development of diagnostic tools to predict incidence of bitter pit during apple storage mehrdad ...

252 Pages·2015·4.2 MB·English

DEVELOPMENT OF DIAGNOSTIC
TOOLS TO PREDICT INCIDENCE OF
BITTER PIT DURING APPLE STORAGE

MEHRDAD MIRZAEE

A thesis submitted in partial fulfilment of the
requirements of the University of Greenwich
for the Degree of Doctor of Philosophy

This research programme was carried out
in collaboration with Landseer Ltd.

August 2015
ACKNOWLEDGEMENTS
My grateful thanks to everyone who helped me during this study, especially my supervisors,
Dr. Richard Colgan and Dr. Deborah Rees, for their kind instructions and valuable guidance
during this research.
Also my grateful thanks to Mr. Mark Tully (Landseer Ltd.) for all his support and collaborating
in this project. My appreciation to all members of staff at Landseer Ltd. especially Mrs. Tracey
Tully, Mr. Tony De Fraine and Mr. Colin Carter who helped me during collecting and
assessments of samples. Also especial thanks to all apple growers in Kent who participated in
this project for their kind cooperation during collecting and monitoring samples over storage
time.
I would like to thank all NRI staff and their support especially laboratory technicians and Mr.
Dudley Farman for their help in different areas in molecular and chemistry laboratories. Also I
would like to thank Prof. Atkinson for his kind instructions and advice. My especial thanks goes
to Mr. Stephen Young for all his help, support and kind instructions in statistics and analysis of
data.
My grateful thanks to Dr. Nazanin Zand and her colleagues at laboratories of School of Science
(University of Greenwich) for all their help and support for providing services for mineral
analysis.
I would like to thank all East Malling Research members of staff who helped me during this
project, especially Mrs. Karen Thurston for all her help and support during storage and
monitoring samples at PQC.
My grateful thanks to AgroFresh and Hansatech Instruments for providing equipment during
this project.
Finally my deepest thanks to my family who encouraged and supported me throughout my years
of study.

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ABSTRACT

Bitter pit is an important physiological disorder of many apple cultivars where the low uptake
and poor distribution of calcium within the cortex of apples pervades. Controlled atmosphere
storage and application of 1-MCP (SmartFreshSM) can delay the onset of bitter pit symptoms
by delaying maturity and senescence; however, significant losses may occur in long-term stored
apples. It is hard to detect internal bitter pit using external examination alone.
Previous studies have focused on improving pre-harvest prediction and curative treatments
before harvest. Present prediction models are based on history of orchards, mineral analysis 2-
3 weeks before harvest and quality assessments and monitoring over storage time.
This study aimed to identify a greater understanding of the storage potential of fruit based on
destructive standard quality assessments, biochemical and molecular analysis, also a non-
destructive monitoring method by chlorophyll fluorescence at the point of harvest and
monitoring during storage for developing more reliable prediction models to improve storage
management. The role of free and conjugated calcium in maintaining cellular integrity and the
relationship between biochemical and fluorescence changes and development of bitter pit were
investigated.
A diagnostic model based on comparison of changes of ascorbic acid during storage was
developed. Another diagnostic model based on changes in the proportion of calcium oxalate
content during storage in comparison with harvest was developed to identify samples with
higher propensity to bitter pit. Also chlorophyll fluorescence was investigated as a non-
destructive method for monitoring fruit during storage and prediction models for detecting
changes in the maturity of fruit and developing bitter pit and reduction of fluorescence during
storage as an alert to identify incidence of bitter pit were developed. Furthermore, changes in
gene expression profiles of a limited number of genes like calmodulin showed the differences
in patterns of transcripts between apples suffering from bitter pit and healthy apples.
All the suggested methods have potential of being commercialised and applied practically to
improve apple fruit store management. It would be possible to build a multi variate model for
predicting the onset of bitter pit development in apple by combination of two or more suggested

