When the calli had grown to about 2–3 square millimeters (mm2) in size they were immersed in 30 ml CCM liquid medium in deep petri-dishes with the Agrobacterium strain containing the binary vector pCAMBIA 2300. Acetosyringone was added at the concentration of 200 μM to enhance the virulence of Agrobacterium ( Dong et al., 2006). Suspension cultures were hand-shaken under sterile conditions for 2–10 min. Infected main calli pieces (devoid of peripherally separated minute friable tissues) were immediately blotted with sterile filter paper and transferred to semisolid CCM medium ( Table 1) for 48 h in the dark.