COMPOSITION AND FLAVONOID LEVELS IN ONIONS
(Allium cepa) GROWN IN HYDROPONICS
IN GREENHOUSES AND GROWTH CHAMBERS
by
JAY L. MORRIS, B.S.
A THESIS
IN
FOOD TECHNOLOGY
Submitted to the Graduate Faculty
of Texas Tech University in
Partial Fulfillment of
the Requirements for
the Degree of
MASTER OF SCIENCE
Approved
December, 2001
ACKNOWLEDGEMENTS
I want to dedicate this to Jerry and Ross, you taught me that hard work and
persistence will get you all good things in life.
Mere words cannot express all the thanks and gratitude I owe Dr. Leslie "Doc"
Thompson. Smce startmg college in 1995, your guidance and wisdom has given me more
opportunities than I could have unagmed and placed me on the right path. For all the
guidance you afforded me about career and educational goals I can never repay you. From
Supplier's Night and College Quiz Bowl competition to settmg up the poultry contest, I
was always appreciative to have those opportunities to work with you and learn. Teaching
all the labs was hard work and made for some late nights, but it taught me more about
science than any textbook. I am grateful to have you as a professor, a mentor, and as a
friend. I am also thankful to Dr. Ellen Peffley, Dr. Paul Pare and Dr. Andy Herring for
servmg on my committee.
Dr. Peffley, I wanted to thank you for introducmg me to Kevin and lettmg me help
hun with his project. I do not thmk that I would have had as good an understanding of the
work involved in a getting a master's degree if it was not for all the work we did. Dr. Pare,
I wanted to thank you for all that you taught me about biochemistry. Your msightfiilness
and teachmg really opened my eyes and taught me a great deal, even though my grades
might not have reflected it. Dr. Herring, I wanted to thank you for all your help m setting
up my statistical analysis. I know the subject matter of this project is different from
anythmg you have probably worked on before but your excellence m statistical knowledge
was appreciated.
ii
To Uncle Maxie, what can I say, from the first time I met you, you made my career
here at Texas Tech the best and one I will never forget. Your caring and kmdness is one of
a kind and will always be somethmg I will remember.
To Jeremy, Chad, Janet and Brent, all your help with the little thmgs have made this
project fun. I do not thmk any other group of people could accomplish what we have, even
when it seems liked we would never reach an endpomt. I wanted to thank Crystal Smith
for the help you gave me. Thanks to Ambika for all her help with the atomic absorption
mstrument, and your constant harassmg made workmg m the lab fun. I do not thmk I could
have kept my head on at tunes if it were not for your constant griping and kiddmg all the
tune.
I wanted to thank Dr. Gary Green and Dr. David Tissue for all your insight mto my
research. Your analysis in the meetings and solutions to problems was extremely helpful.
Dr. Tissue, I also wanted to thank you for allowing us to use C/N analyzer, it saved a great
deal of time.
To my family- Mom, Dad, E and Graimy, your support from day one has been
something that has helped to get where I am today. I know I hardly ever say thanks, but
this time I mean it. Thanks to all myfriendsfor your support and words of encouragement.
At tunes, I questioned why, but all of you made me see that I could do it. To Opie,
Nosmo, and Wolf you are the best set of friends anyone could ever have. One day we all
be through with school and be able to enjoy the fhiits of our labor, hopefully. I wanted to
thank Southwestern Bell corporation for their Summer Thesis Scholarship.
ni
TABLE OF CONTENTS
ACKNOWLEDGEMENTS
ii
ABSTRACT
viii
LIST OF TABLES
xi
LIST OF FIGURES
xiii
LIST OF ABBREVIATIONS
xvii
CHAPTER
L
REVIEW OF THE LITERATURE
1
Introduction
1
Flavonoids, Flavonols, and Quercetm
3
Structure
4
Formation
6
Function
6
Color and Distribution in Bulbs
10
Storage and Processing of Quercetin
11
Beneficial Health Attributes of Quercetm
12
Antioxidation
12
Cancer
14
Anti-Atherosclerosis Attributes and Coronary Heart Disease
16
Antiviral Activity
19
Intestmal Absorption and Bioavailbility of Quercetin
20
Hydroponic Growth and Nutritional Quality
22
IV
IL
Minerals
23
Sulfiir Content
24
Growth Enhancement m Growth Chambers
26
Project Objectives
28
References
29
MATERIALS AND METHODS
41
Greenhouse Experiments
41
Plant Material Experiment 1
41
Plant Material Experiment 2
41
Sample Preparation
42
Extraction for Total Flavonol Determmation
43
Quantification with a Spectrophotometer
43
Dry Matter and Moisture
44
Nitrogen and Carbon Analyses
44
Ash and Mineral Analyses
45
Sulfur Quantification
47
Experimental Design and Statistical Analyses
48
Experiment 1
48
Experiment 2
48
Envkormiental Growth Chamber Experiment
Plant Material
49
49
Envu-onmental Conditions
50
Sample Preparation
50
Experimental Design and Statistical Analyses
50
Individual Growth Chamber Experunent
m.
IV.
51
Plant Material
51
Envkonmental Conditions
52
Sample Preparation
52
Experimental Design and Statistical Analyses
52
References
53
RESULTS AND DISCUSSIONS
54
Greenhouse Experiments
54
Experiment 1
54
Experiment 2
58
Environmental Growth Chamber Experiment
63
Individual Growth Chamber Experiment
72
References
76
CONCLUSIONS AND IMPLICATIONS
116
Greenhouse Experiments
116
Experiment 1
116
Experiment 2
116
Envu-onmental Growth Chamber Experiment
VI
117
Individual Growth Chamber Experiment
117
Overall Implications
118
APPENDIX
A: FLAVONOL STANDARD CURVE
119
B: PLANT VARIABILITY
123
C: SOIL VERSUS HYDROPONICS
127
D: PHOTOPERIOD LENGTH IN ENVIRONMENTAL GROWTH
CHAMBERS
131
E: ELEVATED CO2 GROWING CONDITIONS
138
F: CHEMICAL COMPOSITION
141
G: EXPERIMENTAL LAYOUT AND PLANT LOCATION
144
Vll
ABSTRACT
One-thousand eighty long-day onions (Allium cepa L. 'Purplette') grown
' hydroponically m a greenhouse were analyzed for composition and total edible biomass to
determine the amount of variability as the plant matures. Plants were harvested at 14, 21,
28, 35, 42, 49, 63, 77 and 98 d after sowmg. Plant height mcreased as plants aged, and a
significant mteraction between planting date and plant age was noted. Plant weight and net
number of leaves uicreased with age and again an mteraction between age and planting
date was observed. Percent N decreased from 0.55-0.34% (p < 0.05) as the plants aged.
Percent C (C) decreased (p < 0.05) as the plants aged but after d 77 a significant mcrease
occurred. Ash content increased (p < 0.05) as the plant aged, with means ranging from
0.09 -1.07%. Calcium (Ca) and magnesium (Mg) concentration decreased (p < 0.05) as the
plant aged with means rangmg from 128.7 - 64.2 mg/100 g and 57.1 - 22.0 mg/100 g,
respectively. Potassium (K) concentrations showed an interaction (p < 0.05) between age
and plant age. Totalflavonol(TF) content increased (p < 0.05) from 226.1 - 554.7 mg/100
g, as the plant aged from 14 d to 98 d. Dry matter (DM) content and sulfur (S)
concentration was unchanged (p > 0.05) as the plant aged. Mean values ranged from
10.47-10.70% and 185.2-193.6 mg/100 g for dry matter content and S concentration,
respectively. Biomass production and proximate composition of onions varied
significantly as the plant ages and underwent morphological changes.
Five-himdred-sixty long-day onions {Allium cepa L. 'Purplette') grown
hydroponically and in potting soil hi a greenhouse were analyzed for composition and total
Vlll
edible biomass to determme differences due to growth medium. Biomass measurements
were made at 14, 21, 28, 35, 42, and 49 d of age. An mteraction (p < 0.05) between grov^h
medium and age occurred for plant weight and net leaf number. There were no significant
differences in plant height due to growing medium, however; as expected, plants were
taller (p < 0.05) as the plant aged, with means ranging from 7.64-29.07 cm. Composition
of plants at ages of 28, 35, 42, and 49 d was examined with TF level also being analyzed at
14 and 21 d of age. Percent DM, C, Ca, K, and S did not differ (p > 0.05) among plants
regardless of growing medium or age, with means of 10.39%, 4.15%, 126.1 mg/100 g,
270.0 mg/lOOg, and 185.4 mg/100 g, respectively. Percent N and Mg concentration
decreased (p < 0.05) as plants matured and no effect due to growing medium was observed.
Percent ash increased (p < 0.05) as plants matured. Percent ashfromplants grown in
Oasis™ ranged from 0.98-1.02% and m the pottmg soil ranged from 0.86-0.98%. Total
flavonol concentrations increased (p < 0.05) as plants matured with no growing medium
effect. Growing medium does effect plant weight, net number of leaves, and ash percent.
Forty long-day onions (Allium cepa L. 'Purplette') and short-day onions (Allium
cepa L. 'Redbone') were grown hydroponically in envu-onmental growth chambers (EGC)
to determhie if composition and total edible biomass was effected by two photoperiods, 16
h and 11 h of daylight. Plants were also partitioned mto leaves, bulbs, and roots to
determine nutritional composition of each component. Plant height was higher (p < 0.05)
m the short-day growing condition (EGC2) and the 'Purplette' variety. Plant weight did
not differ between EGC, however 'Purplette' was heavier (p < 0.05) than the 'Redbone'
and an interaction between photoperiod and variety was observed. Net number of leaves
IX
was higher (p < 0.05) m the 'Purplette' variety compared to 'Redbone.' Dry matter content
was different (p < 0.05) between photoperiod, variety and plant part. Percent N and C were
different (p < 0.05) between plant parts with an interaction of EGC and variety. Percent C
was also different between EGC as well. Calcium and K concentration mg/100 g were
different (p < 0.05) between plant parts with an interaction between photoperiod and plant
parts. Totalflavonoland S concentration mg/100 g was different (p < 0.05) between plant
parts with leaves contammg the highest amount, then bulbs, and the lowest amount m the
roots. Ash content and Mg concentration mg/100 g was not different (p > 0.05) between
photoperiod, variety, plant part, and all interactions. Photoperiod and variety due effect
plant nutrient amounts and the concentrations of each nutrient m the different plant parts by
onions.
Twelve long-day onions (Allium cepa L. 'Purplette') grown hydroponically and
gassed for 96 h with three different CO2 concentrations, 370 (ambient), and 1000 and 2000
(elevated) ppm m individual growth chambers (IGC) to determme if composition and total
edible biomass was effected by CO2 level. Plant weight uicreased (p < 0.05) between CO2
treatments with means rangmg from 37.5-44.4 cm. Net leaf number was also different
(p < 0.05) between CO2 treatments. There was no difference (p > 0.05) m plant height,
percent ash, S and TF content. Withmeansof 42.02% ± 3.92, 1.14%±0.06, 124.7± 1.7
mg/100 g, and 443.2 ± 107.2 mg/100 g, respectively. Decreases (p < 0.05) m Ca, Mg, and
K content were observed from ambient CO2 to 2000 ppm of CO2. Dry matter content, N
and C percent were different (p < 0.05) between CO2 levels.
