2 Page Physiology of tuber development and stolon architecture 3 Efstathios Roumeliotis Page Thesis committee Thesis supervisor Prof. dr. R. G. F. Visser Professor of Plant Breeding, Wageningen University Thesis co-supervisor Dr. C. W. B. Bachem University lecturer/ researcher, Laboratory of Plant Breeding, Wageningen University Daily supervisor Dr. ir. B. Kloosterman Researcher, Keygene N. V. Other members Prof. dr. A. H. J. Bisseling, Wageningen University Prof. dr. ir. P. C. Struik, Wageningen University Associate prof. dr. D Vreugdenhil, Wageningen University Associate prof. A. Kanellis, Aristotle Univeristy, Hellas This research was conducted under the auspices of the Graduate School of Experimental 4 Plant Sciences Page Physiology of tuber development and stolon architecture Efstathios Roumeliotis Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof.dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Friday 5 October 2012 at 11 a.m. in the Aula. 5 Page Efstathios Roumeliotis Physiology of tuber development and stolon architecture 125 pages. Thesis Wageningen University, Wageningen, NL, (2012) With references, with summaries in English and Dutch 6 ISBN: 978-94-6173-383-2 Page The researcher of this thesis acknowledges financial support from the Bakalas Foundation and the Stichting Veenhuizen-Tulp fonds. Financial support from Wageningen University for printing this thesis is gratefully acknowledged. 7 Page 8 Page Table of Contents General introduction ............................................................................................................... 13 The potato crop ................................................................................................................... 14 Initiation of potato tuber formation ................................................................................... 15 Photoperiod ......................................................................................................................... 16 Phytohormones ................................................................................................................... 17 Other factors ....................................................................................................................... 19 The role of auxin in plant development .............................................................................. 20 Outline of this thesis ............................................................................................................ 21 References ........................................................................................................................... 23 The effects of auxin and strigolactones on tuber initiation and stolon architecture in potato ................................................................................................................................................. 31 Abstract ............................................................................................................................... 32 Introduction ......................................................................................................................... 33 Materials and methods ....................................................................................................... 35 Plant materials and in vitro tuberization ................................................................................... 35 Detection of strigolactones in potato roots ............................................................................... 35 Assessment of the auxin concentrations ................................................................................... 35 DR5::GUS transgenic plants and GUS staining .......................................................................... 36 Quantitative RT-PCR. ............................................................................................................... 37 Auxin transport assays .............................................................................................................. 37 Results ................................................................................................................................. 38 Auxin measurement in potato plants under inductive and non-inductive conditions ................. 38 Expression of an auxin biosynthesis gene ( StYUC-like1 ) in the potato stolon ............................. 41 The effects of IAA, SL and their inhibitors on stolon axillary bud outgrowth and tuberization in vitro .......................................................................................................................................... 42 IAA transport assays ................................................................................................................. 44 Discussion ............................................................................................................................ 45 Acknowledgements ............................................................................................................. 48 References ........................................................................................................................... 48 The PIN family of proteins in potato and their putative role in tuberisation ..................... 53 Abstract ............................................................................................................................... 54 Introduction ......................................................................................................................... 55 Materials and methods ....................................................................................................... 56 Identification of StPIN gene sequences ..................................................................................... 56 Cloning and analysis of STPINV and StPINVII promoter sequences........................................... 57 Expression analysis of StPIN genes ........................................................................................... 57 Results and discussion ......................................................................................................... 58 Identification of the PIN gene family members in potato .......................................................... 58 9 Tissue specificity expression of potato PIN family members ..................................................... 59 Page PIN expression during tuber development ................................................................................ 62 StPINV/VII promoter GUS staining ............................................................................................ 63 Conclusion ........................................................................................................................... 64 Acknowledgements ............................................................................................................. 65 References ........................................................................................................................... 65 Down regulation of StGA3ox2 gene in potato results in altered GA metabolism and effect plant and tuber growth characteristics ................................................................................... 68 Abstract ............................................................................................................................... 69 Introduction ......................................................................................................................... 70 Materials and methods ....................................................................................................... 71 Construction of the StGA3ox2 RNAi and promStGA3ox2 ::GUS plasmids ................................... 71 Plant material ........................................................................................................................... 72 Quantitative RT-PCR ................................................................................................................ 72 GA measurements .................................................................................................................... 73 Results ................................................................................................................................. 73 Expression levels of the StGA2ox2 gene in the RNAi clones. ..................................................... 73 StGA3ox2 RNAi transformed clones have shorter plants with smaller leaves and smaller average tuber weight. ............................................................................................................................ 74 Localisation of StGA3ox2 expression. ....................................................................................... 75 Discussion ........................................................................................................................... 80 References ........................................................................................................................... 82 Over-expression of a YUCCA-like gene results in altered shoot and stolon branching and reduced potato tuber size ....................................................................................................... 85 Abstract ..............................................................................................................................