A reliable peanut root transformation system would be useful to study the functions of genes involved in root biology and disease resistance. The objective of this study was to establish an effective protocol to produce composite plants mediated by Agrobacterium rhizogenes transformation. In total, 75% of transformed peanut seedlings produced an average of 2.83 transgenic roots per plant. Peanut seed had the highest germination rate after treatment in a chlorine gas chamber for 8 h compared with 16 h in chlorine gas or Clorox and mercuric chloride immersion treatments. High transformation efficiency was achieved when the wound site for A. rhizogenes inoculation was covered with vermiculite instead of enclosing the whole plant in a high humidity chamber. On average, 2.5 galls from Meloidogyne arenaria infection were formed per transgenic root from susceptible genotype TifGP-2. These data indicate that A. rhizogenes-transformed roots can be used to phenotype the host response to nematode challenge. Transformation of RLP-2, a candidate resistance gene for M. arenaria integrated into a silencing construct, did not alter the resistance response of Tifguard, even though downregulation of endogenous RLP-2 expression was detected in transformed roots. It is likely that RLP-2 is not the gene conditioning M. arenaria resistance in peanut.