Materials and Reagents

1. Grapevine berries from greenhouse-grown fruiting-cuttings of Vitis vinifera L. cv. Cabernet Sauvignon at various developmental stages (e.g. pea size, green berry, veraison, or later stages). The fruiting-cuttings (i.e. only one primary shoot axis with a single cluster per plant) were prepared as described in Mullins and Rajasekaran (1981) and grown in a naturally illuminated and semi-regulated greenhouse (mean seasonal temperature amplitude 20-35 °C) with fungicide treatments every two weeks (Figure 1).
   Note: Efficient fungicide treatments are essential to maximally exclude pathogen infection to grape berries and minimize the sterilization step for the in vitro culture. For example, we continuously provide sulfur contained in sulfur diffusion hotbox (Nivola Sulphur fl 220 v 3 m, Figure 1B) in the greenhouse and treated the vines with armicarb^® at 5 g/L every two weeks.
   
   
   Figure 1. Fruit-bearing cuttings grown in a semi-controlled greenhouse (A) and the sulfur diffusion hotbox (B)
   
   
2. 70% ethanol
   
3. NaClO with available chlorine 2% (Sigma-Aldrich, catalog number: 425044-1L )
   
4. MS (Murashige & Skoog) medium (Duchefa Biochemie, catalog number: M 0221 )
   
5. Sucrose (Duchefa Biochemie, catalog number: S0809 )
   
6. N-Z-Amine A (Sigma-Aldrich, catalog number: C7290 )
   
7. Vitamines
   Myo-inositol 100 mg/L (Sigma-Aldrich, catalog number: I5125 )
   Nicotinic acid 1 mg/L (Sigma-Aldrich, catalog number: N0765 )
   Pantothenic acid 1 mg/L (Duchefa Biochemie, catalog number: C0604 )
   Biotin 0.01 mg/L (Sigma-Aldrich, catalog number: B4639 )
   Pyridoxine HCl 1 mg/L (Sigma-Aldrich, catalog number: P9755 )
   Thiamine HCl 1 mg/L (Sigma-Aldrich, catalog number: T4625 )
   
8. 0.5 M NaOH
   
9. Agar (Kalys, catalog number: HP 696 )
   
10. EDTA (Sigma-Aldrich, catalog number: E5134 )
    
11. Sterile water
    
12. MS medium with sucrose and vitamins (see Recipes)
    
13. 200x vitamin (see Recipes)
    
14. 20 mM EDTA (see Recipes)
    

Equipment

1. Laminar flow cabinet (Steril-Helios, Figure 2A)
   
2. Shaker (Dragon lab, sk-330-pro, Figure 2A)
   
3. Autoclave machine
   
4. Growth room with constant temperature of 26 ± 0.5 °C, light period 16 h/8 h day/night, and light ~50 μmol m^-2 s^-1 (Figure 4H)
   
5. Sterilized 6-well plates (Dutscher, catalog number: 353046 , Figure 5B)
   
6. Sterilized plastic boxes with filter (Dutscher, catalog number: E 1650 , Figures 3F, 4G, and 5A)
   
7. Tip plate (Figure 3A)
   
8. Polystyrene (Figure 3C)
   
9. Culture dish (145/20 mm, Greiner Bio-One GmbH, Figure 4E)
   
10. Sterilization solution container (Figure 2C-D)
    
11. Colander, forceps, scissors, and blade (Figure 2B)
    
    
    Figure 2. Equipment used in grape berry culture. A. Laminar flow cabinet with a shaker. B. colander, forceps, scissors, and blade. C. solution container, culture boxes, and small materials just after autoclave. D. Sterilized containers for ethanol and NaClO.
    

Procedure