A method for preparing metaphase chromosomes from tapetal cells of maize
--Maillet, DS; Walden, DB

Most studies of maize metaphase chromosomes utilize root-tip cells. With this approach it has been possible to detect evidence of somatic association of homologous chromosomes during interphase (Horn and Walden, Genetics 88:181-200, 1978). Recent studies of the arrangement of chromatin in differentiated mammalian neurons indicate that the different classes of cells have different characteristic patterns of chromatin (Manuelidis, Science 250:1533-40, 1991), suggesting that the developmental state of a cell may be related to the form/pattern in which the chromatin is arranged.

It is possible that different cell types of maize may exhibit characteristic arrangements of chromatin. The tapetum of maize is composed of highly specialized cells that are responsible for a number of processes that are necessary for the production of viable pollen. The tapetum is an ideal tissue within which to examine chromatin organization. Our goal is to determine if there is order within tapetal nuclei by examining them during different stages of their development, including metaphase and interphase, and to determine if there are differences / similarities between root-tip and tapetal nuclei organization. An anther can provide hundreds of synchronized cells that can be studied in metaphase using the following protocol for the recovery of tapetal nuclei in metaphase:

1. The upper portion of a sexually immature plant was removed and the leaves were trimmed to make the plant a manageable size.
2. The explant was kept at 4 C for 24 h with the stalk immersed in dd H2O.
3. The tassel was dissected out of the explant and fixed in 3 : 1 ethanol : acetic acid overnight.
4. Anthers were staged with a standard propionocarmine smear technique (Burnham, Maize for Biological Research, pp. 107-118, 1982).
5. Anthers were then placed in dd H2O for 3 X 10 min followed by 20 min in 0.01 M citrate buffer, pH 4.7.
6. The anthers were cut in two and digested with 0.25% (w/v) beta glucuronidase (Sigma G0251) and 0.002 % (v/v) pectinase (Sigma P9179) in citrate buffer for 2-3 h at 37 C.
7. Anther pieces were rinsed in citrate buffer, placed in dd H2O for 15 min then transferred to a microscope slide in a drop of fixative.
8. After maceration the cells were spread and allowed to dry for 2 h and placed in 100 % ethanol overnight.
9. Slides were dried for 1 h followed by staining with propionocarmine (wet mounted) or C-banded (Jewell et al., The Maize Handbook, pp. 484-93) followed by mounting in Pro-Texx mounting media (America Scientific Products M7635-1).

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