Low CO2 Levels Are Detrimental for In Vitro Plantlets through Disturbance of Photosynthetic Functionality and Accumulation of Reactive Oxygen Species

Photosynthesis of plantlets in tissue culture containers is not considered important, compared to photosynthesis of ex vitro plants, due to the exogenous source of carbohydrates present in tissue culture media. However, CO2 starvation can generate a burst of reactive oxygen species (ROS). We examined this phenomenon in tissue culture, since CO2 levels may become very low during the light period. The research was carried out with lily scales, regenerating adventitious bulblets, and with Arabidopsis seedlings. CO2 starvation was achieved by placing a small vial of concentrated KOH solution in the culture container. CO2 removal reduced the growth of regenerated lily bulblets by 33% or 23%, with or without sucrose in the medium, respectively. In Arabidopsis seedlings, CO2 removal decreased growth by 50% or 78% in the presence or absence of sucrose in the medium, respectively. Therefore, the addition of sucrose as a replacement for photosynthesis resulted in only partial recovery of growth. Staining with nitroblue tetrazolium (NBT) showed little to no ROS in ex vitro growing seedlings, while abundant ROS were detected in seedlings grown under in vitro CO2 starvation. Seedlings grown under normal tissue culture conditions (no CO2 withdrawal) showed low levels of ROS. In lily tissue culture, CO2 starvation decreased the maximum quantum efficiency of photosystem II (F-v/F-m) from 0.69 to 0.60, and in Arabidopsis from 0.76 to 0.62. F-v/F-m of ex vitro lily and Arabidopsis seedlings was 0.77 and 0.79, respectively. This is indicative of a disturbance in photosynthesis functionality and the occurrence of in vitro stress under reduced CO2 concentrations. We conclude that poor growth, in the absence of CO2, was partly due to strongly reduced photosynthesis, while the detrimental effects were most likely due to a burst of ROS.