A root tip squash technique for maize mitotic chromosome spreads was recently published (P. J. Sallee, in Sheridan, ed., Maize for Biological Research, p. 119, 1982). Our protocol, based on Sallee's method, gives a higher frequency of countable figures, primarily because cycloheximide greatly shortens prophase chromosomes. Cycloheximide also allows some visualization of the heterochromatic and knobbed regions of the chromosomes (J. Tlaskal, Stain Tech. 54:313-320, 1979).
1. Germinate, in a 30 C incubator, fungicide-treated kernels in a drainable, nearly covered container of moist, coarse sand until roots are 3-4 cm long. Germination of small or old seed is better in sand than in petri dishes (C. R. Burnham, MNL 49:122, 1975), and metaphases are more frequent in sand-grown root tips from all classes of seed. We use window screen to remove fine grains from river sand. After a few plantings, the sand should be sterilized or replaced.
2. Root tips (1 cm long) are collected in glass vials and prefixed in the following solution for 4 1/2 hours at room temperature: Thoroughly dissolve 7 mg cycloheximide in 100 ml tap water. Warming the water will help dissolve the cycloheximide. Add 4 drops of monobromonaphthalene and mix vigorously by squirting back and forth with an eyedropper. Add one drop of dimethyl sulfoxide (DMSO) and mix again. It is important to add the chemicals in the order specified. It is best to have two labeled eyedroppers, one for monobromonaphthalene and the other for DMSO. For consistent results, use these eyedroppers each time the mixture is prepared. All glass materials can be cleaned by rinsing in acetic acid and water.
3. Pour off the prefixative and replace with glacial acetic acid, cap the vial, and let stand overnight at room temperature. Fixation in acetic acid can be reduced to 1 hour, if necessary.
4. To hydrolyze the tips, pour off the acetic acid and replace with hot (60 C) 1N HCl and incubate at 60 C for 5-10 minutes.
5. Pour off all HCl, rinse in distilled water, and stain in basic fuchsin for 10-15 minutes or until tips become bright purple.
6. To prepare a squash, cut off about 1 mm of the tip of the stained root, place on a slide, and add a drop of propionic orcein. Place a plastic cover slip adjacent to the pool of stain, then cover the root tip with a second plastic cover slip so that one edge rests on the edge of the first slip. Tap the second slip many times with a pencil or dull needle to break up the tip and disperse the cells, then slide the first cover slip out from under the second cover slip and blot excess stain. Place slide between two pieces of folded filter paper and apply moderate thumb pressure to flatten the cells. This squash method, which eliminates the need to treat root tips with an enzyme, is commonly used by wheat cytogeneticists here and abroad.
If it is necessary to keep root tips for more than two days, complete Step 5, decant the basic fushsin solution, add distilled water, cap and refrigerate. Both the distilled water and root will turn purple, but the root tip will remain dark purple. We have noted no deterioration after three weeks of storage, and R. O. Flagg reported (Stain Tech. 36:95-97, 1961) that root tips can be safely stored in the refrigerator for eight months. The usually recommended storage in 70% ethyl alcohol after Step 3 gives, in our experience, blurred, poorly stained chromosomes.
B. K. Kindiger and J. B. Beckett
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