Metabolite Production in Callus Culture of Burdock (Arctium lappa L.) THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Bizhen Hu Graduate Program in Horticulture and Crop Science The Ohio State University 2012 Master's Examination Committee: Professor John Cardina, Advisor Professor John J. Finer Professor Joseph C. Scheerens Copyrighted by Bizhen Hu 2012 Abstract Burdock (Arctium lappa L.) is a biennial plant that is generally regarded as a weed in the U.S. However, in the Amish community, it has been used for treating burns by placing burdock leaves on the wounds after the application of salve. This folk medicine is believed to be effective to speed healing, relieve pain, reduce scars, and prevent infection. More knowledge of the mechanism of burdock treating burns and practical methods for its application is needed before burdock can be recommended as a conventional burn treatment. Few studies have been conducted to determine the basis for burdock treatment for burns. The objectives of this research were to develop callus culture technique for phytochemical production from burdock for eventual medical usage, and analyze these potential medicinal metabolites. The metabolites in burdock we were most interested in were phenolic compounds because of their high content in burdock and their well-known antioxidant bioactivities and related properties which are likely to be useful for treating burns. The methods we used included plant tissue culture and biochemical analysis. We first developed procedures for burdock callus culture by testing different explant types and culture conditions, with emphasis on the type and concentrations of growth regulators in the culture media. After burdock callus culture was developed, we added different ii concentrations of methyl jasmonate and sucrose to the culture media and assessed their effects on callus growth and phenolic production. To identify and quantify the phenolic components in burdock callus, we analyzed the phenolic extraction from callus by the Folin-Cuicaktey assay and reversed-phase HPLC-DAD methods. Burdock callus could be induced from burdock cotyledon or leaf sections in the dark on media with 1 mg/L BA and 2 mg/L 2, 4-D, and then maintained over the long term on media with 1.5 mg/L NAA and 1 mg/L 2, 4-D. Various phenolic compounds were detected by HPLC and preliminarily identified as hydroxycinnamic acid derivatives based on their retention times and UV-spectra. Addition of methyl jasmonate up to 1 mM and 60 g/L and 90 g/L sucrose increased the content of total phenolic and some major individual phenolic compounds in burdock callus, but decreased callus growth. The optimum concentrations were 1 mM or 0.1 mM MeJA and 30 g/L sucrose for high total phenolic production (total phenolic content × callus relative growth). Our results provide a foundation for further studies on burdock metabolite production. The callus culture system can be used as a method for future phytochemical study of burdock and practical production of burdock medicinal metabolites. iii Acknowledgments I would like to thank my advisor Dr. John Cardina, co-advisor Dr. Joseph C. Scheerens and my committee member Dr. John J. Finer for their help and guidance. I would also like to thank Dr. Joshua Blakselee, who gave me advice for the biochemistry studies. Lisa Robbins, a graduate student working in this project with me, helped me a lot with my research and studies. Dr. Ann Chanon, the technician in Dr. Scheerens’ lab and Dr. Blakselee’s lab, and Jinshan Lin in Dr. Blakselee’s lab, both of whom helped me a lot with my lab work in biochemistry area. Catherine Herms in Dr. Cardina’s lab helped me order seeds and grow plants in the greenhouse and field. Members in Dr. Finer’s lab, including Carola De La Torre, Carlos Hernandez-Garcia, Cheri Nemes and Zhifen Zhang, helped me with the plant tissue culture experiments. iv Vita 2010................................................................B.S. Agronomy, China Agricultural University 2010 to present ..............................................M.S. Horticulture and Crop Science, The Ohio State University Fields of Study Major Field: Horticulture and Crop Science v Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iv Vita ...................................................................................................................................... v List of Tables ..................................................................................................................... ix List of Figures ..................................................................................................................... x Chapter 1: Introduction ...................................................................................................... 1 Burdock ........................................................................................................................... 1 Burdock use for burn treatment ....................................................................................... 2 Phenolics in burdock might be responsible for burn treatment ....................................... 3 Use of plant cell culture to produce phenolics ................................................................ 5 Methyl jasmonate and sucrose stimulate in vitro phenolic production ........................... 6 Research objectives ......................................................................................................... 8 References ....................................................................................................................... 9 Chapter 2: Callus Culture of Burdock .............................................................................. 13 Summary ....................................................................................................................... 13 vi Introduction ................................................................................................................... 14 Materials and Methods .................................................................................................. 16 Explants and culture conditions ................................................................................. 16 Callus induction ......................................................................................................... 18 Callus maintenance .................................................................................................... 19 Results ........................................................................................................................... 20 Callus induction ......................................................................................................... 20 Callus maintenance .................................................................................................... 21 Discussion ..................................................................................................................... 23 References ..................................................................................................................... 26 Chapter 3 Effects of Methyl Jasmonate and Sucrose on Phenolic Accumulation, and Phenolic Profiles in Burdock Callus Culture .................................................................... 34 Summary ....................................................................................................................... 34 Introduction ................................................................................................................... 35 Materials and Methods .................................................................................................. 39 Reagent, materials and culture conditions ................................................................. 39 Effects of MeJA on phenolic production in burdock callus culture .......................... 40 Effects of sucrose concentrations on phenolic production in burdock callus culture 41 Methods for phenolic extraction ................................................................................ 41 vii Determination of total phenolic content .................................................................... 43 Analysis of individual phenolic compounds by HPLC ............................................. 43 Statistical analysis...................................................................................................... 44 Results ........................................................................................................................... 46 Effects of MeJA on burdock callus growth and total phenolic content ..................... 46 Effects of sucrose on burdock callus growth and total phenolic content .................. 47 Optimization of MeJA and sucrose treatment for total phenolic production ............ 48 Individual phenolic profiles determined by HPLC.................................................... 49 Effects of MeJA and sucrose on individual phenolic content ................................... 50 Discussion ..................................................................................................................... 52 References ....................................................................................................................