DNA Damage Sensitivity Assays with Arabidopsis Seedlings

Materials and Reagents

1. Arabidopsis thaliana seeds [Wild type and T-DNA insertion lines of arp6-3, swc6-1 and sensitive ku70-2 (Rosa et al., 2013) are used as examples. All mutants are in the Columbia-0 background.]
   
2. Sodium hypochlorite (Sigma-Aldrich, catalog number: 425044 )
   
3. Tween-80 (Sigma-Aldrich, catalog number: 4780 )
   
4. Sterile H₂O
   
5. Solid growth medium
   
6. Liquid plant growth medium (same as solid growth media but without agar)
   
7. Hydroxyurea (Sigma-Aldrich, catalog number: H8627 )
   
8. 70% ethanol
   
9. Seed sterilization solution (see Recipes)
   
10. Bleocin (commercial name for bleomycin) (EMD Millipore, catalog number: 203408 ) (see Recipes)
    
11. Mitomycin C (Duchefa Biochemie BV, catalog number: M0133 ) (see Recipes)
    

Equipment

1. Sterile hood, preferably a biological safety cabinet to avoid exposure to the genotoxins
   
2. Bench top block shaker (e.g. Eppendorf, Thermomixer^®)
   
3. UV crosslinker (254-nm UV light bulbs, 15 watts each) (Stratagene, model: Stratalinker 2400 )
   
4. Gamma-irradiation source (Nordion, model: Co-60- Gamma-cell 220 )
   
5. Forceps
   
6. Box or aluminum foil
   
7. Petri dishes for plant culture with solid growth medium (see Notes) (round 200 x 15 mm and 55 x 15 mm, and square 100 x 100 x 15 mm)
   
8. 1.5 ml Eppendorf tubes
   
9. Plant growth facilities with 16-h-light/8-h-dark cycles, at 21 °C
   

Procedure