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).