diagnostic tools.
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CONTENTS
ACKNOWLEDGEMENTS ....................................................................................................... ii 
ABSTRACT .............................................................................................................................. iii 
CONTENTS .............................................................................................................................. iv 
ABBREVIATIONS ................................................................................................................... ix 
CHAPTER 1 .......................................................................................................................... 1 
INTRODUCTION ...................................................................................................................... 1 
1.1 Apple cultivars and history: ............................................................................................. 3 
1.1.1 Bramley’s Seedling: .................................................................................................. 3 
1.1.2. Cox's Orange Pippin: ................................................................................................ 4 
1.2 Anatomy of apple fruit: .................................................................................................... 4 
1.3 Physiological disorders of apple: ..................................................................................... 6 
1.3.1 Bitter pit: ................................................................................................................... 6 
1.4 General physiology of fruit ripening: .............................................................................. 9 
1.4.1 Ethylene biosynthesis: ............................................................................................. 10 
1.4.2 Role of ethylene and other factors in fruit ripening: ............................................... 12 
1.4.3 Methods to control ethylene biosynthesis and perception: ..................................... 13 
1.4.4 The use of controlled atmosphere to extend the storage life of fruit: ..................... 16 
1.5 Pre-harvest and climate factors: ..................................................................................... 17 
1.5.1 Rootstock: ............................................................................................................... 18 
1.5.2 Orchard management, cropping level and fruit size: .............................................. 19 
1.5.3 Weather and environmental effects: ........................................................................ 20 
1.5.4 Harvest date: ............................................................................................................ 21 
1.6 Calcium: ......................................................................................................................... 21 
1.6.1 Balance of calcium and other nutrients: .................................................................. 25 
1.7 Methods for Predicting Bitter Pit: .................................................................................. 26 
1.7.1 Nutritional Status Methods: .................................................................................... 27 
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1.7.2 Maturity Acceleration Methods: ............................................................................. 27 
1.7.3 Mg infiltration: ........................................................................................................ 27 
1.7.4 Vegetative Growth Methods: .................................................................................. 27 
1.9 Molecular diagnostics and transcriptomic analysis: ...................................................... 32 
CHAPTER 2 ........................................................................................................................ 41 
MATERIALS AND METHODS ............................................................................................. 41 
2.1 Sample collection: .......................................................................................................... 41 
2.2 Fruit quality assessments: .............................................................................................. 48 
2.2.1 Ethylene measurement: ........................................................................................... 48 
2.2.3 Background colour: ................................................................................................. 49 
2.2.4 Total soluble solids (%Brix): .................................................................................. 49 
2.3 Analysis of minerals, organic acids and sugars: ............................................................ 50 
2.4 Chlorophyll fluorescence: .............................................................................................. 52 
2.5 Transcriptomic analysis of genes regulating calcium homeostasis: .............................. 53 
2.5.1 RNA extraction (method 1): .................................................................................... 54 
2.5.2 RNA extraction (method 2): .................................................................................... 55 
2.5.3 RNA extraction (method 3): .................................................................................... 55 
2.5.3.1 RNA extraction (method 3-stage 1): .................................................................... 56 
2.5.3.2 Final RNA extraction (method 3-stage 2): ........................................................... 56 
2.5.3 Quantification of RNA: ........................................................................................... 57 
2.5.4 Qualification of RNA by gel electrophoresis: ......................................................... 58 
2.5.5 cDNA library: .......................................................................................................... 58 
2.5.6 Primers: ................................................................................................................... 59 
2.5.7 Real Time PCR (qPCR): ......................................................................................... 60 
2.6 Statistics and experimental design:..................................................................................... 61 
CHAPTER 3 ............................................................................................................................. 62 
FRUIT QUALITY ASSESSMENTS ....................................................................................... 62 
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3.1. Introduction: ...................................................................................................................... 62 
3.2. The effect of the days taken to load stores, storage regimes, length of storage and the effect
of SmartFreshSM on incidence of bitter pit: .............................................................................. 62 
3.3. Picking date and storage regimes: ..................................................................................... 64 
3.4. Fruit firmness (N): ............................................................................................................. 67 
3.5 Background colour: ............................................................................................................ 70 
3.6 Total soluble solids (%Brix): .............................................................................................. 72 
