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Brent Tisserat, Christopher Herman, Robert Silman and Rodney J. Bothast

A continuous CO2 flow system was used to study the growth of carrot (Daucus carota L.), citrus (Citrus macrophylla L.), kale (Brassica oleracea L.), lettuce (Lactuca sativa L.), radish (Raphanus sativus L.), and tomato (Lycopersicum esculentum L.) cultures in vitro under photoautotrophic, photomixotrophic, and heterotrophic conditions. Lettuce plantlets were grown on Murashige and Skoog medium with 0%, 0.3%, 1%, and 3% sucrose within flow chambers containing 350, 750, 1500, 3000, 10,000, 30,000, and 50,000 μL·L−1 CO2. Increasing the levels of CO2, especially at the ultra-high levels (i.e., ≥3,000 μL·L−1 CO2), increased fresh weight, shoot length, leaf number, leaf length, leaf width, root number, and root length for plantlets grown regardless of sucrose levels tested compared to plantlets grown at normal atmospheric CO2 levels, i.e., 350 μL·L-1. For example, fresh weights of lettuce plantlets grown on medium containing 0% or 3% sucrose increased 11- and 13-fold, respectively, when supplemented with 30,000 μL·L-1 CO2 compared to growth of lettuce plantlets grown on the same media without CO2 enrichment. Similar fold increases in growth responses were obtained with carrot, citrus, kale, radish, and tomato plantlets grown in atmospheres enriched with high CO2 levels, elevated from 3000 to 30,000 μL·L-1. Optimum CO2 concentration varied among species, suggesting a species-related response. Varying the rate of CO2 application between 250, 500, 1500, or 2000 mL·min-1 did not effect the rate of growth of lettuce plantlets. The passive diffusion continuous flow-through system presented in this paper is inexpensive, easily constructed, and allows for testing ultra-high CO2 levels on plant culture growth in vitro.