Metabolic evaluation of cellular differentiation of tobacco leaf explants in response to plant growth regulators in tissue cultures using 1H NMR spectroscopy and multivariate analysis

To investigate the metabolic changes that precede visible organogenesis in tissue culture, tobacco leaf explants were cultured on media supplemented with various plant growth regulators (PGRs) and analyzed with proton nuclear magnetic resonance (1H NMR) spectroscopy. Principal component analysis (PCA) of 1H NMR spectral data was unable to differentiate between leaf explants cultured with α-naphthaleneacetic acid and those cultured with 6-benzyladenine after 4 days of culture; however, a difference was evident after 8 days of culture. A hierarchical dendrogram from PCA analysis could be grouping leaf explants cultured with various auxins separately from those treated by various cytokinins. However, leaf explants cultured with thidiazuron (TDZ) were identified as an outlier group; TDZ appeared to produce pleiotropic metabolic effects that differed from those induced by other PGRs. These results show that dedifferentiation can be initiated by either auxins or cytokinins, which is reflected by similar metabolic changes produced by the two distinct PGRs during the initial incubation period. The subsequent redifferentiation differs according to the PGR treatment, which is reflected by differential metabolic changes, depending on the fate of cells in organogenesis. Glutamine and glutamate levels increased approximately twofold in cytokinin-treated leaf explants compared with auxin-treated explants; however, changes in the levels of sugar compounds did not differ between the two treatments, demonstrating auxin regulation of the carbon/nitrogen ratio in favor of rooting. Taken together, our results suggest that 1H-NMR spectroscopy combined with multivariate analysis is a promising means for the metabolic evaluation of plant growth and differentiation.