Materials and Reagents

1. Pirex^® culture tubes 25 x 150 mm
   
2. Disposable syringes (25 ml)
   
3. 1.5 ml Eppendorf tubes
   
4. Citrus seeds (e.g., from a commercial rootstock)
   
5. Murashige and Skoog (MS) medium (4,302.09 mg L⁻¹) (Duchefa Biochemie, catalog number: M0221 )
   
6. Sucrose (30 g L⁻¹) (any supplier)
   
7. Sterile MiliQ-water
   
8. Agar (European Bacteriological Agar) (Conda, Pronadisa, catalog number: 1800 )
   
9. Tween^® 20 (0.1% v/v) (Sigma-Aldrich, catalog number: P2287 )
   
10. Myo-inositol (100 mg L⁻¹) (Duchefa Biochemie, catalog number: I0609 )
    
11. Pyridoxine-HCl (1.0 mg L⁻¹) (Duchefa Biochemie, catalog number: P0612 )
    
12. Thiamine-HCl (0.2 mg L⁻¹) (Duchefa Biochemie, catalog number: T0614 )
    
13. Nicotinic acid (0.5 mg L⁻¹) (PANREAC QUÍMICA, catalog number: A0963 )
    
14. Glycine (0.2 mg L⁻¹) (PANREAC QUÍMICA, catalog number: 141340 )
    
15. Mix A (see Recipes)
    
16. Sodium hypochlorite solution (50.0%, v/v) (see Recipes)
    
17. NaOH (0.1 N) (any supplier) (see Recipes)
    

Equipment

1. Pirex^® media solution bottles
   
2. Autoclave (Raypa, model: Steam Sterilizer AES-75 )
   
3. Laminar flow hood (Bioquell, ASTEC MICROFLOW, model: Microflow Laminar Flow Workstation M50546 )
   
4. Hooked tweezers
   
5. Growth chamber (Snijders Scientific, model: Economic Lux Climate Chamber )
   
6. Long-handled scissors with curved tip
   
7. pH meter (HACH LANGE SPAIN, model: pH meter Basic 20 )
   

Procedure