Establishment of a model system for studying polyacetylene biosynthesis in Asteraceae and studies on transformation and cryopreservation of carrot cells Von der Fakultät für Lebenswissenschaften der Technischen Universität Carolo-Wilhelmina zu Braunschweig zur Erlangung des Grades einer Doktorin der Naturwissenschaften (Dr. rer. nat.) genehmigte D i s s e r t a t i o n von Nargis Farag Abdelhadi Elgahme aus Benghazi/Libyen 1. Referentin: Prof. Dr. Ute Wittstock 2. Referent: Prof. Dr. Ludger Beerhues eingereicht am: 16.11.2016 mündliche Prüfung (Disputation) am: 04.05.2017 Druckjahr 2018 Table of Contents Table of contents Table of contents ......................................................................................................................... I List of Figures .......................................................................................................................... IV List of Tables ........................................................................................................................... VII Abbreviations ........................................................................................................................ VIII 1 Introduction ............................................................................................................................ 1 1.1 Specialized metabolites in plant tissue culture ................................................................ 1 1.2 Polyacetylenes in plants ................................................................................................... 3 1.2.1 Definition, properties and biological activity of polyacetylenes .................................. 3 1.2.2 Biosynthesis of polyacetylenes ........................................................................................ 8 1.2.3 Polyacetylenes in Asteraceae.......................................................................................... 11 1.2.4 Polyacetylenes from Tagetes patula .............................................................................. 14 1.2.5 Polyacetylenes from Arctium lappa ............................................................................... 20 1.2.6 Polyacetylenes from Carthamus tinctorius ................................................................... 22 1.3 Glucosinolates ................................................................................................................ 26 1.3.1 The glucosinolate-myrosinase system ........................................................................... 26 1.3.2 Glucosinolate biosynthesis ............................................................................................. 28 1.3.3 Genetic engineering of glucosinolate biosynthesis in heterologous hosts................. 32 1.3.4 The viral 2A polycistronic open reading frame as a tool to express multiple genes in eukaryotic cells................................................................................................................. 32 1.4 Aim of this work ............................................................................................................ 35 2 Experimental ........................................................................................................................ 36 2.1 Chemicals and biochemicals........................................................................................... 36 2.2 Material and methods for polyacetylene analysis .......................................................... 36 2.2.1 Seed source ....................................................................................................................... 36 2.2.2 Soil-grown plants ............................................................................................................. 36 2.2.3 Aseptically grown plants ................................................................................................. 37 2.2.3.1 Seed sterilization and treatment ........................................................................ 37 2.2.3.2 Plant growth and maintenance .......................................................................... 39 2.2.4 Tissue culture .................................................................................................................... 39 2.2.4.1 Callus culture ..................................................................................................... 39 2.2.4.2 Callus suspension culture .................................................................................. 40 2.2.4.3 Root culture ....................................................................................................... 40 2.2.4.4 Hairy root culture .............................................................................................. 41 2.2.5 Elicitation treatment ......................................................................................................... 44 2.2.5.1 Elicitors ............................................................................................................. 44 I Table of Contents 2.2.5.2 Elicitor treatment of suspension cultures ......................................................... 45 2.2.5.3 Elicitor treatment of root cultures .................................................................... 45 2.2.5.4 Elicitor treatment of hairy root cultures ........................................................... 45 2.2.6 Phytochemical analysis of polyacetylene content ....................................................... 46 2.2.6.1 Extraction of polyacetylenes ............................................................................ 46 2.2.6.2 Identification of polyacetylenes ....................................................................... 46 2.2.6.3 Quantification of polyacetylenes ...................................................................... 47 2.3 Material and methods for carrot cell suspension culture as transgenic production platform ......................................................................................................................... 48 2.3.1 Bacterial strains, plasmid vectors, and PCR primers .................................................. 48 2.3.2 Cultivation of carrot callus suspension culture ............................................................ 49 2.3.3 Synchronization of carrot callus suspension culture ................................................... 50 2.3.4 Agrobacterium-mediated transformation of carrot suspension culture ..................... 50 2.3.4.1 Method Ӏ ........................................................................................................... 50 2.3.4.2 Method II .......................................................................................................... 52 2.3.5 Isolation of genomic DNA from transformed carrot cells .......................................... 53 2.3.6 Cryopreservation of suspension-cultured cells and transgenic cell lines .................. 53 2.3.7 Transformation of A. tumefaciens C58C1 .................................................................... 54 2.3.8 Transformation of E. coli ............................................................................................... 56 2.3.9 Molecular biology techniques ........................................................................................ 57 2.3.9.1 PCR .................................................................................................................. 57 2.3.9.2 Agarose gel electrophoresis and DNA purification from the gel ..................... 58 2.3.9.3 DNA Digestion ................................................................................................. 58 2.3.9.4 Ligation of DNA fragments ............................................................................. 59 2.3.9.5 Isolation of plasmid DNA from E. coli ............................................................ 59 2.3.9.6 DNA-sequencing .............................................................................................. 60 2.3.10 Protein biochemical methods ....................................................................................... 60 2.3.10.1 SDS-PAGE ..................................................................................................... 60 2.3.10.2 Western blot ................................................................................................... 62 2.3.10.3 Plant extracts for detection of myrosinase ..................................................... 63 2.3.10.4 Myrosinase assay ............................................................................................ 63 3 Results ................................................................................................................................. 64 3.1 Polyacetylene composition in tissue cultures and soil-grown plants of the Asteraceae 64 3.1.1 Polyacetylene composition of Tagetes patula ............................................................. 64 3.1.1.1 Identification and quantification of five thiophenes from T. patula ................ 64 3.1.1.2 Profile of thiophenes in different organs of soil-grown T. patula .................... 66 3.1.1.3 Thiophene content in T. patula root cultures ................................................... 68 II Table of Contents 3.1.1.4 Thiophene content in T. patula callus culture .................................................. 69 3.1.1.5 Thiophene content in T. patula hairy root cultures .......................................... 70 3.1.1.6