Kansas State University

AG–NOR STAINING

1. Germinate seeds on moist filter paper in petri dishes at 20°C.
2. Collect root tips when they are about 1-cm long and pretreat with ice water for 24–48 h.
3. Fix in Carnoy's I.
4. Squash in 45% acetic acid.
5. Remove cover slips by placing on dry ice until frozen and air dry.
6. Drop fixative onto the preparations and flame dry.
7. Dissolve 1 g of silver nitrate (AgNO₃) in 1 mL of 0.02 g sodium citrate (C₆H₅Na₃O₇·2 H₂O) per 500 mL distilled water, adjusted to pH 3.0 with formic acid.
8. Add 1 or 2 drops of the above solution (needs to be prepared fresh each time) onto the slides and place a cover slip over the preparation.
9. Incubate slides in a moist chamber at 55–60°C for 30 min to several h. Monitor the silver precipitation under the microscope.
10. When chromosomes are stained brown to yellow and Ag-NOR-bands appear black, staining is complete. Rinse slides thoroughly in distilled water.
11. Soak in xylene and mount in Depex.