In vitro physiology and carbon metabolism can be affected by the sink-source relationship. The effect of different sucrose concentrations (10, 30, and 50 g L −1 ), light intensities (80 and 150 μmol m −2 s −1 ), and CO 2 levels (375 and 1,200 μmol mol −1 ) were tested during plantain micropropagation in temporary immersion bioreactors. Activities of pyruvate kinase, phosphoenol pyruvate carboxylase, and the photosynthesis rate were recorded. From the morphological and practical point of view, the best results were obtained when plants were cultured with 30 g L −1 sucrose, 80 μmol m −2 s −1 light intensity, and 1,200 μmol mol −1 CO 2 concentration. This treatment improved leaf and root development, reduced respiration during in vitro culture, and increased starch level at the end of the hardening phase. In addition to that, the number of competent plants was increased from 80.0% to 91.0% at the end of the in vitro phase and the survival percentage from 95.71% to 99.80% during ex vitro hardening.