| I. Research Notes Visualization of wheat chloroplast DNA in situ with the DNA fluorochrome Takaji IKUSHIMA Research Reactor Institute, Kyoto University, Kumatori-cho, Sennan-gun, Osaka 590-04, Japan Genetic information of plant cells is separately pooled among their nuclei, mitochondria, and plastids. It is well known that such organelle DNAs are predominant determinants of cytoplasmic inheritance1). The contribution of cytoplasmic genome to speciation on the wheat genus Triticum and its related genus Aegilops has been revealed from analyses of genetic characters of the cytoplasm by the nucleus substitution2-5). Molecular biological analyses of the problem are needed at present6). Since WILLIAMSON and UNNELL7) showed that a trypanocide, 4'6-diamino-2-phenylindole (DAPI) can be a probe for detection of mitochondrial DNA in yeast by fluorescence microscopy, DAPI has been employed to visualize mycoplasm8), kinetoplast DNA9), and chloroplast DNA in algae10) and in isolated chloroplasts of higher plants11). This paper reprots a simple and rapid method for cytological detection of chloroplast DNA in situ of wheat mesophyll protoplasts using the DNA fluorochrome, DAPI. The first foliage leaves of 10-day-old wheat seedlings (Triticum aestivum L., cv. Chinese Spring) grown under a normal diurnal light regime, 16 h light (5000 lux)-8 h dark, at 25C were used throughout this study. The mature regions of the leaves were finely chopped with a razor blade, and suspended in the enzyme solution containing 10% (w/v) mannitol and 10 mg/ml Cellulysin (Calbichem, B grade) in 0.2 M phosphate buffer (pH 5.7). The suspension was incubated at 37C in the dark for 4-5 h. Released protoplasts were collected by centrifugation at 1000 rpm for 10 min after being filtered through two layers of Miracloth. The sedimented protoplasts were fixed in 0.5% glutaraldehyde on a microscope glass slide for 10 min. A drop of 15 microg/ml DAPI solution was added to the fixed protoplasts and it was kept standing for 10 min, followed by being placed a cover slip over. Preparations were examined with a Nikon fluorescence microscope under epifluorescent illumination by UV light from a 200 W mercury lamp. The observation of the DAPI fluorescence was done with a UV-F100 objective, using a U excitation filter in combination with a 420 K suppression filter. Within chloroplasts numerous blue-white fluorescent dots were discretely observed, while nuclei were overall brightly stained as shown in Fig. 1. |
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