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The term in vitro photomixotrophism refers to the ability of explants to obtain metabolic energy from the culture medium and as a product of photosynthesis. The objective of this research was to study the physiological and biochemical mechanisms of V. planifolia during in vitro multiplication using a photomixotrophic system with different sucrose contents (0, 15 and 30 g L–1) and CO2 supply levels (500, 800 and 1200 ppm) using a Temporary Immersion Modular System (SMIT®). After 45 days of multiplication, response percentage, number of shoots per explant, shoot length, number of leaves per shoot, stomatal index (%), percentage of closed stomata, and chlorophyll, β-carotene, Phosphoenolpyruvate (PEP) and Rubisco contents were evaluated. In addition, the survival rate during acclimatization at 60 days was evaluated. For the multiplication stage, the highest response percentage was obtained in the treatments with 15 g L–1 sucrose with 500 and 800 ppm CO2, and 30 g L–1 sucrose with 500 ppm CO2. In the latter treatment, the best development parameters were obtained, with 14.75 shoots per explant, a shoot length of 2.38 cm and 2.5 leaves per shoot. In general, the highest chlorophyll, β-carotene, PEP and Rubisco contents were observed with 30 g L–1 sucrose + CO2. No effects of the treatments on stomatal index (%) were observed, while the percentage of closed stomata showed differences among treatments. At the acclimatization stage, the highest survival percentages were obtained from the treatments of 30 g L–1 sucrose with 500 and 800 ppm CO2. In conclusion, this study demonstrates physiological and biochemical mechanisms for a better understanding of photomixotrophism during in vitro multiplication in vanilla and may be applied to other species. Key message: In vitro photomixotrophism can be induced by injecting carbon dioxide to promote photosynthesis. [ABSTRACT FROM AUTHOR]

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