Leaf Disk Transformation Using Agrobacterium tumefaciens-Expression of Heterologous Genes in Tobacco

Leaf disk transformation of tobacco is a very simple and robust method. It is used with success in many laboratories. The protocol presented here is a simplified version of that of Horsch et al. (1 ). Basically, it consists of immersing the leaf disks in a liquid culture of Agrobacterium carrying the chosen transformation vector. The plant tissue and Agrobacterium are then cocultivated on regeneration medium for a period of 2 d at the end of which the leaf disks are transferred to regeneration medium supplemented with an antibiotic to kill the bacteria (cefotaxime), and a selective agent against untransformed plant cells. It takes about 2 mo to obtain rooted plantlets that can be transferred to soil. The protocol presented here works well in our hands with Nicotiana tabacum cultivar “petit havana” mutant SR1 (2 ) and Agrobacterium tumefaciens strain LBA4404 (3 ) harboring binary vectors conferring kanamycin resistance (100 mg/L). We have also used pBIB-HYG (4 ), which confers hygromycin resistance (50 mg/L).