensity and area. Mitotic figures which have a known DNA content (2C or 4C) assisted in these determinations. Nuclear area varied greatly in different apical stages of the cell cycle, but this still allowed an accurate identification of cell cycle stage. Loblolly pine seedlings in rapid neoformed growth had 75 % of their cells in Gl, 14.8 % in S, 4.7% in G2, and 4.6 in mitosis, and is correlated with the relative duration of each cell cycle stage. The G 1/S transition appeared to be limiting cell cycle progression in these apices. These findings are discussed in relation to apical zonation and previous studies which used microdensiometry to determine cell cycle stage.