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Kansas State University
Department of Plant Pathology

4024 Throckmorton
1712 Claflin Road
Manhattan, KS 66506-5502
785-532-6176
785-532-5692 fax
wgrc@k-state.edu

Wheat Genetics Resource Center
Kansas Wheat Innovation Center

1990 Kimball Ave
Manhattan, KS 66502
785-320-4383

FLUORESCENT PLUS GIEMSA (FPG) TECHNIQUE

The following protocol is for onions and rye, but also should be applicable to other plant species. InAllium cepaandSecale 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 1xbet online games login 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, 1xbet online games login 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 1xbet online games login freshly-prepared treatment medium. The treatment medium consists of 10-4M BrdU, 5 X 10-8M FdU, and 10-6M Urd, dissolved in tap 1xbet online games login . 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 1xbet online games login fresh medium of the same composition and incubate for 17 h in the dark at 25°C; or to 125 1xbet online games login freshly-prepared medium consisting of 10-4M Thd and 10-6M Urd dissolved in tap 1xbet online games login ; 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 in1xbet bestat 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 1xbet online games login , 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 1xbet online games login .
    11. Rinse briefly in 0.5 X 1xbet online games login .
    12. Stain for 30 min with the fluorochrome Hoechst 33258 (H 33255, bisbenzimide, ICN Biomedicals Inc. #190304) (1 mg H 33258 dissolved in 1 1xbet online games login ethanol, add 0.1 1xbet online games login of this solution to 200 1xbet online games login 0.5 X SSC).
    13. Rinse briefly in 0.5 X 1xbet online games login .
    14. Cover slides with 0.5 X 1xbet online games login 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 1xbet online games login bath for 1 h.
    16. Stain in 3 % Giemsa (Merck 3204) in Soerensen phosphate buffer pH 6.8.
Soerensen's buffer
StockStain
Part Asodium phosphate dibasic (Na2HOP4)9.47 g / L H2O 58 1xbet online games login
Part Bpotassium dihydrogen phosphate (KH2PO4)9.07 g / L H2O
42 1xbet online games login
  1. Rinse briefly in 0.5 X 1xbet online games login and air dry (slides can be made permanent by soaking in xylene and mount in Euparal or Permount).

Solutions

  • 10-4M BrdU (5-bromo-2'-deoxyuridine, Sigma B-5002) : 30.7 mg/L distilled 1xbet online games login .
  • 10-4M Thd (2'-deoxythymidine, Sigma T-5018) : 24.2 mg/L distilled 1xbet online games login .
  • 10-6M Urd (uridine, Sigma U-3750) : stock solution is 1.2 mg/100 mL distilled 1xbet online games login ; add 20 mL stock solution per liter distilled 1xbet online games login .
  • 5 X 10-8M FdU (5-fluoro-2'-deoxyuridine, Sigma F-0503) : stock solution is 1.2 mg/100 mL distilled 1xbet online games login ; add 1 mL stock solution per liter distilled 1xbet online games login .
  • 0.5 X 1xbet online games login (prepare a 20 X 1xbet online games login stock solution that is 88.2 g tri-sodium-citrate-2-hydrate (Na3C6H5O7·2H2O) plus 175.3 g sodium chloride, NaCl/L distilled 1xbet online games login . This solution can be stored for several months; dilute 1:39 with distilled 1xbet online games login before use for a 0.5 X SSC treatment solution.