University of Illinois

Chromosome knob numbers of somatic cells of five inbreds

--Y. Wan and J. M. Widholm

Maize chromosome knobs consist of constitutive heterochromatin which remains condensed throughout the mitotic cell cycle. The knob compositions can be determined by pachytene analysis. However, the position of heterochromatin can be detected by mitotic chromosome C-banding techniques (Hadlaczky and Kalman, Heredity 35:371-374; Rayburn et al., Amer. J. Bot. 72:1610-1617). The pachytene knobs, for the most part, are the same heterochromatin detected by C-banding.

To rapidly evaluate the effectiveness of some antimicrotubule agents in inducing chromosome doubling of anther-derived callus (Wan et al., Theor. Appl. Genet. 81:205-211), we tried to determine the ploidy level of the callus cells by counting the knob numbers of interphase or nondividing cells. There could be at least two advantages to this approach: 1) the knobs should be visible in these cells; and 2) interphase and nondividing cells are more abundant than the cells at other stages. Although we later used flow cytometric analysis to determine the effectiveness of the chromosome doubling treatments, we did determine the knob numbers of root tip cells of five inbreds (B73, FR16, H99, Mo17 and Pa91) when we tested the approach. We felt that the approach may be useful for determining the ploidy level in some cases.

Root tips were collected from germinating seeds and were treated in cold water (4 C) for 24h and were fixed in a solution of 3:1 ethanol:glacial acetic acid for another 24h. After being stained with 1% acetocarmine and squashed on a glass slide, root tip meristem cells were examined under a light microscope. The visible heterochromatin regions in well spread interphase or nondividing cells were counted.

As shown in Figure 1, dark stained heterochromatic regions could be clearly seen in interphase root tip cells. Mo17 had 4 such regions and FR16 had 8, whereas H99, Pa91 and B73 all had 12. Since in mitotic interphase or nondividing cells it is impossible to distinguish the heterochromatic knobs from the heterochromatic nucleolus organizing region (NOR) of chromosome 6, the heterochromatic regions we observed include the 2 NORs. Thus Mo17 should have 2 knobs in the diploid somatic cells, FR16 has 6, and H99, Pa91 and B73 should have 10. Previously reported knob numbers in some inbreds were determined by pachytene analysis (Chughtai and Steffensen, Maydica 32:171-187), where the NORs could be recognized and be excluded. Also in pachytene cells the homologous chromosomes are paired and two homologous knobs appear as one. The reported knob numbers of Mo17 and B73 were 1 and 4, respectively (Chughtai and Steffensen, Maydica 32:171-187), so the 2 knobs of Mo17 we observed is consistent with the reported 1 knob in the pachytene cell. However the 10 knobs of B73 we found would be 2 more than the expected number. The difference could be caused by counting 2 chromomeres as small knobs. The knob numbers of the other three inbred lines have not been presented previously.

Figure 1. Heterochromatic regions including knobs and two NORs observed in the root tip meristem cells of maize inbreds. a) Mo17, 4 such regions; b) FR16, 8; c) Pa91, 12. Bar represents 5µm.

Among these 5 inbred lines, the knobs of Mo17 were relatively larger, so were more apparent on the nuclear background (Fig. 1). The knobs of FR16 were also easily distinguished from the diffuse euchromatin (Fig. 1). We found that the knobs of B73, H99 and Pa91 are smaller compared with those of Mo17 and FR16.

The results indicate that maize heterochromatin knobs are visible in interphase or nondividing somatic cells using a simple method, especially for the strains with few or larger knobs such as Mo17 and FR16. The dark stained knobs and NORs are also visible in well spread maize callus cells using the same method, so the ploidy level of the callus cells could be determined by counting the knob number.

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