Abstract Biotechnology tools based on tissue culture techniques allow mass clonal propagation of plants with desired genetic and sanitary features. In the present work a pineapple micropropagation protocol was established based on nodule cluster cultures (NC) associated with different culture systems. Leaf segments from in vitro -grown plantlets cultivated in culture medium supplemented with 2 μM NAA and 8 μM 2-iP or BAP resulted in the highest rates of NC induction. NC fresh weight increased ratio was more efficient in a culture medium supplemented with 2 μM NAA and 2 μM BAP in twin-flasks temporary immersion system (TIS-TF). This culture medium was also efficient for the microshoots development in RITA ® temporary immersion system (TIS-RITA ® ). Microshoots elongation in permanent immersion system (PIS) was more efficient in a culture medium supplemented with 10 μM GA 3 . Shoots longer than 2 cm showed 92% survival rate when acclimatized. Free polyamine endogenous levels was measured and showed higher contents for the TIS-TF, followed by TIS-RITA ® , and PIS, respectively. Putrescine levels also followed this pattern, being absent in PIS, which, on the other hand, showed higher levels of spermine and spermidine. The results indicate the establishment of efficient protocol for pineapple micropropagation, with a high multiplication and regenerative rates and great potential for agriculture application. The biochemical analysis suggests that besides culture medium composition, flask design and culture system also drastically affects the plant's metabolism toward multiplication and plant regeneration.