Photoautotrophic Arabidopsis Culture (PAC): Development of In Vitro Experimental Platform to Assure Normal Plant Physiology

Plant tissue culture serves both as a propagation and a research tool to grow small plants under aseptic conditions. Plant biological studies using model plant species (i.e., Arabidopsis spp.) sometimes employ tissue culture as the research platform to test the biological hypothesis of interest. Especially for Arabidopsis spp., tissue culture has been employed as a preferable contained experimental system assuring uniform growth, presumably due to the environmentally sensitive nature of Arabidopsis plants.  The most widely used tissue culture technique employs full or reduced strength of Murashige and Skoog salts with 10-30 g/L sucrose and agar or alternative gelling agent inside a closed vessel placed under relatively low light intensity. MS medium has 4-5 times greater nitrogen concentration (one third of which is in the form of NH₄-N) than a typical hydroponics solution. Sucrose has been known as a transcriptional factor affecting gene expression involving hexokinase activities or flowering. Sucrose has been also shown to reduce Rubisco activities. Limited ventilation of tissue culture vessels results in CO₂ concentrations as low as the compensation point during photoperiod. Therefore, simple removal of sugar from the medium would not achieve normal photoautotrophic plant growth, and other environmental conditions need to be improved all together. To address this issue, we demonstrated photoautotrophic Arabidopsis culture (PAC) using A. thaliana (Col-0), a widely used genotype. Plants were germinated in medium containing half strength MS and 10 g/L sucrose for one week and transferred to vented vessels containing sugar-free medium with hydroponic salts under 160 mmol m^-2 s^-1 PAR.  Photoperiod was set at 10 hours throughout the experiment to avoid flowering. Agar and rockwool were examined as substrate. Biomass of photoautotrophic Arabidopsis plants was 3 times greater than that of plants grown under conventional conditions in petri dish. Photoautotrophic plants developed more normal morphology and greener leaves with more trichomes than conventional ones. PAC will be advantageous when one wants to test transgenic lines or materials that are difficult to test in greenhouse. A user-friendly PAC manual was developed based on this demonstration. Methodological ideas to test environmental factors such as humidity and CO₂ concentration will be presented. This controlled environment culture platform that allows whole-plant experimentation using transgenic or tissue culture materials in vitro will be a major contribution, with increasing demand for translational plant biology using other crop species beyond Arabidopsis.