Mycorrhizas providea symbiotic associations in which the fungus obtains carbon from the plant, while providing the plant with a supply of phosphorus. On the other hand, periwinkle plants (Catharanthus roseus L.) have been investigated due to production of two medicinal alkaloids namely vinblastine and vincristine. In this evaluation, tryptophan was used as precursor of these alkaloids under tissue culture conditions. Inoculation by mycorrhiza induced the growth and survival of several species during acclimatization, which was the most important challenge of tissue culture. For this reason, the regeneration plantlets obtained under different concentrations of tryptophan, were exposed to acclimatization to mycorrhizal colonization and their shoot growth and biochemical factors were evaluated. Results from measurement of length, fresh and dry weights, soluble sugars and total protein content and also, chlorophyll a and b of shoots, indicated the positive role of these two treatments on growth of periwinkle plantlets under acclimatization. The highest amount of height and fresh and dry weight in shoots were obtained in colonized samples with Glomus versiforme and under 0, 150 and 250 mg/l tryptophan treatments, respectively. Also, the highest content of total protein and soluble sugars content was observed in G. versiforme colonized plants under 350 mg/l tryptophan treatment. Finally, evaluation of the chlorophyll a and b contents showed that the highest amount of these factors obtained in the colonized samples with G. versiforme and G. etunicatum, respectively, under 250 mg/l tryptophan treatment.