Procedure for Giemsa staining Zea mays pachytene chromosomes

Giemsa dye, when used according to modified C-band techniques, gives better resolution of the pachytene chromosome knobs of Zea mays than does acetocarmine. With Giemsa the knobs seem to have a more distinct shape and structure than with acetocarmine. These shapes are consistent from cell to cell. The problems of overlapping chromosomes, chromomeres, etc. were overcome by the Giemsa method. Giemsa staining permits heterozygous vs. homozygous knob recognition, a distinction which is difficult using acetocarmine. Also noted was the absence of the nucleolus when using the Giemsa technique.

This method gives better resolution of knobs and their structure yet is still relatively fast and results in a permanent slide. This technique is most useful when dealing with corn lines with characteristics which result in poor acetocarmine preparations, e.g., lines with large numbers of knobs, fused knobs, poor chromosome spreading, etc.

The florets were collected and fixed in cold (4oC) 3:1, Farmers Solution of 3 absolute alcohol:1 glacial acetic acid for 24 hours and stored in 70% alcohol until stained. The squashing and staining series is as follows:

1. Hydrate the florets in distilled water for 30 minutes.

2. Remove the anthers and place on a subbed slide (subbing agent: 5 g gelatin, .1 g chrome alum in 100 ml distilled water.

3. Squash the anthers with a coverslip in distilled water.

4. Place slide on slide warmer at 37 C for 15 minutes. Remove coverslip and return to the slide warmer until dry.

5. Immerse slides in a saturated solution of barium hydroxide (pH 13.5) for 170 minutes.

6. Remove slides and rinse 3 times in distilled water.

7. Place in Giemsa stain (2% Gurr improved Giemsa stain R66 in .1 M phosphate buffer, pH 6.8) for 7 minutes.

8. Rinse twice in distilled water, air dry and mount coverslips over permount.

A. L. Rayburn III, J. D. Smith, and H. J. Price
Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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