Isolating and Measuring the Growth and Morphology of Pro-embryogenic Masses in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae)

Materials and Reagents

1. Falcon tube rack (Kasvi, catalog number: K30-1552 )
   
2. 12-well cell culture plates - disposable (TPP, catalog number: 92012 )
   
3. Manual pipette 200 µl tips (Corning, Axygen^®, catalog number: T-200-Y )
   
4. Manual pipette 1,000 µl tips (Corning, Axygen^®, catalog number: T-1000-B )
   
5. Aluminum foil
   
6. Glass slides (Kasvi, catalog number: K5-7101 )
   
7. Cover slips (Kasvi, catalog number: K5-2450 )
   
8. Falcon tubes, 50 ml (Kasvi, catalog number: K19-0050 )
   
9. Embryogenic suspension cultures of A. angustifolia, induced according to the methodology established by Steiner et al. (2005)
   
10. Cellulase (Sigma-Aldrich, catalog number: 22178 )
    
11. Potassium nitrate (KNO₃) (Sigma-Aldrich, catalog number: V000944 )
    
12. Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: V000199 )
    
13. Magnesium sulfate heptahydrate (MgSO₄·7H₂O) (Sigma-Aldrich, catalog number: V001861 )
    
14. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: V000104 )
    
15. Potassium dihydrogen phosphate (KH₂PO₄) (EMD Millipore, catalog number: 104873 )
    
16. MnSO₄·H₂O (Labsynth, catalog number: S2036 )
    
17. ZnSO₄·7H₂O (Labsynth, catalog number: S1072 )
    
18. Boric acid (H₃BO₃) (Sigma-Aldrich, catalog number: 31146 )
    
19. Potassium iodide (Kl) (Sigma-Aldrich, catalog number: V000130 )
    
20. Cobalt(II) chloride hexahydrate (CoCl₂·6H₂O) (Sigma-Aldrich, catalog number: V000213 )
    
21. CuSO₄·5H₂O (Labsynth, catalog number: S1054 )
    
22. Sodium molybdate dihydrate (Na₂MoO₄·2H₂O) (Sigma-Aldrich, catalog number: M1651 )
    
23. FeSO₄·7H₂O (Labsynth, catalog number: S1057 )
    
24. Na₂EDTA (Labsynth, catalog number: E2005 )
    
25. Myo-inositol (Sigma-Aldrich, catalog number: I17508 )
    
26. Nicotinic acid (Labsynth, catalog number: A1043 )
    
27. Pyridoxine (Sigma-Aldrich, catalog number: P9755 )
    
28. Thiamine (Sigma-Aldrich, catalog number: T4625 )
    
29. L-glutamine (Labsynth, catalog number: G1011 )
    
30. Sucrose (Labsynth, catalog number: 2731 )
    
31. MSG culture medium (see Recipes)
    

Equipment

1. Cell dissociation sieve-screens, 150 mesh (Sigma-Aldrich, catalog number: CD1-1KT ) sterilized by autoclave (121 °C, 30 min)
   
2. Chamber flow (or its equivalent) (Pachame, model: PA 220 )
   
3. Adapted glass Erlenmeyer flasks (custom-made) (Figure 1), sterilized by autoclave (121 °C, 30 min). This adjustment to the flask can be performed by a company that produces laboratory glassware, fusing a glass tube to an Erlenmeyer flask
   
4. Ruler
   
5. Orbital shaker (or its equivalent) (Cientec Equipamentos para Laboratório, model: CT-165 )
   
6. Analytical balance (or its equivalent) (Shimadzu, model: BL3200H )
   
7. Spatula sterilized by autoclave (121 °C, 30 min) (VWR, catalog number: 231-2233 )
   
8. AxioPlan light microscope (Carl Zeiss, model: AxioPlan )
   
9. Manual pipettes (or their equivalent) (Eppendorf, catalog numbers: 3120000062 and 3120000054 )
   
10. Forced air circulation drying oven (or its equivalent) (Ethik Technology, model: 420-6D )
    
11. AxioCam MRC5 digital camera (Carl Zeiss, model: AxioCam )
    
12. Desktop computer
    
    
    Figure 1. Adapted Erlenmeyer flasks (100 and 50 ml) used in SCV analyses. For an adapted Erlenmeyer flask of 100 ml capacity, we used 25 ml of culture medium and 500 mg of fresh cells. For an adapted Erlenmeyer flask with 50 ml capacity, we used 10 ml of culture medium and 200 mg of fresh cells, with a ratio of 20 mg fresh cells to 1 ml of culture medium.
    

Software

1. AxioVision Rel. 4.8 software (Carl Zeiss, AxioVision)

Procedure