A non-destructive method for secondary metabolite determination in hairy root cultures

Traditional analytical methods of determining secondary metabolite levels ( e.g., chromatography, spectrophotomety) are based on extraction of the test ed tissue and involve its destruction, As a result, it is very difficult to determine such levels in a small localized area. Moreover, because measure ments performed in the course of culture necessarily refer to different sam ples of tissue, levels recorded could reflect variability in the hairy root s themselves, rather than variation as a function of time or morphological location. In this communication we report on a method developed to measure local and overall secondary metabolite levels in a non destructive manner u sing image analysis and apply it to determine metabolite concentrations in hairy root cultures of Beta vulgaris. To develop the method, after scanning a root under sterile conditions and replacing it in the growth vessel, we converted the image data from red-green-blue to the hue-saturation-intensit y coordinate system. Hue and saturation values served respectively for thre sholding and determination of pigment level. Root diameter, simulated by th ickness of root extract films, was found to be proportional to saturation v alues, and thus is taken into account when pigment concentration is determi ned. The data obtained by image analysis were used to determine local and o verall accumulation of betacyanin and betaxanthin pigments over time and re sults were then validated by spectrophotometric analysis. It was concluded that the proposed non destructive method enables accurate determination of changes in local secondary metabolite level at any point in the root, as we ll as monitoring of changes in overall levels in the entire root system thr oughout the growth period.