An Easy Method for Agrobacterium tumefaciens-Mediated Gene Transfer to Nicotiana tabacum cv. TAPM26

Abstract

Research in Agrobacterium tumefaciens mediated gene transfer has advanced and opened new avenue for plant improvement via introduction of various beneficial genes. Current study investigates transformation parameters during co-cultivation to improve T-DNA delivery into Nicotiana tabacum cv. TAPM 26 by monitoring GUS expression level. The techniques involved basic plant tissue culture and establishment of plant transformation systems. Conditions assessed were bacterial inoculum concentration, infection period, wounding and pre-culture of explants, acetosyringone (AS) concentration and co-cultivation temperature. Optimized conditions resulted in high transformation efficiency at transient level were as follows; Agrobacterium tumefaciens growth phase of A600nm 0.8, infection period of 30 min, pre-culture of wounded explants prior to infection, addition of acetosyringone (AS) in bacterial growth culture (100 µM) and in co-cultivation medium (200 µM) and co-cultivation temperature of 22°C. Although higher density bacterial culture used for the infection process gave higher transformation rate, however, it compromised the viability of the explants. On the other hand, dilution of bacterial suspension reduced necrosis in explants and improved transformation events greatly. The transformation efficiency was increased 9 fold when the infection process was carried out at low temperature of 22°C. Current study has proven among the parameters assessed, temperature was the critical factor during co-cultivation process in Agrobacterium tumefaciens mediated gene transfer.