Use of photoautotrophic cultures for highthroughput screening of toxic compounds by fluorescence imaging
Research output: Book/Report › Ph.D. thesis › Research
With increasing pollution, herbicide application and interest in plant phenotyping, sensors capturing early responses to toxic stress are required for screening of susceptible or resistant plant varieties. However, standard toxicity tests on plants are laborious, demanding in terms of space and material and measurement of growth-inhibition-based endpoints requires a relatively long time. The aim of this study was to establish a multi-sensor system based on fluorescence imaging for high-throughput screening of early toxic stress. In order to minimize the duration and space requirements of plant toxicity screening tests, the potential of photoautotrophic (PA) cell suspensions for such setup was tested. As biomarkers of toxic stress, parameters and ratios of multicolor fluorescence and chlorophyll fluorescence induction were measured. The selected methods proved to be suitable candidates for toxicity screening as the stress was detected non-invasively, earlier than inhibition of plant growth and they also allowed detection of mild and transient stress responses. Based on the measured parameters, it was possible to construct stress signatures specific for three toxic compounds with different modes of action. The study also confirmed that the PA cell suspension reacts to tested toxicants faster and in lower concentrations than whole plants, that it is compatible with testing in microplates and thus could be useful for rapid high-throughput pre-screening of toxic stress.Earlier studies on the subject ha ve established that PA cell suspensions can be used for obtaining interesting and relevant data also in fields other than toxicology. Nevertheless, one of the reasons the use of PA cell suspensions is not more widespread is the necessity of special cultivation settings. The traditional cultivation setup based on a batch carbonate-bicarbonate buffer system providing the desired CO2 concentration for the culture consists of a rather elaborate structure of custom-made glass vessels. Moreover, as our study indicates, the provided CO2 concentration is not stable. Therefore, improvements in the cultivation setup were made. A small-scale cultivation setup alternative to the conventional one was established in a photoincubator with adjustable ambient CO2 level. The growth and biomass characteristics of the PA cell suspensions in this setup were comparable with the conventional one and the biomass accumulation rate was even more stable throughout the cultivation period. The second improvement to the cultivation setup consists in employment of a laboratory scale photobioreactor. This setup provides a semi-upscale, online monitoring of relevant cultivation parameters and settings and allows for employment of dynamic cultivation protocols. Optimization of growth of PA cell suspension in a photobioreactor gave satisfactory results, although the biomass characteristics did not reach those of the conventional setup. Probable causes of such insufficiency as well as possibilities for further improvement were listed. The innovated cultivation setups provide stability and adjustability of cultivation conditions, ensuring full control of such factors for experimental use and broaden the range of possible applications of PA cell suspensions. On top of that, all components and devices are commercially available, thus making not only the cultivation of PA cell suspensions available but also contributing to standardization of research employing PA cell suspensions.
|Publisher||Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen|
|Number of pages||156|
|Publication status||Published - 2020|