Anal Bioanal Chem (2005) 383: 576–586 DOI 10.1007/s00216-005-0028-3 ORIGINAL PAPER Jiří Petřek . Jan Víteček . Helena Vlašínová . René Kizek . Karl J. Kramer . Vojtěch Adam . Bořivoj Klejdus . Ladislav Havel Application of computer imaging, stripping voltammetry and mass spectrometry to study the effect of lead (Pb-EDTA) on the growth and viability of early somatic embryos of Norway spruce (Picea abies /L./ Karst.) Received: 10 January 2005 / Revised: 8 July 2005 / Accepted: 13 July 2005 / Published online: 12 October 2005 # Springer-Verlag 2005 Abstract Image analysis (IA) was used to determine the areas and circumferences of clusters of early somatic em- bryos (ESEs) of the Norway spruce (Picea abies /L./Karst.). Results obtained from IA were compared with the fresh weights of the ESE clusters and their esterase activities. The areas of the ESE clusters correlated well with both the in- creases in fresh weight (R 2 =0.99) of the ESEs and their esterase activities (R 2 =0.99). In addition, we studied the viability of the ESEs, which was determined by (a) double staining with fluorescein diacetate and propidium iodide (the resulting fluorescence was quantified by IA) and (b) deter- mining esterase activity using a spectrofluorimetric detector. The results obtained with IA and esterase assay were com- parable (the deviation between the tangents of the bisectors was 6.4%). IA was also used to study the effect of Pb–EDTA chelate (50, 250 and 500 μM) on the viability of the ESEs and on the growth of clusters. The presence of Pb–EDTA markedly slowed the growth of ESEs clusters (by more than 65% with 250 μM of Pb–EDTA after 288 h of cultivation) and decreased the viability of ESEs (by more than 30% with 500 μM of Pb–EDTA after 288 h of cultivation). The lead concentration in the ESEs was determined by differential pulse anodic stripping voltammetry and increased with the external lead concentration and the time of treatment from 100 to 600 pg Pb/100 mg of fresh weight of ESEs. Gluta- thione is a diagnostic marker of the influence of Pb–EDTA on ESEs and its content was determined by high–perfor- mance liquid chromatography coupled with mass spectrom- etry. The glutathione content changed linearly with treatment time and the applied external lead concentration. The highest glutathione content was obtained at 250 μM of Pb–EDTA after 192 h of cultivation. Keywords Early somatic embryo of Spruce . Computer image analysis . Lead . Glutathione . Differential pulse anodic stripping voltammetry Abbreviations IA: computer image analysis . ESE: early somatic embryo . CCD: charge-coupled device . Pb-EDTA: lead (II)-ethylene diamine tetra-acetic acid . FDA: fluorescein diacetate . PI: propidium iodide . GSH: glutathione . HPLC-MS: High-performance liquid chromatography mass spectrometry . DPASV : Differential pulse anodic stripping voltammetry Introduction The plant growth curve is a fundamental parameter for a plant physiologist and its measurement in plant tissues and cell cultures can be accomplished using several different methods [1]. The most frequently used methods are based on the counting and/or weighing of cells or tissues [1]. The disadvantages of these methods are the destruction and/or contamination of cultures during analysis. In recent years, J. Petřek . J. Víteček . H. Vlašínová . L. Havel Department of Botany and Plant Physiology, Mendel University of Agriculture and Forestry, Zemědělská 1, 613 00, Brno, Czech Republic R. Kizek (*) . V. Adam . B. Klejdus Department of Chemistry and Biochemistry, Mendel University of Agriculture and Forestry, Zemědělská 1, 613 00, Brno, Czech Republic e-mail: kizek@sci.muni.cz Tel.: +420-5-45133350 Fax: +420-5-45212044 K. J. Kramer Grain Marketing and Production Research Center Agricultural Research Service, US Department of Agriculture, Manhattan, KS 66502, USA V. Adam Department of Analytical Chemistry, Faculty of Science, Kotlarska 2, 611 37, Brno, Czech Republic