THE BASIS OF VARIATION IN THE SIZE AND COMPOSITION OF GRAPE BERRIES John D. Gray, B.Sc. (Hons) A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy Department of Horticulture, Viticulture and Oenology Faculty of Agriculture and Natural Resource Sciences The University of Adelaide Waite Campus Glen Osmond S.A. 5064 Australia May 2002 Table of Contents ii TABLE OF CONTENTS Table of Contents ................................................................................................ ii Statement of Authorship.................................................................................... iv Acknowledgements.............................................................................................. v Abstract ............................................................................................................... vi Abbreviations...................................................................................................... ix Chapter 1 – General Introduction..................................................................... 1 1.1 Introduction .............................................................................................. 1 1.2 Berry Development .................................................................................. 1 1.2.1 Berry Set ...................................................................................................................2 1.2.2 Berry Growth ............................................................................................................3 1.2.3 Ovule and Seed Development.................................................................................10 1.3 Berry Composition ................................................................................. 16 1.3.1 Sugars......................................................................................................................17 1.3.2 Acids .......................................................................................................................19 1.3.3 Phenolic Compounds ..............................................................................................21 1.3.4 Flavour Compounds................................................................................................23 1.4 Variation.................................................................................................. 24 1.4.1 Levels and Components of Variation......................................................................27 1.4.2 When Does Variation Begin?..................................................................................28 1.4.3 Measuring Variation ...............................................................................................29 1.5 Conclusion............................................................................................... 30 Chapter 2 – Variation in Shiraz Berry Size and Composition ..................... 32 2.1 Summary ................................................................................................. 32 2.2 Introduction ............................................................................................ 33 2.3 Experimental Procedures ...................................................................... 34 2.3.1 Biological Materials................................................................................................34 2.3.2 Berry Processing .....................................................................................................34 2.3.3 Calculating the Amount of Malvidin, Quercetin and Catechin...............................35 2.3.5 Statistical Techniques..............................................................................................37 2.4 Results...................................................................................................... 37 2.4.1 Developmental Changes in Berry Physical Characteristics....................................37 2.4.2 Developmental Changes in Berry Compositional Characteristics..........................47 2.5 Discussion................................................................................................ 54 Chapter 3 – Variation in Chardonnay Berry Size ......................................... 58 3.1 Summary ................................................................................................. 58 3.2 Introduction ............................................................................................ 58 3.3 Experimental Procedures ...................................................................... 59 3.3.1 Biological Materials................................................................................................59 3.3.2 Ovary and Berry Mass ............................................................................................60 3.3.3 Berry Growth Rates ................................................................................................62 3.3.4 Combined Seed Mass..............................................................................................62 3.4 Results...................................................................................................... 62 3.4.1 Clonal Variation and Girdling Treatments..............................................................62 3.4.2 Berry Age Classes...................................................................................................66 3.4.3 Variation between Bunches.....................................................................................72 Table of Contents iii 3.4.4 Ovary and Berry Growth Rates...............................................................................85 3.4.5 The Relationship between Ovary/Berry Mass and Ovule/Seed Mass ....................90 3.5 Discussion................................................................................................ 93 Chapter 4 – Confocal Measurement of 3D Cell Size and Shape .................. 96 4.1 Summary ................................................................................................. 96 4.2 Introduction ............................................................................................ 96 4.3 Experimental Procedures ...................................................................... 98 4.3.1 Tissue Preparation...................................................................................................98 4.3.2 Confocal Laser Scanning Microscopy (CLSM)....................................................100 4.3.3 Transmission Electron Microscopy (TEM) of Cell Walls ....................................101 4.3.4 Calibration of Intensity Attenuation with Depth...................................................101 4.3.5 Calibration of Axial Distortion .............................................................................102 4.3.6 Three-Dimensional (3D) Reconstruction..............................................................102 4.4 Results.................................................................................................... 103 4.4.1 Staining and Mounting the Tissue Block..............................................................103 4.4.2 Visualising Cell Walls ..........................................................................................103 4.4.3 Comparison of Cell Wall Thickness (TEM vs CLSM).........................................105 4.4.4 Correcting the Confocal Z-Series for Signal Attenuation and Axial Distortion...105 4.4.5 Reconstructed Three-Dimensional (3D) Models of Cells and Cell Walls............107 4.5 Discussion.............................................................................................. 109 4.5.1 Comments on Techniques Used............................................................................109 4.5.2 Shapes and Sizes of Parenchyma Cells.................................................................110 Chapter 5 – Berry Size and Cell Size in Chardonnay ................................. 113 5.1 Summary ............................................................................................... 113 5.2 Introduction .......................................................................................... 114 5.3 Experimental Procedures .................................................................... 114 5.3.1 Biological Materials..............................................................................................114 5.3.2 Calculating Berry Volume ....................................................................................115 5.3.3 Confocal Laser Scanning Microscopy (CLSM)....................................................116 5.3.4 Calculating Cell Volume.......................................................................................116 5.3.3 Statistical Techniques............................................................................................117 5.4 Results.................................................................................................... 118 5.4.1 Ovary Cell Volumes (1-15 days) ..........................................................................118 5.4.2 Berry Cell Volumes (29-43 days) .........................................................................122 5.5 Discussion.............................................................................................. 128 Chapter 6 – General Discussion..................................................................... 132 6.1 Summary ..............................................................................................