LIST OF TABLES
3.1
Ash percent and Mg concentration of 45 d old
'Redbone' and 'Purplette' onions grown hydroponically m two
different enviroimiental growth chambers
80
A. 1
Isoquercitrin (Q) calculations for standard curve
120
A.2
Calculation of TF concentration using linear regression
121
B. 1
Average phenotypic characteristics of 'Purplette' onions grown
hydroponically m a Texas Tech University greenhouse from March
to July 2001
124
Composite characteristics on a wet matter basis of 'Purplette'
onions grown hydroponically in a Texas Tech University greenhouse
from March to July 2001
125
Average phenotypic characteristics of 'Purplette' onions grown
hydroponically (H) and m potting soil (S) m a Texas Tech University
greenhouse from May to August 2001
128
Composite characteristics on a wet matter basis of 'Purplette' onions
grown in potting soil in a Texas Tech University greenhouse from
May 2001 to August 2001
129
Composite characteristics on a wet matter basis of 'Purplette' onions
grovm hydroponically in a Texas Tech University greenhouse from
May 2001 to August 2001
130
Phenotypic characteristics of 'Redbone' (RB) variety grown
hydroponically in two different environmental grov^rth chambers
(EGC) with two photoperiods
132
Phenotypic characteristics of 'Purplette' (P) variety grown hydroponically
m two different envu*onmental growth chambers (EGC) with two
different photoperiods
133
Composite characteristics on a wet matter basis of 45 d old 'Red Bone'
onions grown hydroponically in two different envhronmental growth
chambers, plants placed in chambers at 24 d of age
134
B.2
C. 1
C.2
C.3
D. 1
D.2
D.3
XI
D.4
E. 1
E.2
F. 1
F.2
Composite characteristics on a wet matter basis of 45 d old 'Purplette'
onions grown hydroponically in two different envkonmental
growth chambers, plants placed in chambers at 24 d of age
136
Phenotypic characteristics of 36 d old 'Purplette' onions grown
at ambient CO2 (370 ppm), acclimated for 48 h m mdividual growth
chambers and then exposed to elevated CO2 levels
139
Composite characteristics on a wet matter basis of 36 d old 'Purplette'
onions grown at ambient CO2 (370 ppm), acclunated for 48 h in
individual growth chambers and then exposed to elevated CO2 levels
139
Chemical composition of Hydro-SoF^ 5-11 -26 (Scotts-Sierra
Horticultural Product Co, Marysville, OH, USA) used as the
primary nutrient source for all onion plants grown for research
142
Chemical composition of Ball Growing Mix 2''"'^ (Ball Seed;
West Chicago, IL, USA) pottmg soU
143
xu
LIST OF FIGURES
1.1
Three major conjugates of quercetm: quercetm aglycone (Qag),
quercetm-4'-0-glucoside (4'-Qmg), and quercetm-3,4'-(9-diglucoside
(3,4'-Qdg)
5
1.2
Biochemical pathway schematic of quercetin formation m plants
7
3.1
Per plant weight (edible portion) of 'Purplette' onions grown
hydroponically in a Texas Tech University greenhouse from
March to July 2001
81
Plant height of 'Purplette' onions grown hydroponically m a
Texas Tech University greenhouse from March to July 2001
82
Number of leaves on 'Purplette' onions grown hydroponically in a
Texas Tech University greenhouse from March to July 2001
83
Nitrogen percent on a wet matter basis m edible portion of
'Purplette' onions grovm hydroponically in a Texas Tech University
greenhouse from March to July 2001
84
Percent C on a wet matter basis in edible portions of
'Purplette' onions grown hydroponically in a Texas Tech University
greenhouse March to July 2001
85
Percent ash on a wet matter basis m edible portions of 'Piuplette'
onions grown hydroponically in a Texas Tech University greenhouse
March to July 2001
86
Calcium content on a wet matter basis in edible portions of 'Purplette'
onions grown hydroponically m a Texas Tech University greenhouse
March to July 2001
87
Magnesium content on a wet matter basis m edible portions of 'Purplette'
onions grown hydroponically m a Texas Tech University greenhouse
March to July 2001
88
Potassium concentration on a wet matter basis in edible portion of
'Purplette' onions grown hydroponically m a Texas Tech University
greenhouse from March to July 2001
89
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
xiu
3.10
3.11
3.12
3.13
3.14
3.15
3.16
3.17
3.18
3.19
3.20
Flavonol content m edible portions of 'Purplette' onions grown
hydroponically in a Texas Tech University greenhouse
March to July 2001
90
Per plant weight (edible portion) of 'Purplette' onions grovm
hydroponically and in pottmg soil m a Texas Tech University
greenhouse from June to August 2001
91
Plant height of 'Purplette' onions grown hydroponically and m potting
soil in a Texas Tech University greenhouse from Jime to August 2001
92
Nimiber of leaves on 'Purplette' onions grown hydroponically and ui
potting soil in a Texas Tech University greenhouse from
June to August 2001
93
Nitrogen percent on a wet matter basis of 'Purplette' onions grovm
hydroponically and in potting soil m a Texas Tech University greenhouse
from June to August 2001
94
Percent ash on a wet matter basis of 'Purplette' onions grown hydroponically
and in potting soil in a Texas Tech University greenhouse from June to
August 2001
95
Magnesium content on a wet matter basis of 'Purplette' onions grown
hydroponically and in potting soil m a Texas Tech University greenhouse
from June to August 2001
96
Flavonol content on a wet matter basis of 'Purplette' onions grown
hydroponically and in pottmg soil m a Texas Tech University greenhouse
from Jime to August 2001
97
Per plant weight (edible portion) of 45 d old 'Red Bone' and 'Purplette'
onions grown hydroponically m two different envu-onmental growth
chambers, plants placed in chamber at 24 d of age
98
Plant height of 45 d old 'Red Bone' and 'Purplette' onions grown
hydroponically in two different envu-omnental growth chambers, plants
placed m chamber at 24 d of age, one with a long-day
99
Number of leaves 45 d old 'Red Bone' and 'Purplette' onions grovm
hydroponically m two different environmental growth chambers, plants
placed m chamber at 24 d of age
100
xiv
3.21
3.22
3.23
3.24
3.25
3.26
3.26
3.28
3.29
3.30
3.31
Dry matter percent of 45 d old 'Red Bone' and 'Purplette' onions grown
hydroponically m two different envu*ormiental growth chambers, plants
placed in chamber at 24 d of age
101
Nitrogen percent on a wet matter basis of 45 d old 'Red Bone' and
'Purplette' onions grown hydroponically in two different envu*onmental
growth chambers
102
Carbon percent on a wet matter basis of 45 d old 'Red Bone' and
'Purplette' onions grown hydroponically hi two different environmental
growth chambers
103
Calcium content on a wet matter basis of 45 d old 'Red Bone' and
'Purplette' onions grown hydroponically m two different environmental
growth chambers
104
Potassium content on a wet matter basis of 45 d old 'Red Bone' and
'Purplette' onions grown hydroponically in two different envu-omnental
growth chambers
105
Sulfur content on a wet matter basis of 45 d old 'Red Bone' and
'Purplette' onions grown hydroponically hi two different envu-onmental
growth chambers, plants placed in chamber at 24 d of age
106
Flavonol content on a wet matter basis of 45 d old 'Red Bone' and
'Purplette' onions grovm hydroponically in two different envu-onmental
growth chambers
107
Per plant weight (edible portion) of 36 d old 'Purplette' onions grown
at ambient CO2 (370 ppm), acclunated for 48 h in mdividual growth
chambers and then exposed to elevated CO2 levels
108
Number of leaves 36 d old 'Purplette' onions grown at ambient CO2
(370 ppm), acclimated for 48 h in individual growth chambers and then
exposed to elevated CO2 levels (1000 and 2000 ppm)
109
Dry matter percent of leaves 36 d old 'Purplette' onions grown at
ambient CO2 (370 ppm), acclunated for 48 h in individual growth chambers
and then exposed to elevated CO2 levels
110
Nitrogen percent on a wet matter basis of 36 d old 'Purplette' onions
grown at ambient CO2 (370 ppm), acclunated for 48 h m individual growth
chambers and then exposed to elevated CO2 levels
111
XV
3.32
3.33
3.34
Carbon percent on a wet matter basis of 36 d old 'Purplette' onions
grown at ambient CO2 (370 ppm), acclunated for 48 h m mdividual growth
chambers and then exposed to elevated CO2 levels
112
Calcium content on a wet matter basis of 36 d old 'Purplette' onions
grown at ambient CO2 (370 ppm), acclunated for 48 h m mdividual
growth chambers and then exposed to elevated CO2 levels
113
Magnesium content on a wet matter basis of 36 d old 'Purplette' onions
grown at ambient CO2 (370 ppm), acclimated for 48 h in mdividual growth
chambers and then exposed to elevated CO2 levels
114
3.35
Potassium content on a wet matter basis of 36 d old 'Purplette' onions grown
at ambient CO2 (370 ppm), acclunated for 48 h in mdividual growth chambers
and then exposed to elevated CO2 levels
115
A. 1.
Mean spectrophotometer values of isoquercitrm for each level of the
standard curve after 10 readings; concentration vs. absorbency (AU)
122
Experunental diagram of 'Redbone' and 'Purplette' onions grown
ui EGCl with a photoperiod of 16 h. S = 'Redbone' and
P = ' Purplette' with bold letters mdicatmg plants sampled and partitioned
145
Experunental diagram of'Redbone' and 'Purplette' onions grown in
EGC2 with a photoperiod of 11 h. S = 'Redbone' and P = ' Purplette'
with bold letters indicatmg plants sampled and partitioned
146
G. 1.
G.2.
XVI
LIST OF ABBREVIATIONS
A
Antioxidant
APS
Adenosme-5'-phosphosulphate
AU
Absorbance unit(s)
BC
British Columbia
C
Carbon
Ca
Calcium
CELSS
Controlled ecological life support system
CGS
Closed growth system
CHD
Coronary heart disease
CRD
Completely randomized design
CT
Connecticut
Cu
Copper
cv
Cultivar(s)
CVD
Cardiovascular disease
CHD
Coronary heart disease
D-H2O
Distilled water
DM
Dry matter
DNA
Deoxyribonucleic acid
EGC
Environmental growth chamber(s)
EGCl
Environmental growth chamber 1
EGC2
Environmental growth chamber 2
xvii
EtOH
Ethanol
Fe
Iron
GLM
General linear model
h
Hour(s)
HDL
High-density lipoprotein
HIV
Human unmunodeficiency virus
HSE
Horizontal system equatorial cells
HSF
Heat shock transcription factors
I
lodme
lA
Iowa
IDL
Intermediate-density lipoprotein
IGC
Individual growth chamber(s)
IL
Illinois
L
Lipid
LDL
Low-density lipoprotem
LF
Lachrymatory factor
LOOH
Lipid hydroperoxides
MA
Massachusetts
ME
Maine
Mg
Magnesium
Mn
Manganese
MI
Michigan
XVlll
mon
Month(s)
MO
Missouri
MS
Mississippi
N
Nitrogen
Na
Sodium
NFT
Nutrient fihn technique
NC
North Carolma
NJ
New Jersey
OH
Ohio
OR
Oregon
ORAC
Oxygen-radical absorbance capacity
P
Phosphorus
PAH
Polycyclic aromatic hydrocarbons
PPF
Photosynthetic photon flux
Q
Quercetin
Qag
Quercetui aglycone
4'Qmg
Quercetin-4'-0-glucoside
3,4'Qdg
Quercetm-3,4'-0-diglucoside
rep
Replication(s)
RH
Relative humidity
RNA
Ribonucleic acid
s
Seconds
xix
S
Sulfiir
SAS
Statistical analysis system
SEM
Standard error of the mean
TE
Trolox equivalent
TNC
Total nonstructural carbohydrates
TX
Texas
VLDL
Very low-density lipoprotein
wk
Week(s)
Zn
Zinc
XX
CHAPTER I
REVIWE OF THE LITERATURE
Introduction
Flavonoids are ubiquitous throughout the plant kuigdom. Flavonoids are low
molecular weight polyphenolics that accumulate hi the ariel as well as below ground
portions of the plant. Over 4000 different flavonoids have been identified (Bors and
others 1996; Shahidi and Naczk 1995) and Prior and Cao (2000) with biological
properties mcludmg plant pigmentation, defense against pathogens (Shahidi and Naczk
1995), antiplatelet activity (Gryglewski and others 1997; Lanza and others 1987), tumorgrowth suppression (Verma and others 1988; Deschner and others 1991), mhibitory
effects on mutagenesis and carcinogens (Shahidi and Wanasundara 1992; Stavric and
others 1990), and almost all exhibit antioxidant activity (Terao and others 1994; van
Acker and others 1996; Huang and Ferraro 1992; Shahidi and Wanasundara 1992).
Many flavonoids have the ability to scavenge active oxygen species and free radicals,
inhibit nitrosation, reduce bioavailable carcinogens, chelate metals, modulate certain
enzyme activities (Stavric 1997) as well as, having hypocholesterolemic activity, and
anti-mflammatory properties (Greenfield and others 1993; Ramptom and Collms 1993).
In addition to their activity within cells, flavonoids play a role as chemopreventers
in foods serving as antioxidants preventmg the rancidity development in lipids before
consumption or during digestion processes. They accelerate intestinal transit time, protect
intestinal microflora, can mcrease uptake of certain beneficial constituents from the diet,
and reduce the availability of food mutagens and carcinogens (Stavric 1997). Flavonoids
also posses antivu-al activity and mcrease capillary permeability (Havsteen 1993).
Despite all of these positive attributes, flavonoids are considered nonessential dietary
nutrients (Prior and Cao 2000).
One structural classification of the flavonoids are the flavonols, which by
definition contam a hydroxyl group at position 3 in the C-rmg and a double bond at C 2
(Figure 1.1). Quercetm, myricetm, and kaempferol are examples of flavonols. Quercetm
was first isolated in 1936 by Szent-Gyorgyi and labeled it vitamin P (Formica and
Regelson 1995). Quercetm, the most abundant flavonoid m the human diet (Duthie and
Dobson 1998), is foimd mainly in onions. Onions contain the highest amount of quercetm
among commonly consumed fruits and vegetables (Hertog and others 1992a).
Researchers have postulated that onions have been in cuhivation for 5000 years or
more (National Onion Association 2001). Farmers in the U.S. plant about 145,000 acres
of onions aimually resultmg in $800 million dollars at the farm gate and $3-4 billion at
retail (National Onion Association 2001). Many studies have provided direct favorable
evidence that a high intake of fruits and vegetables have an important protective effect
against a wide range of human cancers, such as colon, lung, cervix, esophagus, stomach,
ovarian and bladder (Block and others 1992). Consumption of fruits and vegetables,
which are high hi phytochemicals, has shown to protect agamst, cardiovascular disease,
stroke, cataracts and diabetes (Block and others 1992; Stemmetz and Potter, 1996).
Growmg plants in environmental chambers can optunize the environmental
conditions to enhance growth rates. Light intensity, photoperiod, nutrient solution
composition, temperature and nutrient concentration can all be controlled allowing for
growth rate enhancement. By developing these units and opthnizmg growth rates
consumers can have vegetables year round from the same location. Additionally this
control can allow for a more uniform product. Understanding the changes in onion
composition grown m environmental chambers and the use of hydroponics could enhance
vegetable crop availability and quality for consumers.
In the late 1980's NASA developed a regenerative life support system to develop
systems for long-term space flight (Barta and Heimmger 1996). The controlled
ecological life support system (CELSS) program's purpose was to develop a facility for
large-scale, integrated testmg bed for plant growth with physiochemical life support
subsystems to provide au: and food production for astronauts (Barta and Henninger
1996). This test allowed for plants grovm under opthnal growmg conditions to provide
food for the crew as well as providmg oxygen for them to breathe (Barta and Hennmger
1996).
Flavonoids, Flavonols, and Quercetin
Flavonoids are classified mto 13 different categories (Croft 1998). These
compounds have a wide range of functions includmg antioxidant, antihistamme,
anticarcmogenic and anti-inflammatory properties (Bravo 1998; Erlund and others 2000).
Flavonols consist mainly of quercetm, kaempferol, myricetin, morm and fisetm (Hertog
and others 1992a, Madsen and Jorgensen 2000), with other less known flavonols
catechms, taxifolm, narmgenm, leteolin, apigenm and baicalein (Formica and Regelson
1995). Quercetm, the most abundant flavonoid in the human diet (Duthie and Dobson
1998), is found mamly m onions. Onions contain the highest amount of quercetm among
commonly consumed fiiiits and vegetables (Hertog and others 1992a), but other foods
such as red wine, apples, kiwifioiit, kale, and green and black teas also contribute
quercetin to the diet (Frankel and others 1995; Goldberg and others 1998; Ahmad and
Mukhtar 1999; Dawes and Keene 1999). Other foods contaming quercetm are lettuce,
leeks and cranberries (Hertog and Hollman 1996, Hertog and others 1992a). Hertog and
Hollman (1996) have said that average consumption of quercetm is 23 mg/d and that
daily consumption of quercetm can sustam a bioactive level in the human body
(McDonald and others 1998).
