A new transformation-regeneration procedure in the model legume Lotus japonicus. Root explants as a source of large numbers of cells susceptible to Agrobacterium mediated transformation.

This paper described a simple and efficient transformation procedure for the production of transgenic Lotus japonicus plants. In this new procedure the dedifferentiated root explants, used as starting material, provided a source of a large number of cells, competent for the regeneration procedure, with a high susceptibility to the Agrobacterium infection. This protocol provided a ten-fold increase in the number of transformants produced by a single plant compared to the widely used hypocotyl transformation procedure. Furthermore, the described procedure allowed to start with intact plant stored for a long time at 4°C hence providing a potential continuous supply of explants for transformation experiments. The overall time of incubation in the tissue culture conditions required to have a plant transferable in soil is four months. The transgenic nature of the transformants was demonstrated by detection of GUS activity in the primary transformants and by their molecular analysis. Stable transformation was indicated by mendelian segregation of the hygromycin selectable marker and of the gusA activity after selfing of transgenic plants.