3.7. Fruit size: ........................................................................................................................... 73 
3. 8. Discussion: ....................................................................................................................... 74 
CHAPTER 4 ............................................................................................................................. 82 
METABOLIC CHANGES IN BRAMLEY APPLE DURING STORAGE ............................ 82 
4.1 Introduction: ....................................................................................................................... 82 
4.2 Organic acids and sugars: ................................................................................................... 82 
4.2.1 Relationship between incidence of bitter pit and organic acids: ..................................... 94 
4.2.2 Relationship between incidence of bitter pit and sugars: .............................................. 100 
4.3 Mineral analysis: .............................................................................................................. 100 
4.4 Discussion ......................................................................................................................... 121 
CHAPTER 5 ........................................................................................................................... 131 
CHLOROPHYLL FLUORESCENCE ................................................................................... 131 
5.1 Introduction: ..................................................................................................................... 131 
5.2 Determination of the best characteristics correlated to bitter pit: ..................................... 131 
5.3 Determination of the threshold of ((cid:2162)(cid:4667) for incidence of bitter pit: .................................... 136 
5.4 Changes of chlorophyll fluorescence in different seasons: .............................................. 138 
5.5 Final determination of threshold for incidence of bitter pit: ............................................ 139 
5.6 Discussion ......................................................................................................................... 145 
CHAPTER 6 ........................................................................................................................... 150 
MOLECULAR ANALYSIS .................................................................................................. 150 
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6.1 Introduction: ..................................................................................................................... 150 
6.3 Expression of Ca-ATPase and Lipoxygenase genes: ....................................................... 152 
6.4 Analysis of calmodulin expression in apple: .................................................................... 154 
6.5 Real Time quantitative PCR: ............................................................................................ 157 
6.6 Discussion ......................................................................................................................... 166 
CHAPTER 7 ........................................................................................................................... 170 
CONCLUSIONS AND FUTURE WORK ............................................................................. 170 
7.1 Preharvest factors, storage conditions and quality assessments during storage: .............. 170 
7.2 Biochemical analysis: ...................................................................................................... 172 
7.3 Chlorophyll fluorescence: ............................................................................................... 176 
7.4 Molecular diagnostics: ...................................................................................................... 179 
7.5 Overall Conclusions ......................................................................................................... 181 
REFERENCES ....................................................................................................................... 182 
APPENDICES ........................................................................................................................ 200 
Appendix I: Final list of frozen samples in 2010/11 which were used for chemical and
molecular analysis (88 samples). ............................................................................................ 200 
Appendix II: Final list of frozen samples in 2011/12 which were used for chemical and
molecular analysis (96 samples). ............................................................................................ 203 
Appendix III: Final list of frozen samples in 2011/12 which were collected from two orchards
(EMR-EE193) and (HOO-Top) for comparing picking dates and air stored (4-4.5°C). ........ 206 
Appendix IV: Final list of frozen samples in 2012/13 which were collected from 4 orchards
(EMR-EE193), (HOO-Top), (CAR) and (NEW) at the same time and air-stored in air regime
(4-4.5°C). ................................................................................................................................ 208 
Appendix V (a): Comparison of temperature changes in spring (March, April and May) 2010
to 2013: A) 2010, B) 2011, C) 2012 and D) 2013. (Source: Met Office, 2015) .................... 212 
Appendix VI (a): Organic acid and sugars content of samples in (2011/12) air stored. ....... 214 
Appendix VI (b): Organic acid content of samples in (2012/13) CA storage (9%CO , 12%O )
2 2
and (5%CO2, 1%O2). .............................................................................................................. 215 
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Appendix VI (c): Organic acids content of samples collected from 2 orchards (EMR) and (Hoo)
in (2013/14) air stored for 3 months. ...................................................................................... 216 
Appendix VII: Multiple regression analysis for the influence of mineral constituents Ca2+, P,
N, K, Mg, B, Zn on the incidence of bitter pit of selected samples. ...................................... 218 
Appendix VIII (a): List of samples in 2011-12 air stored which were used for mineral analysis
(30 samples). ........................................................................................................................... 219 
Appendix VIII (b): List of samples in 2011-12 air stored which were used for mineral analysis
(30 samples). ........................................................................................................................... 220 
Appendix IX: Multiple regression analysis for the influence of mineral constituents Ca2+ total,
Ca2+ oxalate, Mg, K and B on the incidence of bitter pit of selected samples and incidence of
bitter pit. ................................................................................................................................. 222 
Appendix X: The study on chlorophyll flurescence and the results of season 2012/13 were
presented at “V International postharvest unlimited conference” and published in Acta
Horticulture vol. 1079 (2015) p: 235-242 .............................................................................. 223 
Appendix XI (a): Results of CT value for housekeeping primer (ITS) and primer “Calmodulin
b” ............................................................................................................................................ 235 