Structure
Onions contain many nutritional constituents but one compound has come under
much study due to its biological significance. The structure of quercetin is a
diphenylpropane (C6-C3-C6) with a sugar usually bound at the C3 poshion (Hertog and
others 1992b). The backbone of the structure is two benzene rings (A, B) coupled
together with a three-carbon pyran rmg (C) (Rhodes and Price 1996) (Figure 1.1).
Onions mainly contain glucose glycosides of quercetin (Hollman and others 1997).
Quercetin is predominately found in three forms: aglycone, quercetin-3,4'-0diglucoside, and quercetin-4'-0-glucoside (Rhodes and Price 1996) (Figure 1.1).
Quercetin aglycone is hydroxylated at the 5 and 7 position on the A ring, the 3 position
on the C rmg and the 3'and 4' on the B ring (Hollman and others 1997). Quercetin has
five hydroxyl substitutions at 3, 5, 7, 3', and 4' positions and is highly unsaturated with
2,3 double bond and a keto group at C 4 on the C ring (Croft 1998).
OH
i
^ 4
B I
HO
Molecular formula
OH
Molecular Weight
Composition
Surface Tension
Density
= Ci5H,oO,
= 302.236
= C(59.61%) H(3.33%) 0(37.06%)
= 114.8 ± 3.0 dyn^cm
= 1.799 ± 0.06 g/cm^
"^9
A
I C
10
OH
OH
OH
2,^
'^
HO
0
7 / % . . ,/-1 \
0
OH
0
\^ ^4 ' ^ 0
^1'
2
^3^
II
.3'
^
^
OH
Molecular formula
Molecular Weight
Composition
Surface Tension
Density
= C2iH22 0,2
= 466.392
= C(54.08%) H(4.75%) 0(41.17%)
= 123.3 ±5.0 dyne/cm
= 1.82 ± 0.1 g/cm^
HO
Molecular formula
Molecular Weight
Composition
Surface Tension
Density
-C27H30O17
= 626.517
= C(51.76%) H(4.83%) 0(43.41%)
= 137.6 ±5.0 dyne/cm
= 1.89 ± 0.1 g/cm'
Figure 1.1. Three major conjugates of quercetm: quercetin aglycone (Q) (top),
quercetin-4'-0-glucoside (4'-Qmg) (middle), and quercetin-3,4'-0-diglucoside
(3,4'-Qdg) (bottom) (Price and Rhodes 1997; Wegh and Luyten 1997)
Formation
Quercetin is created by biosynthetically by bondmg two sets of derivatives, 1
fi-om the Shikhnic acid pathway and 3fi-omthe malonic acid pathway (Taiz and Zeiger
1998). Couplmg a C6-C3 unitfi-omthe Shikhnic pathway forms the CI5 skeleton with
the addition of 3-malonyl-CoA (2C) unitsfi-omthe malonic acid pathway (Harbome
1973). The A-rmg arises by the head to tail condensafion of the 3-malonyl-CoA units.
The B-rmg and the C3 unit comefi-oma cmnamic acid precursor created by the Shikhnic
acid pathway (Harbome 1973). The molecule then under goes several condensation steps
followed by the addition of five hydroxyl groups at the 3, 5, 7, 3' and 4' posifions (Taiz
and Zeiger 1998; Croft 1998) (see Figure 1.2). Protection of these hydroxyls groups is
done by glycosidic addition. The most abundant sugar bound is glucose, but galactose,
rhamose and xylose can be foimd (Harbome 1973; Bravo 1998). Hydroxyl groups and
sugars increase the water solubility, making quercetin water-soluble and thus plants store
them in the vacuoles of the cell (Harbome 1973).
Function
One of the majorfijnctionsof quercetui in plants is to act as a protective barrier
against deleterious effectsfi-ommsect andfiingaldamage. Bohm (1998) observed an
increase in quercetin in European beech (Fagus sylvatica) after an attack of beech bark
disease. Quercetm alsofianctionswithm microorganisms and insects. In lycaenid
°v^"
H-
-OH
H-
-OH
O^^^OH
H.C^'^O
HO
H'
Shikimic Acid Pathway
-O
P
O-
P
0-
Phosphoenolpyruvate
°*=^°"
Erythose-4-phosphate
r*===N
OH
Shikimic acid
p-Cowcmic acid
3 Malonyl CoA
molecules
Addition of 2
Hydroxyl molecules
O
Quercetin aglycone
Figure 1.2. Biochemical pathway schematic of quercetin formation in plants (Taiz and
Zeiger 1998)
butterflies and zebra swallowtail quercetm is a part of the wmg pigmentation, which is
formed during larval feedmg on plants (Bohm 1998; Harbome 1999). Antunicrobial
properties of quercetm have been demonstrated agamst species such as Bacillus cereus
and Cladosporium cucumerinum (Bohm 1998).
Active oxygen species are produced in many aerobic organisms durmg normal
metabolic processes (Schwantz and Polle 2001). Several protective mechanisms agamst
UV-B or photooxidative stress damage has been outlmed for plants. The processes range
from repah- fimction (DNA, free radical scavengmg) (Barabas and others 1998; Britt
1999), to preventative measures like UV-B screenmg, scattermg and reflection (Hoque
and Remus 1999). Quercetm absorbs at different wavelengths but maximum absorption
is m the range of 362-375 nm (Lombard 2000; Jones and others 1998; Hertog and others
1992a; Patil and Pike 1995). A particularly unportant role in this regard has been
attributed to phenylpropanoids, mcludmg hydroxychmamic acid derivatives and
flavonoids with effective absorption in the UV-B spectral region (Reuber and others
1996a; Sheahan 1996; Hoque and Remus 1999). In addkion to UV-screenmg, other
unportant UV-B-protective properties ascribed to flavonoids include antioxidant
activities (Dawar and others 1998), and energy dissipation via intramolecular proton
transfer (Smith and Markham 1998). Flavonoids can increase rapidly with an increase in
UV-B radiation (Jordan 1996) and are frequently found in or on epidermal layers where
exposure to UV-B radiation is the highest (Reuber and others 1996b). Studies with
flavonoid mutants fiirther highlighted the importance of flavonoids for UV-B tolerance
(Lois and Buchanan 1994; Reuber and others 1996a).
8
Recent reports show that highly specific differential UV-B responses between
closely related flavonoids are well conserved m the plant kmgdom. Such differential
responses were demonstrated in liverwort (Markham and others 1998a), in gymnosperms
(Schnitzler and others 1997; Fischbach and others 1999), monocotyledons (Liu and
others 1995; Reuber and others 1996a; Markham and others 1998b), as well as in several
dicotyledons, both herbaceous (Olsson and others 1998; Ryan and others 1998; Wilson
and others 1998), and trees (Lavola 1998). Several of these reports mdicate a shift from
B-rhig monohydroxlyation flavonoids towards their or^/^o-dihydroxylated equivalents
under UV-B. The dihydroxylated flavonoids are seen to confer additional UV-B
protection, which could be mediated by higher relative antioxidant capacity (Montesinos
and others 1995; Cooper-Driver and Bhattacharya 1998). A developmg body of recent
evidence also leads to population-dependant differences in specific flavonoid responses
to UV-B (Lavola 1998; Markham and others 1998b; Olsson and others 1998), suggestmg
a relevance to the ecological theory of stress tolerance, predicting differential degrees of
biochemical stress response depending on the productivity of plant species or population
and of theu- habitat (Diaz and others 1999; Poorter and Gamier 1999). Quercetin
glycosides are able to dissipate potentially harmfiil UV-B radiation through
tautomerizafion (Hofinann and others 2000). Specific quercetin accumulation has been
observed as a response to a number of other forms of stress, ranging from heavy metal
pollution (Loponen and others 1998), N deficiency (Bongue-Bartelsman and Phillips
1995), to electron donating paraquat application (Steger-Hartmann and others 1994).
Color and Distribution m Bulbs
Varieties of onion (Allium cepa) with colored skms have high flavonol content,
mamly quercetin m the aglycone form (Stavric 1997); white onions contam very little if
any quercetm while red and yellow onions contam higher but varyuig amounts (Leighton
and others 1992, Pafil and others 1995). Mizimo and coworkers (1992) found that up to
90% of quercetm is m the first and second layer. In analyzmg three yellow, two red and
one white onion cultivar the dry skm portions contamed a significantly (p < 0.05) higher
amount of quercetui compounds and free quercetm compared to the iimer rings of the
onion (Patil and Pike 1995). The 'Redbone' cv. had the highest amount of total quercetm
m the dry skin at 30.66 g/kg dry weight and as much as, 67% of the total quercetm was m
the aglycone form compared to other red and yellow colored onion varieties. In a similar
study by Hirota and coworkers (1998), 4'-Qmg and 3,4'-Qdg, and Qag were higher in
outer scales and at the upper portions of the scales compared to the lower portion of the
scale. The outer 1-3 rmgs had on average 3 times more quercetin than the cataphylls
middle (5-6) rings (Patil and Pike 1995). Considermg the edible portions of the onion,
the outer rings or cataphylls in all of the cv. contained a higher amount of total quercetm
(Patil and Pike 1995). These correlations suggest a relafionship between red bulb color
mtensity and quercetm content although the red color cannot be attributed to flavonols
since they are colorless.
Compared to other plants onion's quercetin is concentrated in areas just below the
surface. This partitioning of quercetin m the outer surfaces and the prevalence of
different glycosides are affected by its access to light (Patil and Pike 1995). Since outer
10
scales are older and are exposed to more light as the plant matures quercetm content
mcreases in these scales (Hhrota and others 1998).
Storage and Processmg of Quercetm
One concem of growers and processors is quercetm stability in processesing,
shipping, packaging as well the cookmg, commercially and at home. Ewald and others
(1999) stated that approxunately 90% of quercetin in the onion is found m the first and
second scales. Thek study looked at the thermal stability of onions before and after heat
treatments of blanching, water cooking, cooking ui a microwave oven, fiying, and warm
water holding (60 °C for 1-2 h) of the boiled sample. The only significant loss they found
was duruig the peeling and trimming step. They observed a 39% loss m quercetm. Their
findings along with those of Mizuno and others (1992) verified the theory that quercetin
is heat stable (Ewald and others 1999). Hirota and others (1998) stated that the thermal
stability of quercetin, m onions, was due to the absence of the hydroxyl group at the C-3
position.
Even though storage is not considered a processhig step it could affect the
quercetin content. Price and Rhodes (1997) found that total quercetin content did not
change over an extended storage period in 'Albion', 'Rijnsburger', 'Rose', and 'Red
Baron' varieties (8 mon under normal conditions). They mitially found that the stability
of these compounds m onions, finely chopped, suggested that although loss of
conjugation occurs after long periods of storage this will not lead to complete loss of the
11
two major glycosides (3,4'Qdg and 4'Qmg), and the food will contam a mixture of the
two glycosides and a variable amount of the aglycone (Price and Rhodes 1997).
Beneficial Health Attributes of Ouercetm
Antioxidation
The most highly noted benefit from the mgestion and adsorption of quercetm into
the body is quenchmg of free radicals (Hertog and Holhnan 1996; Holhnan and others
1997; McAnis and others 1998; Hollman and others 1996; Madsen and Jorgensen 2000;
Prior and Cao 2000; Ym and Cheng 1998). Oxidative damages by reactive oxygen free
radicals have been hnplicated in the pathogenesis of various diseases, encompassmg
cancer, atherosclerosis, and agmg (Kehrer 1993). Antioxidants help cells to cope with
oxidative stress effectively deactivatmg free radicals, which gives them a positive link to
disease prevention (Gordon 1996). Almost all of the methods used to determine the
antioxidant activities are based on inhibition of free radical induced oxidation reaction or
the deactivation of stable free radicals (Naguib 2000). Reduction of free radicals m the
body has been suggested as a key to reducing cancer risks and decreasing the occurrence
of arteriosclerosis (Hollman and others 1996). A free radical is defined as having an odd
number of electrons. Some examples of oxygen-based free radicals are hydroxyl (0H«),
hydroperoxyl (HOO*), peroxyl (R00«), superoxide (02'*), and alkoxyl (RO»); (Prior and
Cao 2000). These reactive compounds cause many deleterious effects on the body.
Hollman and others (1997) state that reactive species are able to initiate lipid
peroxidation, a chain reaction, and oxidize other cellular components, such as DNA and
12
protems. Heat, mfection, radiation, toxms, and tissue mjury can create free radicals
(Davies 1995). Rock and coworkers (1996) found that extemal factors such as ak-bome
pollutants, ultraviolet radiation, tobacco and alcohol use will produce free radicals durmg
normal cellular activity and oxygen metabolism.
Solomons and coworkers (1999) state that by creatkig a resonance-stabilized
molecule, antioxidants neutralize the reactive free radicals. In dokig this the antioxidant
does not create a reactive secondary species, thus haltkig the propagation of more free
radicals by termmatkig the propagation process (Bors and others, 1990). Prior and Cao
(2000) found that the more -OH substitutions, the stronger the antioxidant capacity of the
compound. Quercetm aglycone has 5-OH substitutions givkig k a trolox equivalent (TE)
of 3.3. As an example some common antioxidants like a-tocopherol, ascorbic acid, pcarotene, uric acid, and bilimbki have TEs of 1.0, 0.52 - 1.12, 0.64 0.92, and 0.84,
respectively (Cao and others 1993). Trolox is a water-soluble analogue of vitamin E.
