This is a protocol to produce stable transgenic plants in Lotus japonicus, which is established based on methods previously reported (Handberg and Stougaard, 1992; Stiller et al., 1997; Thkjaer et al., 1998) with some modifications. In this protocol, hygromycin is used to select transgenic plants.

In L. japonicus, hairy root transformation is the very useful technique to generate transformed root systems in a short term. This protocol was previously described (Kumagai and Kouchi, 2003) with some modifications. After the infection of Agrobacterium rhizogenes, L. japonicus develops not only transformed but also untransformed roots. Thus, transgenic roots need to be identified by certain indications. In this protocol, we use the GFP florescent signals as such indication.