A comprehensive, improved protocol for generating common bean (Phaseolus vulgaris L.) transgenic hairy roots and their use in reverse-genetics studies

Generating transgenic hairy roots has been the preferred strategy for molecular studies in common bean (Phaseolus vulgaris L.), since generating stable knockout lines in this species is challenging. However, the number of plants producing hairy roots following the original protocol published in 2007 is usually low, which has impeded progress. Since its initial publication, the original protocol has been extensively modified, but these modifications have not been adequately or systematically reported, making it difficult to assess the reproducibility of the method. The protocol presented here is an update and expansion of the original method. Importantly, it includes new, critical steps for generating transgenic hairy roots and using them in molecular analyses based on reverse -genetics approaches. Using this protocol, the expression of two different genes, used as an example, was significantly increased or decreased in approximately 30% of the transformed plants. In addition, the promoter activity of a given gene was observed, and the infection process of rhizobia in transgenic hairy roots was monitored successfully. Thus, this improved protocol can be used to upregulate, downregulate, and perform promoter activity analysis of various genes in common bean transgenic hairy roots as well as to track rhizobia infection.