This combkied with the oxygen-radical absorbance capacity (ORAC) activity (3.29)
makes quercetin one the most potent antioxidants ki the human diet (Cao and others
1993). Cao and coworkers (1993, 1995) developed the ORAC assay to provide an
effective method to determine the total antioxidant capacity ki fiuits and vegetables. This
method combines both kihibition time and inhibition degree of the free radical or oxidant
action by an antioxidant into a single quantity using an area under the curve technique for
quantification of the data (Cao and others 1993, 1995). Quercetin may also fimction to
scavenge free radicals by acting as a chain-breaking antioxidant, recycling other chainbreaking antioxidants such as a-tocopherol by donating a hydrogen atom to the
13
tocopherol radical, and a capacity to chelate metal ions (McAnis and others 1998). The
antioxidant capacity of quercetin is key to many of its other functions; the most knportant
is its implication in the reduction of cancer risk.
Cancer
In 1997, 564,800 people died from cancer ki the U.S., making cancer the number
one cause of death. This equates to 1,547 people per d who died from cancer alone
(Landis 1998). In the U.S., epidemiological evidence suggests that 35% (range 10 - 70%)
of cancer deaths are attributable to variation ki diet (Bailey and Williams 1993). Some
food-related carcinogens include mycotoxins produced by the genera Aspergillus,
Penicillium and Fusarium (Hsieh 1989), nitrosamines and nitrosamides found primarily
ki cured meats (Hotchkiss 1989), polycyclic aromatic hydrocarbons (PAHs) (Dipple and
others 1990), and amino acid pyrrolysis products: heterocyclic aromatic amkies mamly
in cooked muscle meats, pork, beef, chicken, lamb, and fish (Bailey and others 1991).
Studies conducted by Wattenberg (1990) and Boone and coworkers (1990) revealed more
than 500 food-derived and synthetic factors that kihibk carcinogen responses ki one or
more protocols. Wattenberg (1985) has also created a classification system that lists
kihibitors into three mam groups: agents that prevent carckiogen formation from
precursors, "blockkig agents" that prevent carckiogen-DNA damage, and "suppressing
agents," which suppress transformation of inkiated cells. Quercetin is classified as a
"blockkig agent" (Bailey and Williams 1993).
14
Quercetm significantly reduces the carcinogen activity of several cooked food
mutagens kicludkig bay-region diol epoxides of benzo [a] pyrene and heterocyclic
amines (Huang and Ferraro 1992). These carckiogens requke activation by cytochrome
P-450 dependent mixed-fiinction oxidases; quercetm mhibks these oxidases in vitro.
Quercetin also kihibks the bkidkig of PAHs to DNA in vitro and ki epidermal and lung
tissues of SENCAR rats (Leighton and others 1992). One mechanism of anticarcmogenic
effectiveness proposed by Shih and coworkers (2000) is that quercetm kihibits the
kiduction of the transcription factor activator proteki-1 (AP-1) activity. However, their
study concluded that quercetin increased the binding of the AP-1, which allowed tumor
cells to proliferate. From this Shki and coworkers (2000) concluded that quercetin may
act through a different mechanism or given its stmcture may act through multiple
mechanisms or in conjunction with other flavonoids or compounds.
Hansen and coworkers (1997) examined the effect of quercetin on heat shock
protein in human breast carcinoma cells. Cells kicubated with quercetin prior to heat
shock inhibited hsp27 and hsp70 induction by 34% and 71%, respectively, as well as
reducing the basal levels of the cells. Treatment of HeLa cells reduced the bkidkig of
heat shock transcription factors (HSF) to the horizontal system equatorial cells (HSE)
under heat induced bkiding condition. Also quercetin reduced the HSF DNA-binding
activity (Hansen and others 1997). Quercetin may block addkional modifications
necessary for activation of HSF. The study of quercetm has reveled some kiteresting
findings. Quercetin can bind to type II estrogen bkiding sites to down regulate signal
transduction in breast MDA-MB-435 cell lines via kihibkion of l-phosphatidylinositol-4-
15
kkiase, which parallels a concomitant decrease of l-phosphatidylkiositol-4-phospahte-5kkiase (Jones and others 1998). Quercetin also has other fiinctions like, kihibition of UV
or chemically-kiduced aberrant ciypt formation, DNA strand breakage, and
tumorigenesis (Duthie and Dobson 1998). Leighton and coworkers (1992) found that
quercetin selectively kihibited the growth of transformed cells (ras/3T3 and H35) and
prevents neoplastic transformation of MIH/3T3 cells with the oncogene H-ras. Quercetm
kihibits cigarette smoke inhalation toxicity transcriptional competence (Leighton and
others 1992). These modifications may kiclude post-translational modifications, release
of a regulatory molecule, conformational changes, kiteractions with other DNA-bkidkig
proteins, and/or mteraction with small lignands or metabolites (Hansen and others 1997).
Just as cancer is prevalent and extremely different case to case, the anticarcinogenic
effects of quercetin are just as widespread. Cancer care has been a huge burden on the
health care kidustry as well as a tremendous emotional tax on the families. When fiiiits
and vegetables are properly harvested, processed, and stored, evidence supports the claim
that they can reduce the incidence of certaki types of cancer.
Anti-Atherosclerotic Attributes and Coronary Heart Disease
Researchers have found that quercetm can delay the onset or even prevent some
forms of coronary heart disease (CHD) such as atherosclerosis. Hertog and Hollman
(1996) found that men who had a high kitake of quercetin had about a thkd of the risk of
developmg CHD than the men who had the lowest intake. In 2 other epidemiological
studies, they found in approximately 800 elderly men there was an inverse correlation
16
between mortality from CHD and quercetm kitake. They also found that in 552 Dutch
men the incident of first stroke event was inversely related to quercetin intake (Hertog
and Holhnan 1996).
There are five main types of lipoprotekis ckculating in semm: high-density
lipoprotem (HDL), "good lipoprotekis" kitermediate density lipoporteins (IDL), lowdensity lipoproteins (LDL), very low-density lipoproteins (VLDL), and chylomicrons
(Voet and others 1999). It is therefore plausible that the consumption of quercetin
because of its antioxidant properties could reduce LDL oxidation and atherogenesis in
vivo.
Lipid peroxidation requires oxygen, a free radical initiating species, and the
presence of an unsatiu-ated double bond in the lipid (L). These products dismpt normal
fimctionkig of a cell and can damage or modify DNA (Williamson and others 1999). The
three steps involved are inkiation, propagation, and termkiation. Propagation of free
radicals is contkiuous and forms lipid hydroperoxides (LOOH) until an antioxidant (A)
donates a hydrogen to form two stable species (Williamson and others 1999). The
reaction is as follows:
LH + R»
r:> L« RH
Inkiation
L» + O2
=> L02»
Propagation
L02» + LH r:>LOOH + L»
Propagation
L02« + AH iz> LOOH + A»
A» + A»
=:> A-A
Termkiation
17
It is believed that oxidative modification of LDLs by free radicals is an essential
early step ki the pathogenesis of arteriosclerosis (McAnis and others 1998; Knekt and
others 1996). When reactive oxygen species oxidize fatty acid side chakis in membranes,
the integrity is compromised (Knekt and others 1996). Oxidized LDL has been detected
ki atherosclerotic lesions and is thought to play a major role ki the kikiation and
progression of atherosclerosis (Sternberg and others 1989). This oxidative modification
of LDL leads to lipid peroxidation, which forms hydroperoxides, lipoperoxide
decomposkion products, and apolipoproteki B modifications (Sternberg and others 1989).
Oxidized lipoproteins are taken up by scavenger receptors of macrophages creating lipidloaded foam cells (Steinbrecher and others 1990). These cells accumulate on the kiterior
walls of the vascular endothelial cells decreasing the blood flow and hardening of the cell
walls, which leads to lesions or breaks ki the vascular tissue, in some cases (Claise and
others 1997).
McAnis and coworkers (1998) found that after subjects ingested 225 g of fried
onions, semm quercetin concentration increased from baseline values of 28.4 ±1.9 ng/ml
to a peak of 248.4 ± 103.9 ng/ml after 2 h, and then decreased to baseline after 24 h.
Despite the increase they found that the quercetm was not found in the LDL or VLDL
fraction but rather in the HDL fraction (McAnis and others 1998). There is still little
information about the absorption, distribution, metabolism, and excretion of quercetin in
humans. Even though quercetin has a high affmity for proteins and some affmity for
HDLs k does not provide a direct antioxidant effect on LDLs. A study done ki Finland
found that daily ingestion offi-uksand vegetables is beneficial. Knekt and coworkers
18
(1996) found that people with very low dietary kitake of flavonoids have an mcrease ki
risks of CHD. The patients who did eat a good variety of fiiiits and vegetables kigested
an average of 3-4 mg/d of flavonoids of which 95% was quercetm (Knekt and others
1996). Flavonoids might still exert an kidirect antioxidant effect because of
kicorporation kito vascular cells or by protectkig other prevalent antioxidants (McAnis
and others 1998).
Antiviral Activity
Quercetm has been reported to have vimcidal activity against enveloped vimses
such as herpes simplex type I, respkatory syncytial, pseudorabies, parainfluenza type 3,
and Sindbis (Kaul and others 1985; Vlietkick and other 1988). Quercetm also protects
agamst macrophage-dependant murine cardiovims infection (Kaul and others 1985;
Vlietinck and other 1988). Antivkal activity of quercetin appears to be related to ks to
bkid a viral protein and interfere with viral nucleic acid synthesis. Castrillo and Carrasco
(1987) found that methylquercetin blocked polio vims replication by kiterfering with the
singled-stranded ribonucleic acid (RNA) replicative intermediate in an association with a
block in cellular proteki synthesis. Quercetm has also shovm an ability to potentiate the
antivkal activity of tumor necrosis factor agamst vesicular stomatitis vims and
encephalomyocardkis vims in a cell culture (Ohnishi and Bannai 1993). The antiviral
activity of quercetm is related to ks ability to bkid a vkal coat proteki, polymerases and
then to damage DNA. This ability to enhance antivkal capackies of interferon and tumor
19
necrosis factor might help with vkal kifections such as HIV (Human Immunodeficiency
Vkais) (Formica and Regelson 1995).
Intestmal Absorption and Bioavailbility of Ouercetm
Absorption of quercetm in its glucoside or aglycone form is an area of kitense
study. A 10-g sample of onion would provide around 4 mg of quercetin, 100 g of green
beans would provide 1.3 mg and 0.2 liters of red wkie provide 0.8-3.2 mg of quercetm.
In comparison only 0.1 mg of a-tocopherol would be ingested from the green beans, less
than 0.01 mg ki onions and none from the red wkie (Hertog and others 1993; Ewald and
others 1999). Human flavonoid absorption has ranged from 9 to 52% depending upon the
flavonoid kigested (Holhnan and others 1995). Formica and Regelson (1995) state that
quercetm has a low rate of absorption, around 0.3-0.5% ingested. Beecher (1999)
indicates the glycosides appear to be the predominant form in plant tissue, but the
aglycone form rather than the glycoside imparts the majority of the biological effects.
Over 135 different glycoside of quercetin have been characterized (Harbome 1999). The
3-, 7-, 3'-, and 4'-monoglucosides and the 3,7-, 3,4'-, and 7,4'-diglucosides of quercetin
are the most common forms (Harbome 1973). Glucose is the most prevalent sugar but
arabinose, xylose, galactose, rhamnose, glucuronic acid, and mtinose are found
(Harbome 1984; Bravo 1998; Harbome and Williams 1988).
Holhnan and others (1997) found that human absorption of quercetin-Pglucosides and aglycone from onions was 52% and 20%, respectively, in the small
intestine. Urkie analysis found that after 24 h excretion of quercetm was ahnost
20
complete, with about 90% excreted around 8-13 h after mgestion. The short tkne k takes
for quercetin to reach peak levels suggests that the absorption takes place ki the stomach
instead of the small intestkie (Hollman and others 1997). This study concluded that the
sugar complex has a major knpact on the absorption and the level of quercetm ki plasma
(Holhnan and others 1997). These glycosides have thek sugar units removed ki the small
kitestkie by the action of glycosidases from bacteria that colonize the termkial ileum.
Most glycosides pass through the small kitestkie and then are hydrolyzed by cecal
microflora yielding the aglycones, which are then absorbed in the colon (Bravo 1998).
However, a study performed by Holhnan and coworkers (1995) foimd that
ileostomy patients absorbed quercetm glycosides better than the pure aglycones
kidicating that the colon is not essential for the absorption of quercetm (Ewald and others
1999; Prior and Cao 2000). Prior and Cao (2000) foimd the absorption of quercetm and
its appearance ki plasma within 60 minutes of ingestion of elderberry (Sambucus nigra
L.)fi-uitsin healthy human subjects. A recent study done by Gee and coworkers (1998)
implies quercetin glucosides have the capability to react with sodium-dependant glucose
transport receptors in mucosal epithelium, thus can be absorbed by the small intestkie.
A study conducted by Hollman and coworkers (1996) found that quercetin plasma
levels reached a maxknum amount of 196 ng/ml 3 h after ingestion of cooked onion
samples containing 64.2 mg of quercetin. This shows that quercetin is absorbed rather
quickly and slowly depleted ki the body throughout the d. From this evidence, it can be
postulated that flavonoids circulate ki plasma after ingestion and absorption, but the exact
mechanism and location of absorption is still unknown.
21
Hvdroponic Grovyth and Nutritional Quality
When uskig hydroponic systems support, water, nutrients and root aeration factors
must be accounted for skice the plants are without soil (Jones and others 1998). To
accomplish this special media is used to hold the plants. In North America Oasis^^
Hortcubes are used, which are 2.54 cm x 2.54 cm x 3.8 cm blocks to create a medium
density plant stand (Resh 1991). These cubes are used because they provide good
drakiage, are sterile, easy to handle and have a stable pH (Resh 1991). Hydroponic
solutions are used in plant studies for controlled envkonment because high plant growth
rates can be maintained in a relatively small root zone that produces an abundant and
constant yield (Steinberg and others 2000). Controlling and maintakiing nutrient levels
and pH are the maki challenges in using a hydroponic solution.
