Since the decade of 1970, the use of pesticides and fertilizers became essential for agricultural high-yield production systems development. However, they generate significant costs and environmental damage. The inoculation of arbuscular mycorrhizal fungi (AMF) is an alternative to reduce phosphate fertilizers application. These obligatory root symbionts enhance water and nutrients uptake, especially phosphorus. Axenic cultivation in roots transformed by the Ti plasmid of Agrobacterium rhizhogenes as a host demonstrated to be effective for propagules multiplication, with higher homogeneity between batches and contamination control. Still, current systems can be optimized in terms of production time, the number of propagules, and costs. For this purpose, this study evaluated the effects of macronutrients content reduction in minimum mineral medium (M), by 75%, 50%, and 25%, on the association between transformed tobacco roots (Nicotiana tabacum L.) and the AMF Rhizophagus clarus. Tobacco root fragments (5 – 7 cm) were transplanted in Petri dishes with M medium and after 10 days inoculated with an M media fragment (2.25 cm²) containing R. clarus propagules. The number of secondary spores (counted in an optical stereoscope) and the length of the extraradicial mycelium were determined using the root length (RL) formula (RL = [π.N.A]/2H; where π = 3.14; N = number of intersections; A = area of the plate; H = sum of the length of the lines). The plates were marked with 0.5 cm separated lines and the numbers of hyphae intersections in the lines were counted, estimating the length (cm), after 30, 45, 100, 120, and 150 days of cultivation, in triplicate. After 45 days, an extraradical mycelium length of 1017 cm was found in the 75% reduction, while 50%, 25%, and 100% obtained lengths of 480.3, 235.6, and 439.8 cm, respectively. Regarding spore production, no differences between treatments were observed, with averages between 238 (control) and 290 (reduction to 25%) spores. The data indicate a relationship between the limitation of macronutrients and fungal development in tobacco roots, and possibly, the change in the phosphorus concentration is responsible for this effect, since its soil availability regulates the association development. The reduction to 25% resulted in lower hyphae growth, suggesting a lower plant investment in the association. Based on the results, we concluded that reducing 75% of macronutrients in M media culminated in a positive effect on the mycelial growth of R. clarus, which can constitute an alternative for optimizing this production system.