Cytokinins are a group of phytohormones discovered about half a decade ago by Miller et al. (1955) and Skoog et al. (1965). Since then they were found to participate in many plant physiological processes, including the regulation of the source/sink transitions, plant growth and organ development, responses to environmental conditions such as light, nutrient and water availability and biotic interactions with mutualists, pathogens and herbivores (Werner and Schmülling, 2009; Giron et al., 2013). To aid the quantification of cytokinins for analyzing their changes after environmental stress conditions, we developed this cytokinin extraction and analysis method. This protocol is based on the cytokinin extraction with an acidic methanol-water solution and purification with a mixed-mode solid phase extraction procedure described by Dobrev and Kamı́nek (2002) and the modifications of Kojima et al. (2009). The protocol was successfully used to verify cytokinin overproduction in transgenic Nicotiana attenuata plants expressing the cytokinin biosynthesis gene Tumor morphology root (Tmr) from Agrobacterium tumefaciens under the control of the chemical inducible expression system pOp6/LhGR in the glasshouse and under field conditions (Schäfer et al., 2013) to study the role of cytokinins in plant-herbivore interactions.
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