Researchers at Tuskegee University (Ahnazan and others 1997) found that sweet
potato greens (Ipomoea batatas (L.) Lam.) groym ki a nutrient film technique (NFT) had
an altered nutrkional quality. The NFT greens had a higher DM, ash, and proteki content
when compared to the same cuhivars grown ki a greenhouse bed. NFT plants had lower
fat, total dietary fiber, tannic acid, and oxalic acid contents compared to the cuhivars
grown ki beds (Ahnazan and others 1997). Steinberg and coworkers (2000) found that
64 d old wheat grown hydroponically at 23 °C, 70% RH at ambient CO2, contained 24.4
± 4.0 mg/g N, 35.8 ± 8.0 mg/g K, 16.6 ± 6.4 mg/g Ca and 9.7 ± 3 mg/g Mg. Wheeler and
coworkers (1993) found that soybeans (Glycine max) grown in a closed growth system
(CSG) had a paradoxical composition. The plants were higher in ash and cmde fiber, but
had decreased digestible carbohydrates. Wheat (Triticum sativum L. cv. Yecora Rojo),
22
soybeans (Glycine max [Merr] L. cv. McCall) lettuce (Lactuca sativa L. cv. Waldmann's
Green), and potatoes (Solanum tuberosum L. cv. Norland) were grovm in a closed
grov^h system (CGS) and conventionally in the field to determine compositional
diflferences (Wheeler and others 1996). Wheeler and his coworkers (1996) found that
CGS lettuce, wheat and potatoes had higher proteki levels. Soybeans were higher ki
cmde fiber and ash content as well as lettuce and potatoes bekig higher ki ash.
Mkierals
Consumption offiruksand vegetables links the human food chain to nature and a
viable source of minerals (Fischer and others 1997). Factors affectkig the mineral
composition of the plant are genetic make-up, soil conditions and salkiity, weather
conditions durkig the growmg season, use of fertilizers and maturity of the plant at
harvest (Lee 1990). Mkieral content of foods is of great interest to dieticians and cell
biologists. Mertz (1982) showed that optimal kitakes of Na, K, Mg, Ca, Mn, Cu, Zn, and
I could reduce some individual risk factors for chronic diseases such as cardiovascular
disease (CVD). Human health is greatly affected by the foods consumed, due to organic
compounds that could impak the bioavailability of trace elements for intestinal
absorption and retention (Sanchez-Castillo and others 1998). Deficiencies of some
elements, includmg Ca, Fe, Mg, and I create well-defined symptoms of ilhiesses ki
humans. Selenium has recently been recognized as playing a protective or proactive role
ki reduckig risks involved with some types of cancer (Sanchez-Castillo and others 1998).
23
Nitrogen availability to onion plants can mcrease yields (Brewster 1990), but ki
later stages of the plant development can create soft bulbs and prolong the maturation
process affectkig the product handling and post harvest quality of the bulb (Randle 2000).
Increased N in hydroponic solutions caused a decrease ki bulb weight and firmness, but
mcreased yield (Randle 2000). Increaskig N content ki hydroponic solutions used to
propagate onions mcreased total N content, decreased B, Ca, Mg, and mcreases K
contents (Randle 2000). Mkierals such as Cu, Fe, P, and Zn were unaffected by N
content in hydroponic solutions (Randle 2000).
Sulfiir Content
Sulfiir constitutes one of the macronutrients necessary for the plant life cycle.
Sulfiir uptake and assimilation in higher plants is one of the critical factors ki determining
plant growth and vigor, crop yield, and resistance to stress and insect attack (Hofgen and
others 2001). In a series of enzymatic steps S is converted to nutritionally valuable Scontakiing amino acids and flavor compounds (Hell 1997; Hell and Rennenberg 1998;
Saito 1999; Block and others 1997). Sulfur is taken from the soil through sulfate
transporters and moved to the plastids or vacuole. Randle (1992) stated that onions can
amass up to 100 mg of S per g of fresh onion tissue. The kitemal sulfate is converted
to thiosulfates or sulfite through an intermediate compound adenoskie-5'-phosphosulfate
(APS). The products are then converted to cystekie, which is kicorporated into vitamins,
cofactors, hormones, glutathiones, polyamines, and secondary metabolites (Hofgen and
others 2001).
24
Ingestion of S-containkig compounds can serve 2 non-nutritional purposes: to
impart flavor and health benefits. These health benefits kiclude antibacterial, antifimgal,
antitumor ackivity; lipid biosynthesis kihibkion; and antithrombotic activity (Koch and
Lawson 1996; Block 1996). The organosulfiir compounds are class by number of C
atoms. They are Ci and C3 kitermediates, C3 lachrymatory factor (LF), C2, C4, and Ce
thiosulfinates, the Ce LF dimer, zweibelanes and bisulfine and C9 ajoene and cepaenes,
with non-protein S and selenium amino acids and volatile selenium compounds (Block
and others 1997). Cuttkig an onion will combine alliinase enzymes with the organosulfiir
compounds and following several biochemical reaction the LF is produced. LF is the
compound that cause one's eyes to tear when cuttkig onions by creating a small amount
of sulfiiric acid ki the eye (Block and others 1996).
Lancaster and Boland (1990) stated that onions accumulate large amounts of S,
which is metabolized prknarily through the flavor precursor biosynthetic pathway. An
onion's ability to partition S as S04'^ is important in overall flavor development, although
total bulb S correlates poorly with overall flavor mtensity in a broad range of onion
varieties (Randle 1992). Gamiely and coworkers (1991) found that kicreasing NH4^ as a
percent of the total N correlated with mcreased total bulb S amounts.
Grovyth Enhancement ki Grovyth Chambers
Growth chambers allow for control of basic condkions wkhin the chamber
environment such as, temperature, light and relative humidity (van lersel and Bugbee
2000). This control allows for creation of optimal growth condkions. Increased CO2
25
levels of 1200 ppm and photosynthetic photon flux (PPF) Iknits increases grovvth.
Development rate is most related to photoperiod (increaskig photoperiod mcreases the
PPF) and temperature, which is optimal ki the range of 15-25 °C (Bugbee 1995).
In controlled environment, to achieve maxknum yield, two short life cycles are
generally preferred over one long cycle (Bugbee 1995). Several studies found that
mcreased CO2 levels affect maintenance respkation by changkig carbon-partitionkig
rates. Baker and coworkers (1992) in long-term CO2 experknents with rice found that
canopy dark respiration mcreased (per unk ground area) as CO2 concentrations mcreased
to 500 lamol/mol from 160 f^mol/mol. However, specific canopy respkation decreased
(per unk dry mass). Higher specific respkation rates ki subambient CO2 treatments were
attributed to higher makitenance respiration. In a study conducted by Thomas and Griffin
(1994) on C02-eiu-iched soybeans makitenance respkation mcreased by 34%, while the
growth respkation did not change. The mcrease was attributed to a 33% increase ki leaf
total nonstmctural carbohydrates (TNC) in the enriched leaves, causing greater starch and
sucrose synthesis and more sucrose export. Increased C use efficiency was caused by a
decrease ki maintenance respiration durkig early vegetative growth, although
maintenance respkation costs were larger in C02-enriched wheat canopies (Bugbee
1995).
Super-elevated CO2 can be detrknental to plants, but some environments have
CO2 levels as high as 6000 ^mol/mol such as the space shuttle cabin while in orbit
(Wheeler and others 1999). Levels this high can impede plant grovs^h by reducing
stomatal opening and conductance. Wheat (Triticum aestivum L. 'Yecora Rojo') and
26
potatoes (Solanum tubersum L. 'Denali') were grown at 400, 1000, and 10,000 ppm CO2
for 50 d ki an EGC ki a reckculatkig NFT at 300 fxmol/m^s with 12 h of daylight. These
plants showed no signs of stress or kijury, and the plants grown 1000 ppm CO2 had
higher stomatal conductance rates and the highest water use efficiency with 3.8 g/kg for
the potatoes and 3.1 g/kg for wheat (Wheeler and others 1999).
Accumulation of ethylene gas in a growth chamber is a concem due to the fact
that plants naturally produce ethylene during normal biological growth and high levels
can cause deleterious effects (Mattoo and Suttle 1991; Abeles and others 1992). An
ethylene level of 0.1 to 10 ppm can cause stem swelling, seedlkig hook opening, leaf
epinasty, floral abortion, leaf abscission andfi-uitripening (Wheeler and others 1996).
The highest concentration of ethylene ki CGS, for wheat, soybeans and potatoes, is
during the first 20-30 d after planting while the plants are young and ethylene is first
produced (Wheeler and others 1996). Chronic exposure to ethylene levels of 50 to 100
ppb has reduced growth of lettuce and lilies (Mortensen 1989; Blankenship and others
1993).
27
Project Objectives
The objective of this set of experknents was to assess total flavonol (TF) levels ki
onions (Allium cepa L.) grown ki an envkonmental controlled growth chambers with a
soil-less growmg media. The nutrkional constituents of onions grovm ki growth
chambers were compared to onions grown under greenhouse conditions ki potting media.
Elevated CO2 level effects on nutrkional content and edible biomass production ki onions
(Allium cepa L.) were also investigated.
Variability of growth rates and composition of onions (Allium cepa L. 'Purplette')
grown hydroponically ki Oasis^'^ Hortcubes and in grown in Ball Growing Mix 2^^
potting soil in a greenhouse was determined. Total flavonol levels and composition
measurements were made to determine the withki species variation exhibited over a
growth cycle. Phenotypic measurements were made to determine total edible biomass.
Effect of photoperiod and environmental conditions on the growth and
composkion of long-day (Allium cepa L. 'Purplette') and short-day (Allium cepa L.
'Redbone') onions grown ki environmental growth chambers was determined. Total
flavonol levels and composition ki the leaves, bulbs, and roots of the onions were
compared. Phenotypic measurements were made to determine total edible biomass.
Effects of elevated CO2 levels on the composition and growth rate of onions
(Allium cepa L. 'Purplette') placed in individual growth chambers (IGC) was determined.
Three different gassing levels of 370 ppm (ambient), 1000 and 2000 ppm (elevated) for a
duration of 96 h with a 48 h acclknation period to the IGCs were used. Total edible
biomass was measured along with composite analysis and TF level in each of the onions.
28
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40
CHAPTER II
MATERIALS AND METHODS
Greenhouse Experknents
Plant Material for Experknent 1
Two-hundred long-day onions (Allium cepa L. 'Purplette') (Johimy's Selected
Seeds; Albion, ME, USA) were planted in Oasis Hortcubes^M (Smithers-Oasis, Kent,
OH, USA) at a depth of 0.75 mm at one-week kitervals begkming March 22, 2001, for 7
consecutive weeks. Seeds were sown and held ki the Horticultural Gardens greenhouse,
Texas Tech University, Lubbock, TX, USA. After 1 week to allow for germkiation,
plants were watered daily with Hydro-SoF'^ (Scotts-Sierra Horticultural Product Co,
Marysville, OH, USA). For chemical composition of Hydro-SoF'^ refer to Appendix F
(Table F.I.). Twenty plants within each of 7 rep were harvest weekly at 14, 21, 28, 35,
42, 49, 63, 77 and 98 d of age. Only 6 rep were obtakied at 63 d, 4 rep at 77 d, and 98 d
of age due to kifections of plants with fungus gnats.
Plant Material for Experknent 2
Four-hundred long-day onions (Allium cepa L. 'Purplette') (Johnny's Selected
Seeds; Albion, ME, USA) were planted in Oasis Hortcubes™ at a depth of 0.75 mm
(Smithers-Oasis, Kent, OH, USA) to create 2 rep within the hydroponic treatment.
Another four-hundred long-day onions (Allium cepa L. 'Purplette') (Johnny's Selected
Seeds; Albion, ME, USA) were planted in Ball Growmg Mix 2™ (Ball Seed; West
41
Chicago, IL, USA) pottmg soil at a depth of 0.75 mm with row and seed spackig
matching that of the Oasis™ plantkigs (3.8 cm x 3.8 cm) to create 2 rep withki the
pottmg soil treatment. Plants were planted on June 6, 2001. Composkion of pottmg soil
is listed ki Appendix F (Table F.2.) Seeds were sown and held ki the Horticultural
Gardens greenhouse, Texas Tech University, Lubbock, TX, USA. After 1 week to allow
for germkiation, plants were watered daily with Hydro-Sol™ (Scotts-Sierra Horticultural
Product Co, Marysville, OH, USA). For chemical composkion of Hydro-Sol™ refer to
Appendix F (Table F.l.). Forty plants (20 plants per rep) from each growmg medium
were harvested at 14, 21, 28, 35, 42, and 49 d of age.
Sample Preparation
In order to have enough samples for analyses, young plants were composked
together. Ten plants from harvest at 14 d of age and 21 d of age were composited to
make two samples representative of the respective age. Five plants were composited for
ages 28 d and 35 d, 4 plants were used for ages 42 d and 49 d, and 2 plants were at 63 d
of age. Plants that were 77 and 98 d of age were large enough to constitute a single
sample. Each plant at each harvest date was measured for height, weight and net number
of leaves before composkkig. Biomass was determined using plant weight, excluding
any root material below the basal plate. The plants were removed from the Oasis^M
below the basal plate and the remakikig roots were removed at the basal prior to
weighing. After weighkig, plants were chopped mto pieces no longer than 5 cm and
knmediately frozen ki liquid N. After freezkig, plant material was ground ki a coffee
42
grkider (Braun®; Boston, MA, USA; Mr. Coffee®; Hattiesburg, MS, USA). A maxknum
of 2 g of ground onion powder was used knmediately for flavonol extraction and the
remakiing powder was placed ki WhklpackTM bags and placed ki a -20 °C-freezer, for
use ki fiirther analyses.
Extraction for Total Flavonol Determmation
Extractions were carried out uskig 80% ethanol (EtOH) and followkig the method
outlkie by Lombard (2000).
Quantification with a Spectrophotometer
After extracts were warmed to room temperature and vortexed, a 0.5-ml sample
was diluted with 4.5 ml of 80% EtOH and vortexed for 5 s on a Vortex Genie (Fisher
Scientific, Houston, TX, USA). Samples were read on a Bausch & Lomb Spectronic 20
Colorimeter/Spectrophotometer Model 95 (Richmond, BC, Canada) at 362 nm in 1-cm
cuvettes (Fisher Scientific; Houston, TX, USA; Bausch & Lomb; Richmond, BC,
Canada) (Lombard 2000). Duplicate readings were preformed and averaged.
