Materials and Reagents

1. A. thaliana seeds (background ecotype and mutant or transgenic line to be analysed)
   Note: Seeds should be collected at the same time to avoid problems of differential germination rate.
   
2. Murashige and Skoog (MS) media including vitamins and MES buffer (Duchefa M0255.0050)
   
3. Sucrose
   
4. Potassium hydroxide (KOH)
   
5. Bacto Agar (BD Bioscience, catalog number: 214010 )
   
6. Ethanol
   
7. Tween-20
   
8. Brassinazole (TCI America, catalog number: B2829 )
   Note: Stock solution is prepared at 2 mM in DMSO and kept at -20 °C. The appropriate volume of BRZ stock solution should be added in the fume hood to autoclaved media. The media should not be too hot (let it cool down to 55-65 °C before adding BRZ). BRZ concentration should range from 0.1 to 2 µM. We routinely use 0.1, 0.2, 0.4, 0.8 and 1.6 µM as a starting point.
   
9. Growth media (see Recipes)
   
10. Sterilization solution (see Recipes)

Equipment

1. Plant growth chamber
   
2. Sterile fume hood
   
3. Autoclave
   
4. Round (9 x 9 cm) and square (12 x 12 cm) polystyrene sterile Petri dishes
   

Software

1. ImageJ software

Procedure