The following protocol is for onions and rye, but also should be applicable to other plant species. In Allium cepa and Secale cereale, the cell cycle time at 25°C is approximately 12 h. Therefore, treating root tips for 24 h in a medium containing BrdU results in a sufficient number of metaphases where chromosomes show incorporation of BrdU in three out of four DNA polynucleotid strands, and permitting differential staining of sister chromatids after FPG-staining. On the other hand, incubation for 12 h (7 h is adequate) in the presence of BrdU, followed by a 17 h treatment with Thd and Urd, results in chromosomes that contain only one BrdU-substituted DNA strand in the pair of sister chromatids, also permitting the differential staining of sister chromatids. Because the cell cycle time is species-specific and also depends on the temperature, treatment times for other species and temperatures need to be adjusted accordingly.
- Germinate seeds on moist filter paper in petri dishes at 20°C.
- Transfer 15 seedlings to large petri dishes (> 20 cm) when root tips are about 1-1.5-cm long, containing 125 ml freshly-prepared treatment medium. The treatment medium consists of 10-4 M BrdU, 5 X 10-8 M FdU, and 10-6 M Urd, dissolved in tap water. Incubate root tips for 7 h in the dark at 25°C (FdU is added because it suppresses the incorporation of Thd into DNA by inhibiting the synthesis of thymidine; uridine is added to avoid negative effects of FdU on RNA synthesis).
- Transfer root tips to either 125 ml fresh medium of the same composition and incubate for 17 h in the dark at 25°C; or to 125 ml freshly-prepared medium consisting of 10-4 M Thd and 10-6 M Urd dissolved in tap water; and incubate for 17 h in the dark at 25°C.
- Add colchicine (final concentration 0.05 %) for the last 3 h.
- Fix in Carnoy's I at least overnight at 5°C.
- Transfer root tips to 45 % acetic acid for 2-3 min, or digest with pectinase/cellulase or cytase to soften of the tissue.
- Make squash preparations in 45 % acetic acid. Check quality of spreading under phase contrast and stain acoording to the FPG-technique described below.
- Remove cover slips with dry ice.
- Pass slides through a 96 %, 70 %, 50 %, and 30 % ethanol series, then distilled water, and finally air dry.
- Treat slides for 1 h with 0.01 % ribonuclease A (Sigma R-4875, from bovine pancreas) dissolved in 0.5 X SSC.
- Rinse briefly in 0.5 X SSC.
- Stain for 30 min with the fluorochrome Hoechst 33258 (H 33255, bisbenzimide, ICN Biomedicals Inc. #190304) (1 mg H 33258 dissolved in 1 ml ethanol, add 0.1 ml of this solution to 200 ml 0.5 X SSC).
- Rinse briefly in 0.5 X SSC.
- Cover slides with 0.5 X SSC and expose to UV light (Osram HNS, 30W, 254 nm) for 1 h.
- Incubate in 0.5 X SSC at 55°C in a water bath for 1 h.
- Stain in 3 % Giemsa (Merck 3204) in Soerensen phosphate buffer pH 6.8.
Soerensen's buffer Stock Stain Part A sodium phosphate dibasic (Na2HOP4) 9.47 g / L H2O 58 mL Part B potassium dihydrogen phosphate (KH2PO4) 9.07 g / L H2O 42 mL
- Rinse briefly in 0.5 X SSC and air dry (slides can be made permanent by soaking in xylene and mount in Euparal or Permount).
- 10-4 M BrdU (5-bromo-2'-deoxyuridine, Sigma B-5002) : 30.7 mg/liter distilled water.
- 10-4 M Thd (2'-deoxythymidine, Sigma T-5018) : 24.2 mg/liter distilled water.
- 10-6 M Urd (uridine, Sigma U-3750) : stock solution is 1.2 mg/100 ml distilled water; add 20 ml stock solution per liter distilled water.
- 5 X 10-8 M FdU (5-fluoro-2'-deoxyuridine, Sigma F-0503) : stock solution is 1.2 mg/100 ml distilled water; add 1 ml stock solution per liter distilled water.
- 0.5 X SSC (prepare a 20 X SSC stock solution that is 88.2 g tri-sodium-citrate-2-hydrate (Na3C6H5O7·2H2O) plus 175.3 g sodium chloride, NaCl/liter distilled water. This solution can be stored for several months; dilute 1:39 with distilled water before use for a 0.5 X SSC treatment solution.