Isoquercitrm (Extrasynthese; Genay, France) was used as a standard, based on
research conducted by Lombard (2000). Nkie different standard solutions were used to
create a standard curve (AU vs. concentration) using linear regression (Appendix A).
Standard concentration levels used to create the standard curve were obtakied from
Lombard (2000) with the addkion of two more concentrated standards to achieve a range
43
of 0.00625-0.125 mg/ml. Totalflavonollevel was calculated using linear regression
(Appendix A).
Dry Matter and Moisture
Approximately 1 g offrozenonion powder was placed ki a pre-weighed
alumkium pan and placed ki a vacuum oven (National Appliance Co.; Portland, OR,
USA) and dried for at least 16 h at 100 °C followkig AOAC method 934.01 (AOAC
1990). Duplicate samples were tested and averaged. Samples were removed from the
oven and allowed to cool in a desiccator. Upon cooling samples were weighed and
recorded as dry weight. Percent DM was calculated by dividing dry weight by the fresh
weight and reported as a percent by multiplying by 100. Moisture content was
determkied by subtracting DM percent from 100%.
Nitrogen and Carbon Analyses
After samples were dried, the material was placed ki a Wkklpack^"^ bag and
ground further with a pestal. Samples werefinelyground and analyzed with a Carlo Erba
NCS 2500 C/N analyzer (Milan, Italy) to determine total N, C level and C/N ratio.
Approximately 2-4 mg samples were weighed and placed into alumkium vials and loaded
mto the analyzer. Atropkie (CE Elantech; Lakewood, NJ, USA) was used as the
standard. Duplicate samples were tested and averaged.
44
Ash and Mkieral Analyses
Approxknately 1 g of frozen onion powder was placed in a pre-weighed ceramic
cmcible. Samples were dried followkig the methodology listed above and then placed ki
a muffle fiimace (Sybran Thermolyne 2000 Fumace, Thermolyne Corp.; Dubuque, lA,
USA; Blue M Lab-Heat Muffle Fumace, Blue M Electric Co.; Blue Island, IL, USA) for
at least 16 h at 500 °C followkig AOAC method 900.02 (AOAC 1990). Samples were
then placed ki desiccator and upon coolkig were weighed. This weight mkius the
cmcible weight was recorded as ash weight. To calculate percent ash the ash weight was
divided by the fresh weight and mukiplied by 100. Duplicate samples were tested
whenever possible.
To determkie mkieral content specifically, Ca, Mg and K, samples were wet
ashed by addkig 15 ml of 20% HNO3 and procedures were followed according to Perkin
Ehner (1976). The dry ash was allowed to dissolve for at least 5 h followkig AOAC
method 900.02 (AOAC 1990). After dissolvkig the solution was filtered through
Whatman (Clifton, NJ, USA) grade 40 ashless fiher paper and diluted to 100 ml with
distilled H2O (D-H2O) in a volumetric flask. Duplicate samples were prepared whenever
possible. One ml of each sample was placed ki one of two separate tubes and 10 ml of DH2O was added. To one set of the tubes 0.5 ml of 5% lanthanum chloride (LaCb) was
added as releaskig agent. This tube was used for Ca and Mg quantification.
Three standards were made for the Ca and Mg analyses. To make the Ca
standards, 500 |il of Ca atomic absorption standard (995 |Ag/ml in 1% HCl) (SigmaAldrich; St. Louis, MO, USA) and 5 ml of 5% lanthanum chloride was diluted to 100 ml
45
with D-H2O, addkig 400 ^1 and 5 ml of 5% lanthanum chloride and dilutkig to 100 ml
with D-H2O made a senskivity standard. A blank was prepared by dilutkig with D-H2O 5
ml of LaCb to a total volume of 100 ml ki 100-ml volumetric flasks. After the
kistmment was standardized, samples were read ki concentration mode on a Perkki Ehner
2380 Atomic Absorption Spectrophotometer (Norwalk, CT, USA). The lamp used was a
Perkki Elmer (Norwalk, CT, USA) hollow cathode Ca Intensitron run at a wavelength of
422.7 nm with an ak/acetylene flame and an oxidizkig lean, blue flame and samples were
recorded as ng/ml (Perkki Ehner 1976). To determkie mkieral concentration ki mg/100
g, the recorded value was multiplied by 1050 (dilution factor), divided by 1000, divided
by the wet sample weight and finally mukiplied by 100.
To make the Mg standard, 50 |il of Mg atomic absorption standard (1015 ng/ml ki
1% HNO3) (Sigma-Aldrich; St. Louis, MO, USA) and 5 ml of 5% lanthanum chloride
was diluted to 100 ml with D-H2O. Plackig 30 ^l of Mg standard and 5 ml of 5%
lanthanum chloride in a 100-ml volumetric flask and diluting to 100 ml with D-H2O
made a sensitivity standard. A blank was prepared by addkig only 5 ml of 5% lanthanum
chloride and diluting to 100 ml with D-H2O. After the kistmment was standardized
sample were read in concentration mode on a Perkin Elmer 2380 Atomic Absorption
Spectrophotometer (Norwalk, CT, USA). The lamp used was a Perkki Elmer (Norwalk,
CT, USA) hollow cathode Mg Intensitron mn at a wavelength of 285.2 nm with an
air/acetylene flame and an oxidizkig lean, blue flame and samples were recorded as
ng/ml (Perkin Ehner 1976). To determkie mkieral concentration in mg/100 g, the
46
recorded value was mukiplied by 1050 (dilution factor), divided by 1000, divided by the
wet sample weight andfinallymukiplied by 100.
To make the K standard 200 \i\ of K atomic absorption standard (995 ng/ml ki 1%
HCl) (Sigma-Aldrich; St. Louis, MO, USA) and was diluted ki a 100 ml volumetric flask
volume with D-H2O. D-H2O was used as the blank. After the mstrument was
standardized samples were read in concentration mode on a Perkin Ehner 2380 Atomic
Absorption Spectrophotometer (Norwalk, CT, USA). The lamp used was a Perkki Ekner
(Norwalk, CT, USA) hollow cathode Na-K Intensitron mn at a wavelength of 766.5 nm
with an air/acetylene flame and an oxidizing lean, blueflameand samples were recorded
as \ig/m\ (Perkin Elmer 1976). To determine mkieral concentration ki mg/100 g, the
recorded value was multiplied by 1000 (dilution factor), divided by 1000, divided by the
wet sample weight andfinallymultiplied by 100.
Sulfur Quantification
Sulfur quantification followed guidelkies set out ki the AOAC, uskig the
Magnesium Nitrate 923.01 and the Sodium Peroxide 920.10 methods (AOAC 1990).
Approximately 1 g of fresh onion powder was weighed in a ceramic cmcible. This
method is an oxidative gravknetric method. At the completion of the oxidation the
remakikig residue (BaS04) was weighed and mukiplied by 0.1374 to determine total S
content (g) present ki the sample. Sulfiir content was reported in mg/100 g by
mukiplykig by 1000, dividing by the wet sample weight and muhiplying by 100.
47
Experunental Design and Statistical Analyses
Experknent 1
This experknent was an age x plantkig date factorial. There were 9 levels of age:
14, 21, 28, 35, 42, 49, 63, 77 and 98 d. There were 7 plantkig dates 1-7, each planted one
week after the previous. Analyses of variance was conducted uskig general Ikiear models
(Proc GLM) ki SAS 8.0 (SAS Institute, Gary, NC, USA). Plant height, weight, and net
leaf number means were displayed by plantkig date over tkne due to a significant
mteraction. Compositional variables of the onion: DM, N, C, ash, Ca, Mg, K, S and TF
means were separated uskig Tukey's mean separation (p < 0.05) when no significant age
X plantkig date interactions were present. Non-significant interactions were removed
from the statistical model to create a more precise mean square error.
Experiment 2
This experknent is a 6 x 2, age x growmg medium factorial with age levels oft
14, 21, 28, 35, 42 and 49 d and 2 growkig media. Oasis Hortcubes^^* and Ball Growmg
Mix 2TM potting soil. Analysis of variance was conducted uskig general Ikiear models
(Proc GLM) in SAS 8.0 (SAS Institute, Gary, NC, USA). Plant weight and ash percent
means were displayed by growkig medium across age due to a significant interaction.
Plant height was different between age and growing medium Composkional variables of
the onion: DM, C, Ca, K, and S means were not different (p > 0.05) between growing
medium, and age separated and were displayed as 1 pooled mean for each variable.
48
Percent N, Mg and TF levels did not differ by growkig medium however age means were
separated using Tukey's mean seperation (p < 0.05).
Envkonmental Grov^h Chamber Experiment
Plant Material
Short-day onion plants (Allium cepa L. 'Redbone') (Asgrow Seed Co.;
Kalamazoo, MI, USA) and long-day (Allium cepa L. 'Purplette') (Johnny's Selected
Seeds; Albion, ME, USA) were planted ki Oasis Hortcubes™ at a depth of 0.75 mm.
Plants were germinated and grown ki a greenhouse (Texas Tech University, Lubbock,
TX, USA). After 1 week to allow for germkiation to occur, plants were watered daily
with Hydro-Sol™ (Scotts-Sierra Horticultural Product Co, Marysville, OH, USA). For
chemical composkion of Hydro-Sol™ refer to Appendix F (Table F.l.). At 24 d of age,
40 plants of each variety were selected with sknilar heights and the same morphological
state, 3'^' leaf stage, and placed in two different envkonmental growth chambers (EGC)
model GC-15 (EGC-Ltd.; Wmnipeg, Manitoba, Canada) each with different
photoperiods. EGCl had a photoperiod of 16 h of daylight and EGC2 had a photoperiod
of 11 h of daylight. Each chamber was set up with four blocks (Appendix G; Figures G. 1
& G.2) with ten sampling positions in each block. Five plants of each variety were
randomly selected and randomly assigned to one of the ten poskion within in each EGC
block. The plants were grovm for 3 wk in the chambers to afinalage of 45 d.
49
Envkonmental Condkions
Envkonmental condkions withki growth chambers were sknilar with photoperiod
bekig the only variable. The kitended envkonmental parameters for each chamber were
as follows: RH 70%, temperature 24 °C d/21 °C night, light density 563 nmol/m^s and
nutrient pH 6.50. The growth chambers used Hydro-Sol™ solution that ckculated with a
flow rate of at least 1 1/min.
Sample Preparation
In order to have enough sample, for analysis plants were composited together to
create a sufficient quantity of material to sample. Plants parts of the same variety withki
each EGC block were composited together. Bulbs of 'Redbone' from EGC 1, block 1
were combkied to give 1 bulb sample of the 'Redbone' variety from EGCl, block 1.
Each plant was measured for height, weight and net number of leaves before compositing
to determine total edible biomass. The plants were removed from the Oasis^^ and the
roots were removed at the basal plate. The roots remakikig ki the Oasis ^"^ were removed
and combkied with the roots that were protmdkig outside of the cubes. The bulb was
separated from the leaves at the beginnkig of the neck. Analyses of plant samples was
conducted as stated under greenhouse experknents.
Experimental Design and Statistical Analyses
This experiment was a splk plot with 2 photoperiods, with 10 plants of 2 different
varieties ki each of 4 blocks withki each EGC (photoperiod). The whole plot is each
50
photoperiod and splk plots are variety and plant part. Analysis of variance was
conducted uskig general Ikiear models (Proc GLM) ki SAS 8.0 (SAS Institute, Gary, NC,
USA). Nutrkional components of the onion: DM, N, C, ash, Ca, Mg, K, S and TF means
were separated uskig Tukey's mean separation (p < 0.05).
Individual Growth Chamber Experiment
Plant Material
Long-day onions (Allium cepa L. 'Purplette') (Johnny's Selected Seeds, Albion,
ME, USA) were planted in Oasis Hortcubes^^ at a depth of 0.75 mm. After 1 week to
allow for germkiation to occur plants were then watered daily with Hydro-SoF^ (ScottsSierra Horticultural Product Co, Marysville, OH, USA). Plants for this experiment were
germinated and grown ki a growth room with 16 h of daylight. For chemical composition
of Hydro-Sol^^ refer to Appendix F (Table F.l.). At 30 d of age, 4 plants were selected
with sknilar heights and the same morphological state, 3 leaf stage with 4 leaf stage
starting to emerge and placed in an mdividual growth chamber (IGC). Plants were
acclknated for 48 h at ambient CO2 and then gassed for 96 h at 1000 or 2000 ppm CO2.
Four plants were placed ki the IGC and acclknated for 48 h at ambient then gassed for 96
h at CO2 (370 ppm) to establish a baselkie.
51
Environmental Condkions
The IGC envkonmental settkigs were as follows: RH 75%, temperature 25 °C/ 21
°C night, light density 630 nmol/m^s, photoperiod 16 h light/8 h dark, and nutrient pH
6.8-6.9. Fresh Hydro-SoF^ was added to the chambers daily.
Sample Preparation
Each plant was analyzed for biomass production and composition by methods
listed ki greenhouse experiments.
Experimental Design and Statistical Analyses
This experiment was completely randomized design (CRD) with 3 levels of CO2
and 4 rep. Analysis of variance was conducted uskig general Ikiear models (Proc GLM)
ki SAS 8.0 (SAS Institute, Gary, NC, USA). Plant weight, and net leaf number means
were separated uskig Tukey's mean separation (p < 0.05). Nutritional components
of the onion: DM, N, C, Ca, Mg, and K, means were separated using Tukey's mean
separation (p < 0.05). Plant height, percent ash, S, and TF contents were not
significantly (p > 0.05) different.
52
References
AOAC 1990. Official Methods of Analysis. 15* ed. Arlkigton: AOAC Int. 58 p.
Lombard KA. 2000. Investigation of the flavonol quercetin ki onion (Allium cepa L.) by
high-performance liquid chromatography (HPLC) and spectrophometric
methodology [MSc thesis]. Lubbock, TX: Texas Tech University. 41-42 p.
Available from: University Library, Lubbock Texas; AC805.T3 2000 no. 147.
