Developing Innovative Marker Systems for Garlic Post-Harvest Quality Assessment A thesis submitted to Cardiff University for the degree of Doctor of Philosophy By Richard Andrew Ludlow, BSc (hons) September 2019 i DECLARATION This work has not been submitted in substance for any other degree or award at this or any other university or place of learning, nor is being submitted concurrently in candidature for any degree or other award. Signed ……………………………………………………… (candidate) Date ………………….…………….……… STATEMENT 1 This thesis is being submitted in partial fulfilment of the requirements for the degree of Ph.D. Signed………………………………………….…………… (candidate) Date …………………………….…………… STATEMENT 2 This thesis is the result of my own independent work/investigation, except where otherwise stated, and the thesis has not been edited by a third party beyond what is permitted by Cardiff University’s Policy on the Use of Third Party Editors by Research Degree Students. Other sources are acknowledged by explicit references. The views expressed are my own. Signed ……………………………………….……….…… (candidate) Date …………………….………………… STATEMENT 3 I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed ……………………………………………..…..….. (candidate) Date ………………………………………… STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS I hereby give consent for my thesis, if accepted, to be available online in the University’s Open Access repository and for inter-library loans after expiry of a bar on access previously approved by the Academic Standards & Quality Committee. Signed ……………………………………………..……… (candidate) Date ………………………………….……… Word Count………………………………… ii Acknowledgements I would firstly like to thank my supervisors Prof. Hilary Rogers and Dr Carsten Müller for their support, guidance and encouragement. Thanks are also due to Gareth Evans and Michael Graz of Neem Biotech for their advice and support throughout the project. I would also like to thank the members of my lab and friends within BioScience and at Neem for their assistance and friendship. Whether it has been through lunchtime conversations, walks, brewing or weekend BBQs, they have always been there to support me. Finally, but by no means least, I would like to thank my Mum, Trev, Emma and the rest of my family for their never ending encouragement and backing throughout this process. iii Synthesis Work presented here furthers current understanding of the postharvest biology of garlic and identifies volatile organic compounds (VOCs) as markers of quality for culinary and biotechnological uses of garlic. Quality markers were developed for gauging storage time, storage temperature, harvest year, cold stress, incidence of disease and alliinase activity. This work also furthers understanding of interspecific and interseasonal variance in postharvest quality traits of garlic. Significant headway was made in translating the VOC quality markers into techniques for industrial application. Chapter 3 focusses on a single garlic cultivar and discovered improved maintenance of alliinase activity in ambient storage, compared to the industry standard of -2 °C but also found that alliinase activity loss did not correlate with alliinase gene expression. The water content of cloves was stable over time and weight loss in garlic occurred at a 1:2 ratio of DW to FW. VOC profiles varied significantly between storage time and temperature; compounds were identified as putative markers for storage conditions. Chapter 4 explores the interseasonal and interspecific variation in quality and alliinase activity throughout storage, and whether VOC based quality assessment is reliable across years and cultivars. A complex relationship was identified, with quality traits of cultivars changing differently according to season, time and temperature of storage. VOC marker systems correlated with alliinase activity, disease rate and discriminated between seasons. Chapter 5 applies the findings from lab work to the industry. A new thermal desorption sampling method allows passive sampling of VOCs from warehouse air and was trialled over a 6 month period in industry. Cold stress did not affect alliinase activity, iv but caused significant changes to quality parameters and the VOC profile. Finally, a pulsed flame photometric detector was trialled as an alternative tool for VOC assessment. v Contents Index of Figures..................................................................................................................... x 1. Chapter 1: Introduction ................................................................................................ 1 Garlic Background ................................................................................................ 1 Garlic Agronomy................................................................................................... 2 Garlic Dormancy and Storage ............................................................................. 11 Organosulphur Biochemistry ............................................................................... 15 1.4.1. Alliin ......................................................................................................... 15 1.4.2. Alliinase .................................................................................................... 17 Allicin and derivatives......................................................................................... 19 Garlic in medicine ............................................................................................... 20 Garlic Preparations .............................................................................................. 23 Volatile Organic Compounds in Plants: .............................................................. 26 Volatile Organic Compounds in Garlic ............................................................... 28 VOCs as quality markers: .................................................................................... 28 Thesis Aims and Objectives ................................................................................ 31 2. Materials and Methods ............................................................................................... 32 Plant Material ...................................................................................................... 32 Garlic Storage...................................................................................................... 34 Sampling Scheme ................................................................................................ 34 VOC Sample Collection ...................................................................................... 35 Alliinase Enzyme Extraction ............................................................................... 35 Alliinase Activity Assay ...................................................................................... 37 Bradford Protein Assay ....................................................................................... 38 Bulb Water Content Evaluation ........................................................................... 38 Disease Assessment............................................................................................. 38 TD-GC-ToF-MS Analysis ................................................................................... 39 Statistical Analysis .............................................................................................. 40 3. Chapter 3: VOCs as Markers for Postharvest Quality Assessment of Garlic ........ 42 Introduction ......................................................................................................... 42 Materials and Methods ........................................................................................ 46 3.2.1. Plant Material ............................................................................................ 46 3.2.2. Garlic Storage ............................................................................................ 46 3.2.3. VOC Sample Collection and Analysis ....................................................... 46 3.2.4. Alliinase Enzyme Extraction ..................................................................... 48 3.2.5. Protein Concentration and Alliinase Activity Assay .................................. 48 3.2.6. Bulb Water Content ................................................................................... 48 vi 3.2.7. Disease Assessment ................................................................................... 49 3.2.8. RNA Extraction ......................................................................................... 49 3.2.9. DNase and cDNA Synthesis ...................................................................... 50 3.2.10. PCR ........................................................................................................... 51 3.2.11. qPCR ......................................................................................................... 52 3.2.12. Statistical Analysis .................................................................................... 52 Results ................................................................................................................. 53 3.3.1. Percent Disease.......................................................................................... 53 3.3.2. Water Content...........................................................................................