Agrobacterium tumefaciens-mediated Transformation of Walnut (Juglans regia)

Materials and Reagents

1. Biological materials
   1. Juglans regia somatic embryo cultures
      
   2. Disarmed A. tumefaciens strain (e.g. EHA101) harboring binary vector of interest
      For this protocol it is assumed that the vector harbors the nptII selectable marker gene and the uidA scorable marker gene (e.g. pDE00.0201 from Escobar et al., 2002).
   
   
2. Chemicals
   1. Driver Kuniyuki walnut (DKW) medium with vitamins (PhytoTechnology Laboratories^®, catalog number: D2470 )
      
   2. Kanamycin sulfate (PhytoTechnology Laboratories^®, catalog number: K378 )
      
   3. Timentin^® (PhytoTechnology Laboratories^®, catalog number: T869 )
      
   4. 4-Hydroxy-3',5'-dimethoxyacetophenone (Acetosyringone) (PhytoTechnology Laboratories^®, catalog number: A104 )
      
   5. Gelzan^TM CM (Sigma-Aldrich, catalog number: G1910 )
      
   6. SeaKem^® LE agarose (Lonza, catalog number: 50004 )
      
   7. Bacto^® Agar/SuperPure^TM agar (BTS, catalog number: A01PD )
      
   8. Proline (PhytoTechnology Laboratories^®, catalog number: D689 )
      
   9. 5-Bromo-4-chloro-3-indolyl β-D-glucuronide (X-gluc) (Sigma-Aldrich, catalog number: B5285 )
      
   10. 1 M sodium phosphate buffer (pH 7.0)
       
   11. 0.5 M EDTA (pH 8.0) (PhytoTechnology Laboratories^®, catalog number: E 582 )
       
   12. 0.005 M potassium ferricyanide (Sigma-Aldrich, catalog number: 702587 )
       
   13. 0.005 M potassium ferrocyanide (Sigma-Aldrich, catalog number: P3289 )
       
   14. Triton X-100 (Sigma-Aldrich, catalog number: 234729 )
       
   15. Indole-3-butyric acid (IBA) (Sigma-Aldrich, catalog number: B-5386 )
       
   16. 6-Benzylaminopurine (BAP) (Sigma-Aldrich, catalog number: B-3408 )
       
   17. Tryptone (BTS, catalog number: T01PD )
       
   18. Yeast extract (BTS, catalog number: Y02MG )
       
   19. NaCl (Thermo Fisher Scientific, catalog number: S271-3 )
       
   20. Saturated ZnSO₄ or NH₄NO₃ solution
       
   21. 10x PCR buffer (Applied Biosystems)
       
   22. PCR primers
       
   23. dNTPs (Applied Biosystems)
       
   24. Taq DNA polymerase (Applied Biosystems)
       
   25. DNeasy Plant Mini Kit (QIAGEN)
   
   
3. Media and supplements
   Microbiological media
   1. Agrobacterium liquid growth medium (LB liquid medium) (see Recipes)
      
   2. Agrobacterium growth plates (LB solid medium) (see Recipes)  
   
   Plant media
   1. Driver Kuniyuki walnut (DKW) basal medium (see Recipes)
      
   2. Virulence induction medium (IM) (see Recipes)
      
   3. Acetosyringone medium (AS) plates (see Recipes)
      
   4. KAN/Timentin^® selection medium (see Recipes)
      
   5. KAN only selection medium (see Recipes)
      
   6. DKW shoot medium (DKW basal medium with 1 mg/L BAP and 0.01 mg/L IBA) (see Recipes)
   
   
4. Glass and Plasticware
   1. Sterile empty 100 x 15 mm Petri plates (VWR International, catalog number: 25384-342 )
      
   2. Sterile empty 35 x 10 mm Petri plates (BD, Falcon^TM, catalog number: 25373-041 )
      
   3. Sterile disposable 50 ml screw-cap centrifuge tubes (BD, Falcon^TM, catalog number: 352070 )
      
   4. Sterile disposable cotton-plugged 10 ml pipettes
      
   5. Plastic pipette tips (20, 200, and 1,000 µl) (Rainin, model: LTS )
      
   6. Sterile disposable 6-well multiwell plates (BD Biosciences, Falcon^®, catalog number: 08-772-1G )
      
   7. Sterile disposable 96-well multiwell plates (BD Biosciences, Falcon^®, catalog number: 08-772-2C )
      
   8. Filter paper cut to fit in 100 x 15 mm Petri plates and autoclaved
      
   9. Filter paper discs cut to the diameter of the wells of a 6-well multiwall plate and autoclaved
      
   10. 150 mm diameter desiccator (e.g. Nalgene, catalog number: 5313-0150 )
       
   11. Magenta GA-7 vessels (Magenta Corp.)

Equipment

1. Spectrophotometer (e.g. Bio-Rad Laboratories, model: SmartSpec 3000 )
   
2. Laboratory centrifuge (e.g. Sigma-Aldrich, Laborzentrifugen Laboratory Centrifuge 3K 10 )
   
3. Micro-centrifuge (e.g. Denville, model: 260D )
   
4. Continuous power supply units for electrophoresis (e.g. Thermo EC, model: EC 105 and Hoefer, model: HE 33 Mini Submarine unit)
   
5. Electrophoresis units for agarose gels (e.g. Fisher Scientific, models: 03-500-124 , 03-500-132 , and 03-500-134 )
   
6. Laminar flow hood (e.g. EdgeGard, Baker, model: EG3252 )
   
7. Pipetman^® 20, 200, and 1,000 µl (Rainin, model: LTS )
   
8. Analytical balance (e.g. Mettler Toledo, model: AT261 DeltaRange^® )
   
9. Top loading electronic balance (e.g. Mettler, model: PM 2000 )
   
10. pH meter (e.g. Corning Pinnacle, model: 540 )
    
11. Constant temperature incubator (e.g. Napco, model: 301 )
    
12. Water baths (e.g.Thermo Scientific, model: 2870 )
    
13. Vortex mixer (e.g.: Scientific Industries, model: G 560 )
    
14. Freezer (- 80 °C) (e.g. New Brunswick, model: U 700 Premium)
    

Procedure