Agrobacterium Minimal medium:
Dissolve K 2 HPO 4 × 3H 2 O (3.44 g), KH 2 PO 4
(1.45 g), NaCl (0.15 g), MgSO 4 × 7H 2 O (0.5 g), (NH 4 )2SO4 (0.5 g) in 800 mL of water. Adjust the pH to 5.5, and the volume
to 900 mL with water and autoclave. In parallel, dissolve CaCl 2 × 2H 2 O (0.067 g), FeSO 4 × 7H 2 O (0.0025 g), glucose (2 g)
in 100 mL water and filter through a 0.2 m m Whatman filter. Once the autoclaved medium reaches ~50°C add filter sterilized
Dissolve K 2 HPO 4 × 3H 2 O (3.44 g), KH2PO4 (1.45 g), NaCl (0.15 g), MgSO 4 × 7H 2 O (0.5 g), (NH 4 )2SO4 (0.5 g) in 800 mL of water. Adjust the pH to 5.5, and the volume to 900 mL with water and autoclave. In parallel, dissolve CaCl 2 × 2H 2 O (0.067 g), FeSO 4 × 7H 2 O (0.0025 g), glucose (1.8 g) MES (7.8 g) and glycerol (5 mL) in 100 mL water and filter through a 0.2 m m Whatman filter. Once the autoclaved medium reaches ~50°C add filter sterilized medium and mix well. Add 1.5% of technical agar for plates.
Selection medium: Autoclave PDA (39 g/L) and add hygromycin (50 m g/mL) and cefotaxime (200 m g/mL) immediately before pouring the plates.
Cefotaxim (200 mg/mL): dissolve the required amount in demineralized water and filter sterilize through a 0.2 m m Whatman filter ( see Notes 3 and 4 ).
Hygromycin (50 mg/mL): dissolve the required amount in demineralized water and filter sterilize through a 0.2 m m Whatman filter ( see Notes 5 and 6 ).
Kanamycin (50 mg/mL): dissolve the required amount in demineralized water and filter sterilize through a 0.2 m m Whatman filter ( see Note 7 ).
Acetosyringone (200 mM): dissolve the required amount in DMSO ( see Note 8
Buffer: TES (100 mM Tris, pH 8.0, 10 mM EDTA, 2% SDS).
Reagents: 5M NaCl, 10% CTAB, 5M NH 4 Ac, Chloroform: isoamyl alcohol (24:1), Isopropanol and 70% ethanol.
Enzymes: Nco I (six cutters) and Msp I (four cutters), T4 DNA ligase and GO Taq DNA polymerase.
Fungal species: Fusarium oxysporum f. sp. ciceris was used as the recipient species for transformation.
Bacterial strain: A. tumefaciens strain EHA-105 with plasmid pCAMBIA1300 was used as a T-DNA donor strain.
Host plant: Cicer arietinum, JG62 as a susceptible genotype and WR315 as a resistance
Nitrocellulose membrane: Hybond-N + (GE Health Care).
Heat sterilized microcentrifuge tube (1.5 mL).
Sterilized glassware: 300 mL conical fl ask.
Spreader.