Synergistic optimization of CO2 enrichment and air exchange enhances growth of medicinal cannabis plantlets in photoautotrophic micropropagation

Photoautotrophic micropropagation (PAM) replaces sugar with CO 2 as the sole carbon source under stringent environmental control, enabling the production of vigorous, pathogen-free plantlets. This study demonstrates that synergistic regulation of CO 2 concentration and air change rate (ACR) significantly enhances the growth and physiological performance of medicinal cannabis ( Cannabis sativa L.) plantlets in PAM. Two genotypes were evaluated: the short-day cultivar ‘Charlotte’ and the day-neutral cultivar ‘Auto Charlotte’. Cannabis plantlets were cultured at 400 or 800 µmol mol −1 CO 2 under four ACR levels of 0.7, 4.4, 9.2 and 13.6 h −1 . Raising CO 2 from 400 to 800 μmol mol −1 enhanced plantlet height, leaf area, biomass, root vigor, and net CO 2 uptake in both cultivars; however, these benefits were partially offset when ACR exceeded 4.4 h −1 , apparently due to increased evaporative water loss from the substrate medium. The synergistic interaction between 800 µmol mol −1 CO 2 and 4.4 h −1 ACR significantly enhanced plantlet performance beyond the effects of either factor alone. Compared with conventional regime of 400 µmol mol −1 CO 2 concentration with an ACR of 0.7 h −1 , the dry weight rose by 181 % in ‘Charlotte’ and 124 % in ‘Auto Charlotte’, while root vigor and photosynthetic capacity were simultaneously enhanced. Importantly, these gains were achieved using passively vented vessels, avoiding cost and contamination risk of forced aeration. We conclude that combining 4.4 h −1 ACR with 800 µmol mol −1 ambient CO 2 constitutes a practical, industry-ready PAM protocol for medicinal cannabis, delivering robust, sugar-free plantlets that are well-adapted for direct ex-vitro transfer. • CO 2 enrichment to 800 μmol mol −1 enhances plantlet growth and quality. • Excessive ventilation increases substrate water loss, inducing drought stress. • An air change rate (ACR) of about 4.4 h −1 is suitable for medicinal cannabis in PAM. • Combining CO 2 and ACR improves NCEA and maximizes plantlet growth performance in PAM.