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ABBREVIATIONS

μL microlitre
µL L-1 microlitre per litre
μmol micromoles
μE m−2 s−1 microeinsteins per square meter per second
1-MCP 1-Methylcyclopropene
2-MCE 2-mercaptoethanol
AA Ascorbic Acid
AAS Atomic Absorption Spectroscopy
ABA Abscisic Acid
ACC 1-aminocyclopropane-1-carboxylic acid
ACO 1-aminocyclopropane-1-carboxylic acid oxidase
ACS 1-aminocyclopropane-1-carboxylic acid synthase
AGPase ADP-glucose pyrophosphorylase
ANCOVA Analysis of Covariance
ANOVA Analysis of Variance
AOA Aminooxyacetic Acid
Area The area above the fluorescence rise between Fo and Fm
ATP Adenosine Triphosphate
AVG Aminoethoxyvinylglycine
B Boron
bp base pair
BP Bitter pit
C H Ethylene
2 4
CA Controlled atmosphere
Ca Calcium
Ca(No ) Calcium nitrate
3 2
CaCl Calcium chloride
2
CaM Calmodulin
cDNA Complimentary DNA
CO Carbon dioxide
2
CPRs The County ploidy ratios
ix
Ctols Conjugated trienols
CT Constant Threshold
DAA Days After Anthesis
DEPC Diethylpyrocarbonate
DNA Deoxyribonucleic acid
DPA Diphenylamine
EDTA Ethylenediaminetetraacetic acid
EELS Electron Energy Loss Spectroscopy
EMRA East Malling Research Association
EMRS East Malling Research Station
EtBr Ethidium Bromide
EU European Union
F Energy lost from photosystem II as fluorescence
FAO Food and Agriculture Organization
FeSO Ferrous sulphate
4
F Fluorescence Intensity at 50 μs
1
F Fluorescence Intensity at 150 μs
2
F Fluorescence Intensity at 300 μs
3
F Fluorescence Intensity at 2 ms
4
F Fluorescence Intensity at 30 ms
5
Fm Maximum Fluorescence Yield
Fo Minimum Fluorescence Yield
Fluorescence (arb.) Fluorescence absorbance (arb. logarithm unit)
FTA Fruit Texture Analyser
Fv Variable Fluorescence Yield
Fv/Fm Maximum Efficiency of Photosystem II
GC Gas Chromatography
HCl Hydrogen chloride
HPLC High Performance Liquid Chromatography
ICP Inductively Coupled Plasma
ITS Internal Transcribed Spacer
K Potassium
KDa Kilo Dalton
x