Cotton Ovules Culture and Analysis

Materials and Reagents

1. Flowers (-1 to 1 DPA of flowers are easy to manipulate)
   
2. 0.1% (w/v) HgCl₂ (caution: HgCl₂ is very dangerous. Please select an advantageous reagent to sterilize according to your lab condition. Otherwise 75% ethanol and N,N'-Dicyclohexylcarbodiimide (DCCS) could be the alternatives)
   
3. Toluidine blue O (Sigma-Aldrich, catalog number: T3260 )
   
4. Glacial acetic acid-ethanol-water (10:95:5, v/v)
   
5. Vitamins:
   VB1(Vitamin B1, Thiamine) (Sigma-Aldrich, catalog number: T3902 )
   VB6 (Vitamin B6, Pyridoxine) (Sigma-Aldrich, catalog number: P8666 )
   VB3 (Vitamin B3, Nicotinic acid) (Sigma-Aldrich, catalog number: N0765 )
   
6. Inositol (Sigma-Aldrich, catalog number: I3011 )
   
7. IAA (Sigma-Aldrich, catalog number: I2886 )
   
8. GA₃ (Sigma-Aldrich, catalog number: G7645 )
   
9. KH₂PO₄ (Sigma-Aldrich)
   
10. CaCl₂^.2H₂O (Sigma-Aldrich)
    
11. MgSO₄^.7H₂O (Sigma-Aldrich)
    
12. KNO₃ (Sigma-Aldrich)
    
13. H₃BO₃ (Sigma-Aldrich)
    
14. Na₂MoO₄^.2H₂O (Sigma-Aldrich)
    
15. KI (Sigma-Aldrich)
    
16. CoCl₂^.6H₂O (Sigma-Aldrich)
    
17. MnSO₄^.H₂O (Sigma-Aldrich)
    
18. ZnSO₄^.7H₂O (Sigma-Aldrich)
    
19. CuSO₄^.5H₂O (Sigma-Aldrich)
    
20. FeSO₄^.7H₂O (Sigma-Aldrich)
    
21. Na₂EDTA (Sigma-Aldrich)
    
22. BT medium preparation (see Recipes)
    
23. Hormone preparation (see Recipes)
    
24. Working BT medium preparation (see Recipes)
    

Equipment

1. Erlenmeyer flask (50 ml)
   
2. 1.5 ml microcentrifuge tubes
   
3. Spectrophotometer (Beckman Coulter, model: DU 800 ) or microplate reader (Tecan Trading AG, model: infinite^® M200 )
   
4. Clean bench laminar air-flow-hood with burner (Harbin East Electronic Technology, model: HD-1360 )
   
5. Common plant tissue culture equipment (Measuring cylinder/volumetric flask, Beaker, Weighing machine, Autoclave, pH-meter, Scalpel, Fine forceps)
   

Software

1. ImageJ

Procedure