Appendix - Protocol for Staining Chromosomes from Grass Root Tips
This technique is modified from Cytogenetics of the Festuca-Lolium Complex: Relevance to Breeding (Jauhar, 1993).
Plants are best grown in light, well-drained soil mix, or better still in soil-less mix (1 part peat moss, 1 part perlite, and 1 part vermiculite).
Fresh, plump, about 1-cm-long root tips are excised and pretreated in 2 g/L (0.2%) aqueous solution of colchicine for 3 h at room temperature (23°C). If this is unavailable, excised root tips can be pretreated with prechilled, distilled water kept in a refrigerator at 1°C for 16 to 24 h. We have found that using prechilled water gives acceptable results.
The pretreated root tips are fixed in freshly prepared acetic alcohol (3 parts 95% ethanol + 1 part glacial acetic acid) for 3 to 14 d in a refrigerator. The quality of staining may deteriorate if the root tips are stored for more than 3 wk.
The fixed root tips are briefly washed in distilled water, hydrolyzed in preheated 1 N HCl at 60°C for 12 to 16 min in an oven, and stained with warm leuco-basic fuchsin (Feulgen stain) at about 40°C in the dark for about 1 h. Staining for longer periods of time (2-4 h) was found to sometimes be helpful.
The stained, dark-purple tips (~2 mm) are cut off on a slide and squashed well in 1% acetocarmine or 45% acetic acid. We have found that squashing tips with a modified, slightly rusty probe (flatten tip of probe with a hammer) works well. The rust may enhance chromosome staining through the addition of iron. The acetocarmine-root tip mixture should appear "cloudy or milky" with no large unmacerated bits remaining.
Gently add a cover slip to the slide. Solution should spread out nicely and evenly.
Briefly heat by wafting over a flame.
Place slide between two pieces of filter paper and thumb squash. It is important to be forceful when applying pressure and to do so only once. Do not cause cover slip to "slide" as this will likely destroy intact chromosomes, or at least, to disrupt desirable chromosome spreads.
Examine slide at 100´ under a compound microscope.
1% Acetocarmine (Jahier et al., 1996)
- 10 g carmine 1 L acetic water (45% acetic acid)
- In a large Erlenmeyer flask (2 L), boil the acetic water.
- Pour the carmine into it and let solution simmer for 5 min.
- Let settle until it cools.
- Filter solution.
- Store in dark glass container.
Feulgen Stain (Brooks et al., 1963)
- Dissolve 1 g of basic fuchsin in 400 mL boiling distilled water.
- Cool and add 1.0 mL thionyl chloride (SOCl2) in a fume hood.
- Let the solution stand for 12 to 24 h to decolorize.
- Shake the solution for about 1 min.
- Filter rapidly through coarse filter paper.
- Store in dark glass bottle in refrigerator.
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