Kansas State University

FLUORESCENT PLUS GIEMSA (FPG) TECHNIQUE

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.

1. Germinate seeds on moist filter paper in petri dishes at 20°C.
2. 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).
3. 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.
4. Add colchicine (final concentration 0.05 %) for the last 3 h.
5. Fix in Carnoy's I at least overnight at 5°C.
6. Transfer root tips to 45 % acetic acid for 2–3 min, or digest with pectinase/cellulase or cytase to soften of the tissue.
7. Make squash preparations in 45 % acetic acid. Check quality of spreading under phase contrast and stain acoording to the FPG-technique described below.
8. Remove cover slips with dry ice.
9. Pass slides through a 96 %, 70 %, 50 %, and 30 % ethanol series, then distilled water, and finally air dry.
10. Treat slides for 1 h with 0.01 % ribonuclease A (Sigma R-4875, from bovine pancreas) dissolved in 0.5 X SSC.
11. Rinse briefly in 0.5 X SSC.
12. 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).
13. Rinse briefly in 0.5 X SSC.
14. Cover slides with 0.5 X SSC and expose to UV light (Osram HNS, 30W, 254 nm) for 1 h.
15. Incubate in 0.5 X SSC at 55°C in a water bath for 1 h.
16. 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

1. 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).

Solutions

• 10^-4 M BrdU (5-bromo-2'-deoxyuridine, Sigma B-5002) : 30.7 mg/L distilled water.
• 10^-4 M Thd (2'-deoxythymidine, Sigma T-5018) : 24.2 mg/L 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/L 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.