Materials and Reagents

1. Plastic 12-well plates (Denmark, Nunc)
   Note: One for the absorption step, one for the desorption step for each treatment (plant type or culture condition).
   
2. Plastic 20 ml vials for radioactivity measurement (Ratiolab GmbH, Dreieich)
   Note: You will need one vial per plant, or 2 vials per plant if you want to quantify 33P in both roots and leaves. The vials should be numbered from 1 to N before you start the experiment. They also should be placed in order in appropriate racks (PerkinElmer) adapted to the beta counter.
   
3. Tips
   
4. Young in vitro plantlets
   
5. MES hydrate (Sigma-Aldrich, catalog number: M8250 )
   
6. CaCl₂ (Sigma-Aldrich)
   
7. ³³P-PO₄ 5 mCi/ml (185 MBq/ml, 1.48-5.84 TBq/mg, >99% isotopically pure, less than 0.5 μM Pi) (PerkinElmer)
   
8. Scintillation cocktail (PerkinElmer, Ultima Gold^TM)
   
9. MgSO₄
   
10. NH₄NO₃
    
11. KNO₃
    
12. NaH₂PO₄
    
13. Kl
    
14. FeCl₂
    
15. MnSO₄
    
16. ZnSO₄
    
17. CuSO₄
    
18. CoCl₂
    
19. Na₂MoO₄
    
20. Thiamine
    
21. Pyridoxine
    
22. Nicotinic acid
    
23. Inositol
    
24. Sucrose
    
25. Agar
    
26. MS/10 medium (see Recipes)
    
27. Stock solution (see Recipes)
    
28. 1 M KH₂PO₄ (Sigma-Aldrich) (see Recipes)
    
29. Incubation medium (see Recipes)
    
30. Desorption medium (see Recipes)
    

Equipment

1. Liquid scintillation counter (PerkinElmer, Packard Instrument Company, model: TRI-CARB )
   
2. Ice-containing large boxes for the desorption step (all 12-well plates will be placed horizontally on ice for 2 h)
   
3. Tweezers for handling plantlets
   
4. Micropipets
   
5. Shield for protection against radiation (Plexiglas)
   
6. Scanner or camera (Epson America, model: Perfection V850Pro or Canon, model: Powershot SX130 ), respectively but other devices from other manufacturers could suit perfectly
   

Software

1. ImageJ version 1.46r with NeuronJ plugin (http://imagej.nih.gov/ij)
   
2. PRISM 6.0 software (GraphPad)
   

Procedure