Heartwood formation and the chemical basis of natural durability in Eucalyptus bosistoana A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Forestry By Gayatri Mishra School of Forestry University of Canterbury Senior supervisor: Dr. Clemens Altaner School of Forestry, University of Canterbury Co-Supervisor: Dr. Ashley Garrill Biological Sciences, University of Canterbury Associate Supervisor: Dr. David Collings School of Environmental & Life Sciences University of Newcastle Acknowledgements At the outset, I express my deep sense of gratitude and appreciation to my principal supervisor, Dr. Clemens M. Altaner, School of Forestry, University of Canterbury for providing me an opportunity to work in this project. Without his ceaseless supervision, support, motivation and robust knowledge base this task would not have been completed. His guidance has helped me immensely in my research and writing the thesis till its logical end. Besides, I feel extremely fortunate to work with my associate supervisor, Dr. David Collings and Co- supervisor, Dr. Ashley Garrill too. Microscopy being a major part of my research, I feel deeply indebted to Dr. David Collings for introducing me into the realms of advanced microscopic skills coupled with his constant guidance. Even after his leaving the University in the meanwhile, he was considerate enough to bridge the gap through his timely communications via emails or video conversations. His unique concepts and expertise generated my strong interest in microscopy. I also thank Dr. Ashley Garrill for providing me with necessary lab facilities to perform bioactivity tests with fungi. His expertise has been a great source of support to my bioactivity experiments. Proficient sectioning skills have been an important part of my research too. Hence, I gratefully acknowledge the kind assistance of technical staff, Graeme Bull and Riejel Gardiner from the School of Biological Sciences for helping me to develop sectioning skills using sledge microtome. I would also thank Manfred Ingerfeld profusely for assisting me in confocal microscopy. Heartfelt thanks to the technical staff, Lachlan Kirk and Nigel Pink, from the School of Forestry for their unequivocal help extended to me. I am extremely grateful to Meike Holzenkampfer as she taught me to operate the gas chromatograph and her expertise in chemical analyses has been an asset in understanding the gas chromatograph with clarity. Thanks to the authorities and staff of university library for granting me the required permission to utilise their valuable collections for my reference. Last but not the least, I express my heartiest gratitude to my parents and sister for being supportive pillars in my current mission. Above all, I am grateful to the almighty for paving the way in accomplishing my dream. 1 Table of Contents Acknowledgements ................................................................................................................................. 1 Table of Contents .................................................................................................................................... 2 List of Figures ......................................................................................................................................... 6 List of Tables ........................................................................................................................................ 10 Abstract ................................................................................................................................................. 13 Chapter-1 .............................................................................................................................................. 16 1.1 New Zealand Dryland Forests Initiative’s (NZDFI) Eucalyptus breeding programme .............. 16 1.2 Research background .................................................................................................................. 17 1.3 Wood formation .......................................................................................................................... 18 1.4 Wood anatomy ............................................................................................................................ 19 1.5 Microscopy of wood ................................................................................................................... 20 1.5.1 Colour based and fluorescent stains ..................................................................................... 21 1.5.2 Immunolabelling .................................................................................................................. 22 1.6 Natural durability ........................................................................................................................ 23 1.6.1 Fungi .................................................................................................................................... 23 1.6.2 Wood degrading insects ....................................................................................................... 24 1.7 Extractives................................................................................................................................... 25 1.7.1 Extractives variation within trees ......................................................................................... 25 1.8 Extraction methods ..................................................................................................................... 25 1.9 Extractives analysis ..................................................................................................................... 26 1.9.1 Gas chromatography (GC) ................................................................................................... 27 1.9.2 High performance liquid chromatography (HPLC) ............................................................. 27 1.9.3 Thin layer chromatography (TLC) ....................................................................................... 28 1.10 Objectives of the thesis ............................................................................................................. 29 1.11 References ................................................................................................................................. 30 Chapter-2 .............................................................................................................................................. 39 2.1 Introduction ................................................................................................................................. 39 2.2 Materials and methods ................................................................................................................ 40 2.2.1 Wood material and fixation .................................................................................................. 40 2.2.2 Histochemistry ..................................................................................................................... 41 2.2.3 Immunofluorescence microscopy ........................................................................................ 41 2.2.4 Confocal microscopy ........................................................................................................... 42 2.3 Results and Discussion ............................................................................................................... 42 2.3.1 Histochemical observations ................................................................................................. 42 2.3.2 Autofluorescence ................................................................................................................. 45 2 2.3.3 Immunolocalisation of organelles and structures ................................................................. 50 2.4 Conclusion .................................................................................................................................. 51 2.5 References ................................................................................................................................... 52 Chapter-3 .............................................................................................................................................. 57 Physiological changes during heartwood formation in young (6-year-old) Eucalyptus bosistoana ..... 57 3.1 Introduction ................................................................................................................................. 57 3.2 Materials and methods ................................................................................................................ 59 3.2.1 Wood material and fixation .................................................................................................. 59 3.2.2 Wide-field fluorescence microscopy.................................................................................... 60 3.2.3 Immunolabelling nuclei and microtubules ........................................................................... 60 3.2.4 Confocal laser scanning (confocal) microscopy .................................................................. 61 3.2.5 Image processing.................................................................................................................. 61 3.3 Results and discussion ...............................................................................................................