Optimizing an Ex Vitro RUBY-Equipped Method for Hairy Root Transformation of Peanuts: An Efficient Approach for the Functional Study of Genes in Peanut Roots

<i>Agrobacterium</i><i>rhizogenes</i> (<i>A. rhizogenes</i>)-mediated transformation of hairy roots is a favored and flexible method for root gene functional analysis. However, the selection of transformants can be complex and time-consuming. Here, we describe our simplified method for the <i>A. rhizogenes</i>-mediated hairy root induction in young peanut shoots using an expression vector with <i>RUBY</i> for direct visual selection of transformants. Analyses verified that this method provides a high-efficiency gene transformation technique for peanut, with transformant frequencies between 46.2 and 73.7%. To test the utility of this method in gene functional analyses, it was used to overexpress <i>AhLRX6</i> in hairy roots and we present our preliminary results indicating the production of thicker cells walls in root tips relative to the WT.