A high-throughput RNA interference (RNAi)-based approach using hairy roots for the study of plant-rhizobia...

Legumes are major contributors to sustainable agriculture; their key feature is their ability to fix atmospheric nitrogen through symbiotic nitrogen fixation. Legumes are often recalcitrant to regeneration and transformation by Agrobacterium tumefaciens; however, A. rhizogenes-mediated root transformation and composite plant generation are rapid and convenient alternatives to study root biology, including root nodule symbiosis. RNA interference (RNAi), coupled with A. rhizogenes-mediated root transformation, has been very successfully used for analyses of gene function by reverse genetics. Besides being applied to model legumes (Medicago truncatula and Lotus japonicus), this method has been adopted for several other legumes due to the ease and relative speed with which transgenic roots can be generated. Several protocols for hairy root transformation have been published. Here we describe an improved hairy root transformation protocol and the methods to study nodulation in Medicago. We also highlight the major differences between our protocol and others, and key steps that need to be adjusted in order to translate this method to other legumes.