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The <i>Cattleya</i> (Orchidaceae–<i>Laeliinae</i> subtribe) intergeneric hybrids, such as <i>Brassolaeliocattleya</i> (<i>Blc</i>.), have great ornamental value, due to their compact-size, with large and high color diversity of flowers. Artificial induction of polyploidy brings agronomic, ornamental and genetic benefits to plants. Polyploidization efficiency depends on factors, such as the type of antimitotic, polyploidization method, concentrations, exposure times and type of explant. This study aimed to develop a protocol to polyploidize <i>Blc</i>. orchids, by testing two types of explants (seeds and protocorms), concentrations and exposure times to colchicine. The effects of colchicine on the in vitro development of explants were also investigated. The responses of explants to colchicine depended on the concentrations, exposure time and the interaction of these factors. Flow cytometric analysis evidenced high endopolyploidy and allowed the separation of polyploidized (4C, 8C and 16C peaks) from non-polyploidized (only 2C and 4C peaks) plants. The highest percentage of polyploid plants was regenerated from protocorms (16.4%) treated with colchicine instead of seeds (3.2%). Protocorms treated with colchicine at 500–750 μM for 18 h resulted in the best percentage of polyploidization. Additionally, in vitro natural polyploidization using protocorms was reported (11.5%). Cytological analyses allowed an estimation of the number of chromosomes of the parents (≡70), polyploidized (≡140) and non-polyploidized progeny (≡70).