Materials and Reagents

1. Dry oilseed rape (Brassica napus) seed samples
   
2. KCl (Sigma-Aldrich, catalog number: 31248-500G )
   
3. Methanolic-HCl (Sigma-Aldrich, catalog number: 33050-U )
   
4. Hexane (Sigma-Aldrich, catalog number: 10549380 )
   
5. Toluene (Sigma-Aldrich, catalog number: 650579 )
   
6. Pentadecanoin (Nu-Chek, catalog number: N-15-A )
   
7. Heptadecanoin (Nu-Check, catalog number: N-17-A )
   
8. Fatty acid standard (Supelco 37 component FAME mix) (Sigma-Aldrich, Supelco, catalog number: 47885-U )
   
9. 6 ml screw cap glass vial (Schott AG, VWR, catalog number: 391-0140 ).
   
10. Organic solvent resistant caps for 6 ml screw cap vials (SKS-Science, catalog number: 240409 ).
    
11. GC column (BPX70) (SGE Analytical Science, catalog number: 0 54600 )
    
12. 98% ethanol (Sigma-Aldrich, catalog number: 459844 )
    
13. Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S5881 )
    
14. 80% ethanol (v/v) diluted in distilled water
    
15. 50% ethanol (v/v) diluted in distilled water
    
16. 0.1 M NaOH (4 g/L) in distilled water
    
17. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 484016 )
    
18. HEPES (Sigma-Aldrich, catalog number: H3375 )
    
19. Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266 )
    
20. Adenosine-5'-triphosphate (ATP) (Roche Diagnostics, catalog number: 10 127 531 001 )
    
21. Sucrose (Sigma-Aldrich, catalog number: 84097 )
    
22. Nicotinamide adenine dinucleotide phosphate (NADP) (Roche Diagnostics, catalog number: 10 240 35 4001 )
    
23. Glucose-6-Phosphate Dehydrogenase grade II (G6PDHII) (Roche Diagnostics, catalog number: 10 737 232 001 ) (80 μl, centrifuge and resuspend pellet in 200 μl of Buffer B)
    
24. Hexokinase (HK) (Roche Diagnostics, catalog number: 11 426 362 001 ) (120 μl, centrifuge and resuspend pellet in 200 μl of Buffer B)
    
25. Phosphoglucose isomerase (PGI) (Roche Diagnostics, catalog number: 10 128 139 001 ) (60 μl, centrifuge and resuspend pellet in 200 μl of Buffer B)
    
26. Invertase (INV) (Sigma-Aldrich, catalog number: I4504 ) (as much as possible to dissolve in 200 μl of Buffer B)
    
27. 60 mg/ml ATP in distilled water
    
28. 36 mg/ml NADP in distilled water
    
29. Buffer A (see Recipes)
    
30. Buffer B (see Recipes)
    
31. Reagent mix 1 (see Recipes)

Equipment

1. Gas chromatograph (GC) (Agilent, model: 7890A )
   
2. MinispecMQ20 (Bruker Corporation)
   
3. Robotic sample handling system (Rohasys BV)
   
4. Centrifuges
   For the "Sucrose and protein content determination" procedure: Eppendorf Centrifuge 5415 R (Eppendorf, catalog number: 022621408 )
   For the "Determination of seed FA content and composition" procedure: Eppendorf Centrifuge 5810 R (Eppendorf, catalog number: 5811 000.010 )
   
5. Vortex-Genie 2 Shaker (Scientific Industries, Cole-Parmer, catalog number: UY-04724-05 )
   
6. 2 ml Eppendorf tube (Sigma-Aldrich, catalog number: T2795 but not important )
   
7. Microplate 96 wells (Sigma-Aldrich, catalog number: M4561 but not important )

Procedure