BACKGROUND: Recently emerging approaches to high-throughput plant phenotyping have discovered their importance as tools in unravelling the complex questions of plant growth, development and response to the environment, both in basic and applied science. High-throughput methods have been also used to study plant responses to various types of biotic and abiotic stresses (drought, heat, salinity, nutrient-starving, UV light) but only rarely to cold tolerance. RESULTS: We present here an experimental procedure of integrative high-throughput in-house phenotyping of plant shoots employing automated simultaneous analyses of shoot biomass and photosystem II efficiency to study the cold tolerance of pea (Pisum sativum L.). For this purpose, we developed new software for automatic RGB image analysis, evaluated various parameters of chlorophyll fluorescence obtained from kinetic chlorophyll fluorescence imaging, and performed an experiment in which the growth and photosynthetic activity of two different pea cultivars were followed during cold acclimation. The data obtained from the automated RGB imaging were validated through correlation of pixel based shoot area with measurement of the shoot fresh weight. Further, data obtained from automated chlorophyll fluorescence imaging analysis were compared with chlorophyll fluorescence parameters measured by a non-imaging chlorophyll fluorometer. In both cases, high correlation was obtained, confirming the reliability of the procedure described. CONCLUSIONS: This study of the response of two pea cultivars to cold stress confirmed that our procedure may have important application, not only for selection of cold-sensitive/tolerant varieties of pea, but also for studies of plant cold-response strategies in general. The approach, provides a very broad tool for the morphological and physiological selection of parameters which correspond to shoot growth and the efficiency of photosystem II, and is thus applicable in studies of various plant species and crops.

Department of Biophysics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Šlechtitelů 11 Olomouc CZ 78371 Czech Republic

Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Šlechtitelů 11 Olomouc CZ 78371 Czech Republic

Department of Genetic Resources for Vegetables Medicinal and Special Plants Centre of the Region Haná for Biotechnological and Agricultural Research Crop Research Institute Šlechtitelů 11 Olomouc CZ 78371 Czech Republic

Department of Mathematical Analysis and Applications of Mathematics Faculty of Science Palacký University 17 listopadu 12 Olomouc CZ 77146 Czech Republic

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