<p>Arbuscular mycorrhizal fungi (AMF) are obligate root symbionts that play important role in sustainable agriculture restoration. In vitro root organ culture has been commonly utilized for axenic cultivation of AMF. Nevertheless, the restricted biomass production poses a limitation. The study deals with establishment of an optimal in vitro co-culture system of AMF <i>Claroideoglomus claroideum</i> with hairy roots (HR) of <i>Boerhavia diffusa</i> L. For efficient induction of HR, the strain of <i>Rhizobium rhizogene</i>, method of infection, explant type, concentration of acetosyringone and antibiotic cefotaxime were assessed. For optimal HR biomass growth and <i>C. claroideum</i> root colonization the MS media strength, light condition, carbon source and concentration of chemical elicitors salicylic acid, methyl jasmonate, chitosan and chitin were determined. Highest HR transformation efficiency (90%) was obtained in leaf explant infected with <i>R. rhizogenes</i> MTCC 532 through injection method in MS0 agar media (0.8%) augmented with 200 µM acetosyringone and 500&#xa0;µg/L cefotaxime. HR transformation was confirmed by PCR amplification of the rolB gene. Maximum HR biomass growth and <i>C. claroideum</i> root colonization in the in vitro co-culture system was achieved in ½ strength MS agar medium fortified with 3% sucrose under diffused light (15 µmol m⁻²s^− 1 photon flux density) in culture condition of 25 ± 1^o C, 60% relative humidity. The salicylic acid and methyl jasmonate both at 0.1 mM L^− 1, chitosan and chitin both at 100 mgL^− 1 resulted in highest HR biomass growth and AMF root colonization. The in vitro co-culture system of <i>C. claroideum</i> and HR can serve as a model to study AMF and host interaction.</p> Graphical abstract <p></p>