University of Minnesota

Detect DNA contents in microsporogenesis of maize mutant
--Liu, Q; Splett, M

The early work on cellular analysis of maize postmeiotic mutant po and alleles showed that premature chromosome condensation and fragmentation occurs at the end of meiosis II in microsporogenesis in po and alleles. Abnormal cell cycles occurred without chromosome replication in this mutant and its alleles (Liu et al., J. Cell Sci. 106:1169-1178). These chromosomal defects do not affect cell wall matrix formation during microspore development. Fluorescence cytometry has been used to detect DNA contents in microspores of po and wild type plants by using 4',6-diamidino-2-phenylindole (DAPI, a fluorescent DNA label) and absorption cytophotometry with technical help from Dr. R. V. Kowles. Maize samples were collected and fixed with ethanol/acetic acid (3:1) at different stages of anther development. These microspore samples were isolated from anthers by using a dissecting microscope for fluorescence cytometric analysis. Results of the analysis indicate there is no DNA duplication before abnormal cell divisions and DNA content was decreased during cell cycle progression in microspore development (Fig. 1 and 2). Correlated to such DNA content, cell size was also reduced during the formation of abnormal microspores (data not shown). This result will be confirmed by developing a new method using BrdU in vitro DNA labeling.

Figure 1. Fluorescence cytometry has been used to detect DNA content in pollen mother cells and individual meiocytes during tetrad formation in the wild type plant as a control. Samples were then labeled with DAPI stain on the same slide and at least ten cells were detected and average measurements were used for their DNA contents. DNA content was about four times higher in maize pollen mother cells than that of individual meiocytes in tetrads of the wild type plant.

Figure 2. DNA content was measured for the samples on the same slide at different stages of abnormal cell cycles after meiosis in po mutants using the methods described in Figure 4. There was no DNA duplication in po meiocytes before abnormal postmeiotic divisions. DNA contents were gradually decreased from po meiocytes to cells in postmeiotic cell divisions I and II. Cell size was also reduced (data not shown).

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