Two essays were carried out to evaluate the effect of different types of auxins on root formation and the influence of dark and culture substratum on tetraploid hybrid FHIA-01 proliferation (Musa spp. AAAB). The plant material consisted of tissue culture plantlets of FHIA -01 hybrid tetraploid banana (Musa AAAB). The trial, with a total of 10 replicates per treatment was carried out in each pot containing five explants. For both tests, a combination of two cytokinins was enriched in culture substratum. The results obtained show that regeneration was high in culture substratum with light than substratum without meta-methoxytopolin riboside (M2). The medium M2 to the light induced a higher number of the buds compared to medium dose reduced meta-methoxytopolin riboside (M1). Meanwhile, only explants inoculated on the medium M1 in the dark induced callus. The bud proliferation, induction of root, leaf and the broadcast callus induction are significantly influenced by the different substratum and photoperiod, increasing the explant size, the number of emerged leaves, roots and the number of the weight of explant with buds proliferated. Formulating specific culture media cultivars according to group (ABB or AAA) and the choice of culture conditions (light intensity) would avoid consecutive failures and low proliferation in in vitro culture.