A Toolbox for Nodule Development Studies in Chickpea: A Hairy-Root Transformation Protocol and an Efficient...

A Mesorhizobium sp. produces root nodules in chickpea. Chickpea and model legume Medicago truncatula are members of the inverted repeat-lacking clade (IRLC). The rhizobia, after internalization into the plant cell, are called bacteroids. Nodule-specific cysteine-rich peptides in IRLC legumes guide bacteroids to a terminally differentiated swollen (TDS) form. Bacteroids in chickpea are less TDS than those in Medicago spp. Nodule development in chickpea indicates recent evolutionary diversification and merits further study. A hairy-root transformation protocol and an efficient laboratory strain are prerequisites for performing any genetic study on nodulation. We have standardized a protocol for composite plant generation in chickpea with a transformation frequency above 50%, as shown by fluorescent markers. This protocol also works well in different ecotypes of chickpea. Localization of subcellular markers in these transformed roots is similar to the localization observed in transformed Medicago roots. When checked inside transformed nodules, peroxisomes were concentrated along the periphery of the nodules, while endoplasmic reticulum and Golgi bodies surrounded the symbiosomes. Different Mesorhizobium strains were evaluated for their ability to initiate nodule development and efficiency of nitrogen fixation. Inoculation with different strains resulted in different shapes of TDS bacteroids with variable nitrogen fixation. Our study provides a toolbox to study nodule development in the crop legume chickpea.