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Arbuscular mycorrhizal fungi (AMF) establish symbiotic interactions that improve productivity of agricultural crops. This study aims to evaluate the effect of different doses of the fungus Glomus intraradiceson ex vitro development of sugarcane plantlets in the acclimatization stage. In vitro sugarcane (Saccharumspp. cv Mex 69–290) plantlets were inoculated with 0, 50, 100, 200, and 400 spores per plant of G. intraradices. After 60 days of acclimatization in a greenhouse, survival rate, colonization percentage, plant growth, dry matter, total chlorophyll content, and macronutrient and micronutrient contents were evaluated. Mycorrhizae were characterized by bright field and multiphoton microscopy. Effects of mycorrhizae on the different variables evaluated were observed. The doses of 50 and 100 spores per plant, with 30 and 58% colonization, respectively, achieved a symbiotic interaction, while doses of 200 and 400 spores per plant, with the highest colonization percentages (80 and 86%), had negative effects on survival, development, chlorophyll content, and nutritional status. Microscopy demonstrates the symbiotic association between G. intraradicesand Saccharumspp. Early application of adequate mycorrhizal doses in plantlets during acclimatization provides a conditioning advantage prior to transplanting for the establishment of basic sugarcane seedbeds.