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.)

Authors: PETREK, JIRI - MENDEL UNIV, BRNO, CZ; VITECEK, JAN - MENDEL UNIV, BRNO, CZ; VLASINOVA, HELENA - MENDEL UNIV, BRNO, CZ; KIZEK, RENE - MENDEL UNIV, BRNO, CZ; KRAMER, KARL - 5430-05-30 RETIRED; ADAM, VOJTECH - MENDEL UNIV, BRNO, CZ; KLEJDUS, BORIVOJ - MENDEL UNIV, BRNO, CZ; HAVEL, LADISLAV - MENDEL UNIV, BRNO, CZ; Beeman, Richard

Submitted to: Bioanalytical Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/13/2005
Publication Date: 10/12/2005
Citation: Petrek, J., Vitecek, J., Vlasinova, H., Kizek, R., Kramer, K.J., Adam, V., Klejdus, B., Havel, L., Beeman, R.W. 2005. 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.). Analytical Bioanalytical Chemistry 383: 576-586.

Interpretive Summary: Plant growth is a fundamental parameter that must be accurately measured in many kinds of experimental studies investigating abiotic and biotic stresses on plants. There is a need for assessing effects on plant growth and viability using more rapid nondestructive methods such as image analysis (IA). In a collaborative project with scientists in the Czech Republic, we used IA to study the growth and morphology of embryos of the Norway spruce and also to determine the effects of a heavy metal pollutant (lead) on development of the embryos. We proved that IA could be used to analyze basic parameters and to assess the influence of stresses. There was a good correlation between IA and destructive methods commonly used for these purposes. IA may have potential use in quality control assessments of plants derived from traditional breeding and genetic transformation programs.

Technical Abstract: Image analysis (IA) was used to determine the areas and circumferences of clusters of early somatic embryos (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 increases in fresh weight (R2=0.99) of the ESEs and their esterase activities (R2=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) determining esterase activity using a spectrofluorimetric detector. The results obtained with IA and esterase assay were comparable (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. Glutathione is a diagnostic marker of the influence of Pb-EDTA on ESEs and its content was determined by high-performance liquid chromatography coupled with mass spectrometry. 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.