New integrative vectors increase Agrobacterium rhizogenes transformation and help characterise roles for soybean GmTML gene family members

Hairy-root transformation is widely used to generate transgenic plant roots for genetic functional characterisation studies. However, transformation efficiency can be limited, largely due to the use of binary vectors. Here, we report on the development of novel integrative vectors that significantly increase the transformation efficiency of hairy roots. This includes pHGUS7, for promoter::reporter visualisation studies, and pHOG13, for genetic insertion and overexpression studies. These vectors have been designed to simplify cloning workflows, enhance the selection of positively transformed Agrobacterium colonies, and increase the transformation efficiency and ease of selection of genetically modified hairy roots. To demonstrate the efficacy of the new vectors, Too Much Love (TML) encoding genes acting in the Autoregulation Of Nodulation (AON) pathway of soybean were investigated. Both constructs provided significantly higher transformation rates than the binary vector control, often resulting in >70% of the roots being transformed. Overexpression of each individual TML encoding gene ( GmTML1a , GmTML1b and GmTML2 ) using pHOG13 resulted in a significant reduction in nodule number, demonstrating the role of all three in inhibiting nodule organogenesis. Moreover, reporter-fusions with the promoter of each TML encoding gene using pHGUS7 revealed that each exhibits a unique pattern of expression in nodules, with GmTML1b displaying considerably stronger expression than GmTML1a or GmTML2 . Taken together, these results demonstrate the utility and efficiency of the new pHOG13 and pHGUS7 integrative vectors in hairy-root transformation, and improve our understanding of the critical TML- encoding genes in soybean nodulation control.