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Background   Agrobacterium rhizogenes -mediated hairy root transformation provides a powerful tool for investigating the functions of plant genes involved in rhizobia-legume symbiosis. However, in the traditional identification methods of transgenic hairy roots based on reporter genes, an expensive chemical substrate or equipment is required.    Results  Here, we report a novel, low cost, and robust reporter for convenient, non-destructive, and directly visual selection of transgenic hairy roots by naked eye, which can be used in the study of rhizobia-legume symbiosis. The reporter gene  AtMyb75  in  Arabidopsis , encoding an R2R3 type MYB transcription factor, was ectopically expressed in hairy roots-mediated by  A .  rhizogenes , which induced purple/red colored anthocyanin accumulation in crop species like soybean ( Glycine max  (L.) Merr.) and two model legume species,  Lotus japonicas  and  Medicago truncatula . Transgenic hairy roots of legumes containing anthocyanin can establish effective symbiosis with rhizobia. We also demonstrated the reliability of  AtMyb75  as a reporter gene by CRISPR/Cas9-targeted mutagenesis of the soybean resistance to nodulation  Rfg1  gene in the soybean PI377578 (Nod-) inoculated with  Sinorhizobium fredii  USDA193. Without exception, mature nitrogen-fixation nodules, were formed on purple transgenic hairy roots containing anthocyanin.    Conclusions  Anthocyanin is a reliable, user-friendly, convenient, non-destructive, low cost, directly visual reporter for studying symbiotic nitrogen-fixing nodule development and could be widely applied in broad leguminous plants.

plant methods

Anthocyanin, a novel and user-friendly reporter for convenient, non-destructive, low cost, directly visual selection of transgenic hairy roots in the study of rhizobia-legume symbiosis