Perkin Ehner. 1976. Analytical methods for atomic absorption spectrophotometry.
Norwalk: Perkki Ehner.
53
CHAPTER III
RESULTS AND DISCUSSIONS
Greenhouse Experiments
Experiment 1
Onions tend to show an early low relative growth rate when compared to other
vegetables (Tei and others 1996). In Figures 3.1 and 3.2, the plant weights and the
heights show this effect. For the first 21 d the height and weight of the onions varied
slightly from 7.87 cm to 6.84 cm and 0.07 g to 0.09 g, respectively. After the 21 d of
age, the growth rate mcreased and became ahnost linear with the height starting to level
off around 77 d. Brewster (1994) has shown that onions planted ki beds reach thek
maxknum height around 70-85 d of age ki conjunction with the start of bulbkig. At the
start of bulbkig the first and second oldest leaves began to desiccate, wither and fall off
(Brewster 1994). In Figure 3.3, the net number of leaves levels off around 35 d of age at
about 3-4 leaves and gradually mcreased to 4 leaves/plant until plants reached 63-77 d of
age and appeared to stabilize at 4-5 leaves/plant. Leaf cessation occurs around this time
ki occurrence with bulb formation (Tei and others 1996). Tei and coworkers (1996)
found sknilar findkigs, reportkig plants tend to kikiate bulbkig at ahnost 63-70 d of age
with a concurrent stabilization of net leaf number. Bulbing was found to be initiated
between the 49 and 63 d measuring period, earlier than Tei and coworkers. This could be
due to plants bekig exposed to a long-day length shortly after germkiation. However,
after bulbing begins ki onions, uniform distribution of the kiside canopy radiation and
54
cessation of leaf development occurs leadkig to a high radiation use efficiency and large
amounts of DM accumulation. Growth Iknitations are based on low light kiterception,
leaf posture, and a short duration of ground cover compared to length of the bulbkig cycle
(Brewster and Sutherland 1993). Brewster (1994) states at the start of bulbkig onions
reach thek maxknum height and do not produce any new leaves. For complete data refer
to Appendix B (Table B.l).
The DM content of the 'Purplette' onions was 10.60 ± 0.10% (mean ± SEM) and
was not significantly (p > 0.05) different across ages or rep. Tei and coworkers (1996)
showed that DM content accumulation ki onion does not increase until plants reached
about 60 d of age. Also a longer growkig season will mcrease the DM content of onions
as well as certain soil conditions (Kahane and others 2001). However, three different cv.
of sweet potato greens grovm using NFT had higher DM contents when compared to the
same cv. grown in three different fields (Almazan and others 1997).
Percent N in the onion significantly (p < 0.05) decreased with plant agefroma
high of 0.55% to a low of 0.35%fromthe age of 28 d to 98 d (Figure 3.4). On the other
hand percent C slightly decreased from 4.16% to 4.00%from28 to 77 d of age and then
reached its peak of 4.27 ± 0.09 at 98 d of age (Figure 3.5). Given that there were only 4
rep for 77 d age treatment and 2 rep for 98 d treatment could account for the significant
difference, since from d 28 to 63 the percent C was unchanged. Overall C content at 28 d
was not significantly (p > 0.05) different from C at 98 d of age. Carbon was constant up
to 63 d, dropped then rebounded. This could be due to the bulbing process just beginning
and total mcrease C had not finished. Nitrogen content decreases as plant matures and
55
undergoes the begkmkig of bublkig, while the C content should kicrease (Randle 2000;
Brewster 1994). Carbon content mcreases due to accumulation and polymerization of
non-stmctural carbohydrates during bulbing. Fmctose and glucose are the two major
components with sucrose and fructans (fructose polysaccharides formed by addkig a
fhictosyl group to a sucrose molecule) makkig up the remakikig content (Kahane and
others 2001).
Ash content significantly (p < 0.05) mcreased from 0.91 to 1.07% as the plant
matured from 28 to 77 d (Figure 3.6). However, Ca and Mg (Figures 3.7 & 3.8) levels
decreased from early high concentrations at 28 d of age of 123.6 mg/100 g and 56.8
mg/100 g to 64.2 and 22.0 mg/100 g, respectively, at 98 d of age. A sharp, significant
(p < 0.05) decrease in concentration for Ca and Mg occurred from 49 to 63 d of age,
followed with a steady significant (p < 0.05) decrease in content to lows of 64.2 and 22.1
mg/100 g at 98 d of age, respectively. Potassium levels tended to decrease from an
average of 279.1 at 28 d of age to 198.1 mg/100 g at 98 d of age. Because of a significant
age by plantkig date interaction, K means are displayed by rep across age groups (Figure
3.9). Whole spring onions, not yet matured contain on average 18.8 mg/100 g of Ca and
148.2 mg/100 g of K (National Onion Association 2001) and Mg concentrations of 11
mg/100 g (Ensminger and others 1994). Aknazan and coworkers (1997) reported an
increase ki K, Ca and Mg content of sweet potato greens ki beds to NFT. They also
reported an increase ki ash percent ki the same samples (Almazan and other 1997).
Randle (2000) has shown that increases ki N content of the hydroponic solutions causes
reduction ki Ca and Mg and that K content will decrease steadily with kicreasing N ki
56
solution. Another cause ki the reduction could be related to the morphological change ki
the onion. From the 49 to 63 d sampling points the onions seemed to start the bulbing
process based on the slow down ki growth rate. Very little is known about the
biochemical changes that occur when bulbkig occurs. Brewster (1994) has hypothesized
that an increase ki reducing series, sucrose, and fiaictans, which change the osmotic
potential and could then affect the mineral contents (Brewster 1994).
There was no significant (p > 0.05) change ki S content from 28 d to 98 d of age.
The overall mean content of S was 188.0 ± 2.9 mg/100 g. Randle (2000) found that total
bulb S content in onions increased cubically as N content increased ki hydroponic
solutions. Randle (1992) reported S contents rangmg from 42.9 to 52.6 mg/g of dry
weight ki onions grovm hydroponically and onions can accumulate as much as 100 mg of
S/g of dry weight. Bulb S correlates poorly with overall flavor intensity among a broad
range of onion accessions and onions that partition little S as SO4" are more pungent than
those that accumulated larger amounts of SO4' (Randle 2000).
Total flavonol content (Figure 3.10) significantly (p < 0.05) increased throughout
the growth of the onions, from a mean low of 266.1 at 14 d of age to 554.8 mg/100 g at
98 d of age. Lombard (2000) found a linear relationship (r^ = 0.96; p < 0.0001) between
TF content quantified by spectrophotometry and 3,4'-Qdg + 4'-Qmg quantified by HPLC
analysis. Price and Rhodes (1997) also found that over 80% of TF content was accounted
for by 3,4'-Qdg and 4'-Qmg. Patil and Pike (1995) found total quercetki content to be as
high as 3066.83 mg/100 g dry weight in one red colored variety 'Redbone' and as low as
from 9.50 mg/100 g ki a white colored onion 'Contessa' variety. Price and Rhodes
57
(1997) reported quercetki levels rangmg from 1369-1778 mg/kg wmb, Patil and
coworkers (1995) found quercetin ranged from 54.3-286.4 mg/kg wmb and Hertog and
coworkers (1992b) reported mean quercetm content of 347 mg/kg. 'Purplette' onions
contam large amounts of TF and the apparent discrepancies among studies are likely
related to varietal differences. Increases ki light mtensity kicrease flavonol content ki
plants (Barabas and others 1998; Britt 1999). For complete nutritional analysis data refer
to Appendix B (Table B.2).
Experiment 2
In Figures 3.11 and 3.13 the plant weights and the number of leaves mcreased
over tkne and after 21 d and appeared to be slightly greater ki plants grown potting soil.
The weight increased from 0.08 g at 14 d of age to 2.31 g at 49 d of age for plants grown
in potting soil. An increase from 0.07 g to 1.43 g in weight of plants grown
hydroponically was observed from 14 d to 49 d. The plants grown ki potting soil
performed better in terms of weight with a linear increase while the plants in the Oasis^"^
decreased ki weight by 22.7% after d 42 to d 49. The number of leaves increased to
around 42 d of age at about 4 leaves for hydroponically grown onions and 4.5 for soil
grown onions. Both growing media did show a decrease of 1-1.5 leaves per plant from
42 to 49 d of age. Leaf cessation occurs around the tkne the plants start to bulb and given
that a d length of around 16 h, these onions could have begun to bulb. Leaf cessation
occurs with bulb formation (Tei and others 1996). If the onions began to bulb around this
time then the Tei and others data was consistent with our data even with significant (p <
58
0.05) kiteractions between age and time. Plants heights were significantly (p < 0.05)
higher for the plants grown in potting soil with a mean of 21.0 cm, while the plants
grown hydroponically managed to average 20.3 cm (Figure 3.12). Plants heights were
significantly (p < 0.05) different ki age with heights ranging from 7.64 cm to 26.13 cm.
The plant height decrease after d 42 this could be related to a levelkig offstage before
bulbkig or an early effect of the spider mite kiterference. Brewster (1994) has shown that
onions reach thek maxknum height around week 70-85 d of age ki correlation with the
start of bulbing. At the start of bulbkig, the first and second oldest leaves began to
desiccate, wither and fall off (Brewster 1994). Brewster (1994) states at the start of
bulbkig onions reach thek maxknum height and do not produce any new leaves. These
plants were only grovm to an age of 49 d due to kifestation of the greenhouse with spider
mites, which knpeded natural plant growth and left no quality samples to analyze. For
complete data on biomass production refer to Appendix C (C.l).
The DM content of plants was not different between plants grown ki Oasis™ or
soil, and plant maturity did not affect DM content with a pooled mean of 10.39 ± 0.41%
(mean ± SEM). Tei and coworkers (1996) showed that DM content accumulation in
onions does not kicrease until the plant reaches about 60 d of age. A longer growing
season will mcrease the DM content of onions as well as certaki soil condkions (Kahane
and others 2001). However, 3 different cv. of sweet potato greens grown uskig NFT had
higher DM contents when compared to the same cv. grown in three different fields
(Almazan and others 1997).
59
There was no significant difference ki N content between plants grown ki the to
media regardless of age. Percent N ki the onions decreased (p < 0.05) with plant age
from a high of 0.57% to a low of 0.53% from the age of 28 d to 49 d (Figure 3.14). On
the other hand, percent C remakied constant averagkig 4.15 ± 0.01% (mean ± SEM) as
the plant matured from 28 to 49 d of age (data not shown). Nitrogen did decrease with
age but the percent C remakied unchanged mdicatmg onions had not kiitiated bulbkig. N
content decreases as the plant ages and begkis bulbkig while the C content mcreases
(Randle 2000; Brewster 1994). Carbon content mcreases due to accumulation and
polymerization of non stmctural carbohydrates durkig bulbkig. Fmctose and glucose are
the two major components with sucrose andfinictans(fructose polysaccharides formed by
adding a fhictosyl group to a sucrose molecule) makkig up the remakikig content
(Kahane and others 2001). Brewster (1994) says bulbkig begkis about 56 to 77 d of age.
Percent ash content was significantly (p < 0.05) higher in the hydroponically
grown plants rangmg from 0.98% to 1.02%, and 0.86% to 0.98% for plants grown ki the
pottmg soil as the plant aged from 28 d to 49 d (Figure 3.15). Plants also differed (p <
0.05) ki ash content across age. Growing medium does effect ash content. Ahnazan and
coworkers (1997) found that NFT grown cv of sweet potato greens had higher ash
contents ki 2 of 3 cv when compared two correspondkig field treatments. The fact that
the ash content ki plants grown ki potting soil was lower than plants grown in
hydroponics corresponds with thek findings. This could be a resuk of the cation
exchange system within pottmg soil. Soil particles attract the poskively bound mineral
and lower the availability of ions for use by the plants (Taiz and Zeiger 1998). Since
60
Oasis^M is chemically kiert k does not kiterfere with mkieral absorption thus allowkig to
the plant have unlknited uptake of mkierals kicreaskig the overall ash content withki
onion plant (Resh 1991). Radishes and lettuce grown hydroponically showed an mcrease
ki ash content compared to field grown plants (McKeehen and others 1996).
Calcium and K did not significantly changed with age and no grow1;h medium
treatment effect was observed. The Ca and K content across age and growing media was
126.1 ± 1.6 mg/100 g and 270.0 ± 1.7 mg/100 g, respectively. On the other hand, Mg
was significantly (p < 0.05) (Figure 3.16) depleted ki the plant tissue during maturation.
At d 28 the content was 57.0 mg/100 g and dropped to 53.9 mg/100 g at d 49, with a
significant drop between samplkig ages 35-42 d with a 5.25% decrease from 57.1 mg/100
g to 54.1 mg/100 g. Onions contam on average 18.8 mg/100 g of Ca and 148.2 mg/100 g
of K (National Onion Association 2001) and Mg concentrations of 11 mg/100 g
(Ensmkiger and others 1994). Almazan and coworkers (1997) reported an increase in K,
Ca and Mg content of sweet potato greens in beds compared to those plants grown NFT.
They also reported an increase ki ash percent in the same samples (Ahnazan and other
1997). Sternberg and coworkers (2000) showed increased K, Ca and Mg contents in
wheat grown hydroponically and microporous tube irrigation compared to optimal
concentrations (fully expanded flag leaves) compared to filed grown crops. Randle
(2000) has shown that increases ki N content of the hydroponic solutions causes
reduction ki Ca and Mg and that K content will decrease steadily with N increase in
solution. Another cause in the reduction could be related to the morphological change in
the onion. From the 49 to 63 d samplkig point, the onions seemed to start the bulbkig
61
process. Very little is known about the biochemical changes that occur when bulbkig
occurs. Brewster (1994) has hypothesized that an mcreases m reduckig series, sucrose,
and fiiictans, which change the osmotic potential and could then effect the mkieral
contents (Brewster 1994). These results are different but the samples grown ki the
pottmg soil were watered with the Hydro-SoF^ and the waterkig systems were static, not
a dynamic flowkig model as used ki some of the previously reported studies. Even
though no kicrease ki mkierals was observed an kicrease ki total ash percent was
consistent with the reported findings.
