Integrated Application of Physiological and Molecular Methods to Forecast Determinative Morphogenetic Events in Tissue Cultured Tobacco (Nicotiana tabacum L. cv Samsun) Leaf Discs

The purpose of this study was to search for physiological parameters that provide an early indication of the morphogenetic response of leaf disc explants to different tissue culture-level manipulations in order to design an accelerated optimisation process for this technology. Tobacco (Nicotiana tabacum L. cv. Samsun) was chosen as model in our studies, because this is still one of the most widely used species in experimental plant biology and detailed knowledge is available from its tissue culture system. Large numbers of physiological markers at the whole plant level (rates of photosynthesis and respiration) and cellular level ‘fitness’ (degree of DNA methylation) were measured together with the concentrations of the most abundant metabolites in photosynthetic carbohydrate metabolism, ATP and protein synthesis in leaf discs induced either for callus development or for shoot differentiation. As a result of the above examinations, we are able to show that the efficiency of photosynthesis, the rate of respiration and the degree of DNA methylation can be used as early markers for the changes that precede the appearance of in vitro morphogenesis. By examining these parameters, we were able to predict early ontogenesis (within 48 h) and the optimal concentrations of growth regulators needed to achieve either shoot differentiation or callus development.