Abstract
This protocol delivers a method to determine the biosynthetic capability of comfrey leaves for pyrrolizidine alkaloids independently from other organs like roots or flowers.The protocol applies and combines radioactive tracer experiments with standard and modern techniques like thin layer chromatography (TLC), solid-phase extraction (SPE), high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS).
Keywords: Tracer, Radio-HPLC, Radio-TLC, SPE, GC-MS, Alkaloid, Plant-organ specificity
Background
Comfrey roots are known to be able to synthesize pyrrolizidine alkaloids (Frölich et al., 2007) and the key enzyme for biosynthesis, homospermidine synthase (HSS), was localized in the endodermis cells. In addition to this site of synthesis, there have been hints that also leaves of a certain developmental stage might be able to produce pyrrolizidine alkaloids (Niemüller et al., 2012). Therefore, a protocol was developed to determine the biosynthetic capability of comfrey leaves to synthesize pyrrolizidine alkaloids independently from other plant organs.
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
This work was funded by a DFG grant given to Dietrich Ober. We thank Dr. Dorothee Langel for her input and ideas contributing to this method. We thank Dr. Christoph Gelhaus and Maren Hartelt, Kiel University, for help in implementing this method. The authors declare no conflict of interest.
References
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