There was no significant (p > 0.05) change in S content regardless of growing
medium or plant age. The overall mean content of S was 185.4 ± 3.5 mg/100 g. Randle
(2000) found that total bulb S content in onions increased cubically by increases in N
content ki hydroponic solutions. Randle (1992) reported S contents ranging from 42.9 to
52.6 mg/g of dry weight in onions grown hydroponically and onions can accumulate as
much as 100 mg of S /g of dry weight. Bulb S correlates poorly with overall flavor
mtensity among a broad range of onion accessions and onion's that partition little S as
SO4 are more pungent than those that accumulated larger amounts of SO4' (Randle
2000).
Total flavonol content mg/100 g (Figure 3.17) significantly (p < 0.05) increased
throughout the growth of the onions, from a mean low of 262.6 at 14 d of age to 583.1 at
98 d of age. Lombard (2000) found a Ikiear relationship (r^ = 0.96; p < 0.0001) between
TF content quantified by spectrophotometry and 3,4'-Qdg + 4'-Qmg quantified by HPLC
analysis. Price and Rhodes (1997) also found that over 80% of total flavonol content was
62
accounted for by 3,4'-Qdg and 4'-Qmg. Patil and Pike (1995) found total quercetki
content to be as high as 3066.83 mg/100 g dry weight ki one red colored variety
'Redbone' and k ranged from 9.50 mg/100 g ki a white colored onion 'Contessa' variety
to the high ki the 'Redbone'. Price and Rhodes (1997) reported quercetki levels rangmg
from 1369 - 1778 mg/kg vmib, Patil and coworkers (1995) found quercetki ranged from
54.3 - 286.4 mg/kg wmb and Hertog and coworkers (1992b) reported mean quercetki
content of 347 m/kg. 'Purplette' onions contam large amounts of TF and the
discrepancy among studies is likely related to varietal differences. Increases ki light
mtensity mcrease flavonol content ki plants (Barabas and others 1998; Britt 1999). For
complete nutritional analysis data refer to Appendix C (Table C.2 & C.3).
Envkonmental Growth Chamber Experiment
In Figure 3.18, the plant weights were different. In 7 of 8 (87.5%) blocks
'Purplette' variety was heavier, taller, and averaged 1 more leaf per plant compared to the
'Redbone' variety (Figure 3.18-3.20). The plant height (Figure 3.19) differed as a resuh
of photoperiod and variety. The short-day photoperiod (EGC2) had taller plants with a
mean of 27.0 ± 1.01 cm (mean ± SEM), while EGCl (long-day photoperiod) had an
average plant height of 21.0 ± 1.01 cm (mean ± SEM). The 'Purplette' variety was 32%
taller at 28.4 ± 1.01 cm (mean ± SEM) than the 'Redbone' at only 19.5 ± 1.01 cm (mean
± SEM) tall. Net number of leaves was not affected by photoperiod but was higher in
'Purplette' compared to the 'Redbone' (p < 0.05) (Figure 3.20). To see complete edible
biomass production, refer to Appendix D (Tables D.l & D.2). Overall the 'Purplette'
63
onion showed enhanced edible biomass production regardless of photoperiod. Leaf
cessation occurs with bulb formation (Tei and others 1996). Brewster (1994) has shovm
that onions reach thek maxknum height around week 70-85 d of age ki correlation with
the start of bulbkig. Durkig this tkne, the first and second leaves began to desiccate,
wither and fall off (Brewster 1994). Brewster (1994) also states that photoperiod does
affect rate and start of bulbkig as well as higher temperatures increaskig bulbing rates.
Onions must be exposed to contkiuously bulbkig kiductive photoperiods to start and
complete bulbkig (Brewster 1994). Studies have shown that bulbkig can be reversed and
green leaves will resume a pre-bublkig grovy^h if the plants are exposed to non-inductive
photoperiods (Brewster 1994). This is the case for the plants heights, for the 'Purplette'
onions ki EGC2 (short-day photoperiod), which were taller mdicatmg more leaf growth.
For complete date set on biomass production refer to Appendix D (Tables D.l-2)
The DM content of the onions (p < 0.05) differed depending on photoperiod,
variety, and plant part (Figure 3.21). 'Purplette' onions had 13.7% more DM than the
'Redbone' with 9.79 ± 0.27% (mean ± SEM) and 8.45 ± 0.40% (mean ± SEM) percent,
respectively. Onions grown under short daylight conditions accumulated 21% less DM
at 8.05 ± 0.33% (mean ± SEM) percent compared to 10.19 ± 0.32% (mean ± SEM) for
the long daylight treatment. The leaves averaged 10.8 ± 0.32% (mean ± SEM) percent
DM, bulbs had 8.93 ± 0.36% (mean ± SEM) percent DM, and the roots were the lowest
at 7.59 ± 0.53% (mean ± SEM) a 10 and 20% percent difference, respectively. Tei and
coworkers (1996) showed a relationship that at early stages of growth the fraction of DM
partkioned to the leaves was about 73% of the total amount of DM produced. The total
64
constitutes leaves, bulbs, and sheaths. Around 63 d of age, about 53% of the DM is
partkion to leaves and at 85 d of age DM partkionkig was towards the bulbs only (Tei
and others 1996). So as onions get older the DM percent of the leaves decrease with a
co-commkiant kicrease ki DM content of the bulb. From measurements observed, the
onions did not startmg to bulb or they were ki the early stages and partitionkig of the DM
did not reflect the morphological change. Tei and coworkers (1996) showed that DM
content accumulation ki onions does not kicrease until the plant reaches about 60 d of
age. DM content ki onions is a function of the plant age (Tei and others 1996). A longer
growing season will increase the DM content of onions as well as certain soil conditions
(Kahane and others 2001). Three different cv. of sweet potato greens grovm uskig NFT
had higher DM contents when compared to the same cv. grovm ki three different fields
(Almazan and others 1997).
There was a significant interaction (p < 0.05) between photoperiod and variety in
N content (Figure 3.22). The leaves contained 0.38 ± 0.02% (mean ± SEM) percent N,
while the bulbs and roots contamed only 0.24 ± 0.02% (mean ± SEM) and 0.21 ± 0.03%
(mean ± SEM) percent, respectively. This 9.2% difference in concentration in the tissues
indicates that onions are not initiatkig bulbing. Nitrogen content decreases as the plant
ages and undergoes the begirming of bubling while the C content increases (Randle 2000;
Brewster 1994), but before that the leaf tissue accumulates most of the N (Tei and others
1996). This is consistent with findkigs from this experknent given that the onions were
only 45 d old and had just begun to bulb. Higher N contents in soil decreases bulb
weight, firmness, stimulate foliar growth at the expense of bulb size (Randle 2000).
65
There were significant (p < 0.05) differences ki C between photoperiods, plant
part and an interaction between photoperiod and variety (Figure 3.23). Plants grown
under long-day photoperiod condkions had an average of 4.3% C compared to only 3.0%
for the short-day length. The concentration of the C among the plant tissues also
differed. Leaves had 4.1 ± 0.18% (mean ± SEM), bulbs had 3.5 ± 0.20% (mean ± SEM),
and roots had 2.77 ± 0.29% (mean ± SEM) C. 'Redbone' varieties ki both chambers
partitioned on average 48.7% more C ki the leaves as compared to the bulbs. This
indicates the onions were not bulbkig. The 'Purplette' onion portioned the least amount
of C into the roots. Under long-day conditions, the C concentrations in the leaves were
31.1% more than the bulbs. Half of all the C was located in the leaves. Under short-day
condkion the bulbs contamed 11.7% more C ki the leaves compared to long-day,
indicatmg a shift ki C concentration to the bulbs. Carbon content increases due to
accumulation and polymerization of non stmctural carbohydrates durkig bulbkig.
Fmctose and glucose are the two major components with sucrose and fructans (fructose
polysaccharides formed by addkig a fhictosyl group to a sucrose molecule) making up
the remakikig content (Kahane and others 2001). However, the 'Purplette' onions grown
under long-day condkions did not begki bulbkig because the C concentrations had not
shifted, but the plants were only 45 d old. The 'Redbone' onions grown under long-day
condkions did have higher concentrations of C ki the bulb compared to the leaves
indicatmg the kikiation of bulbing.
Percent ash content and Mg concentration was not significantly (Table 3.1)
different at any experknental level. The overall ash content for the leaves was 1.14%,
66
bulbs 1.10%, and 0.98% for the roots. Ahnazan and coworkers (1997) showed that NFT
3 different cv of sweet potato greens had higher ash contents ki 2 of 3 treatments when
compared two correspondkig field treatments but was not significantly different between
the 3 varieties. The Mg content of the leaves was 26.3 mg/100 g, 10.4 mg/100 g for the
bulbs, and 16.8% for the roots. The root means are only from the 'Purplette' due to the
'Redbone' variety not producing enough root mass to sample. Onions contam Mg
concentrations of 11 mg/100 g (Ensmkiger and others 1994). Randle (2000) has shovm
that mcreases in N content of the hydroponic solutions causes reduction in Mg content
and will decrease steadily with N increases in solution. McKeehen and coworkers (1996)
reported findkig lower Mg contents ki the roots of radishes grown hydroponically ki
growth chambers compared to the leaf portion. Another cause in the reduction could be
related to the morphological change in the onion. Very little is known about the
biochemical changes that occur when bulbkig occurs other than an mcreases in reducing
sugars, sucrose and finctans which changes the osmotic potent and could then effect the
mkieral contents (Brewster 1994).
Calcium content was significantly (p < 0.05) (Figures 3.24) different ki each plant
part and an mteraction between plant part and photoperiod was also noted. Ca content
was highest ki the leaves at 74.9 ± 5.5 mg/lOOg, lowest ki the bulbs at 39.1 ± 5.9 mg/100
with 70.6 ± 7.6 mg/100 g ki the roots. The percent Ca in the 3 different plant parts was
different for each photoperiod. In EGCl, the concentration of Ca ki the leaves was
45.7% and 54.8% higher than the bulbs and roots, respectively. In EGC2, the
concentration of Ca in the roots was 27.3% and 63.4% higher than the bulbs and leaves.
67
respectively. McKeehen and coworkers (1996) found that radish leaves contam
anywhere from 85.0% - 93.3% more Ca than the roots. Ahnazan and coworkers (1997)
found that sweet potato greens grown hydroponically accumulated higher Ca than do
field grown crops of the same variety. Calcium is a part of many biochemical pathways
ki plants so the high concentrations found ki the leaves and roots can be attributed to
higher growth rates ki the cells ki those areas. Given the plants were 45 d old and
bulbkig was not yet occurrkig or just begkmkig to occur the normal growth of the onion
would be ki the leaves and the roots (Taiz and Zeiger 1998; Brewster 1994). Onions
contam on average 18.8 mg/100 g of Ca (National Onion Association 2001). Randle
(2000) has shovm that mcreases ki N content of the hydroponic solutions causes
reduction ki Ca and very little is known about the biochemical changes that occur when
bulbkig occurs other than an increases ki reduckig sugars, sucrose and finctans which
changes the osmotic potential and could then effect the mkieral contents (Brewster 1994).
Potassium contents (Figure 3.25) were significantly (p < 0.05) different among
plant parts and with an interaction between plant part and photoperiod. The leaves and
roots also had a significantly (p < 0.05) higher K content compared to the bulbs. The
plants grown under short-day conditions had K concentrations 63.7% and 31.4% lower in
the bulbs and leaves compared to the roots. However, under a long-day condkions K
concentrations were 54.6% and 78.0% lower ki the bulbs and roots compared to the
leaves. The K content exhibited the same pattem in decrease in concentration, as did the
Ca. Onions contain 148.2 mg/100 g of K (National Onion Association 2001). The
overall averages of the bulb plus the leaf K content for long-day and short-day
68
photoperiods were 305.6 mg/100 g and 336.3 mg/100 g, respectively. Sweet potato
greens have contained higher K amounts (Ahnazan and others 1997), as well as, radish
leaves and lettuce (McKeehen and others 1996) grown hydroponically ki chambers and
compared to field grown crops. Given that K function ki plants is stomatal openkig
(osmoregulation), activation of enzymes for photosynthesis and respkation as well as
playkig a role ki starch and proteki synthesis k not surpriskig that the contents are the
highest ki the photosynthetically active portions of the plant (Taiz and Zeiger 1998).
Randle (2000) has shown that increases in N content of the hydroponic solutions causes
reduction in K and very little is knovm about the biochemical changes that occur when
bulbkig occurs other than an increases in reducing series, sucrose and fhictans which
changes the osmotic potent and could then effect the mineral contents (Brewster 1994).
There were significant (p > 0.05) differences ki S content mg/100 g (Figure 3.26)
ki the different plant partitions. The leaves were 41.9% and 92.5% more concentrated
compared to the bulb and the roots. Sulfur is taken form the soil through sulfate
transporters and moved to the plastids or vacuole. Randle (2000) found that total bulb S
content ki onions increased cubically with increases in N content of hydroponic solutions.
Randle (1992) reported S contents rangmg from 42.9 to 52.6 mg/g of dry weight ki
onions grown hydroponically and onions can accumulate as much as 100 mg of S /g of
dry weight. Bulb S correlates poorly with overall flavor intensity among a broad range of
onion accessions and onion's that partkion little S as S04'^ are more pungent than those
that accumulated larger amounts of S04'^ (Randle 2000). With the young age of these
plants at 45 d, the occurrence of high levels of S ki the leaves is not uncommon. Sulfur
69
content in the bulb in not uncommon given k storage capacity for use ki flavor precursor
pathways (Block 1992).
Total flavonol content mg/100 g (Figure 3.27) significantly (p < 0.05) higher in
plant leaves as compared to the bulbs and roots, with the bulbs bekig significantly higher
than the roots. The leaves contamed 589.3 ± 43.4 mg/100 g of TF, while the bulbs had
362.6 ± 43.4 and the roots contamed 121.3 ± 48.1 mg/100 g. Lombard (2000) found a
Ikiear relationship (r^ = 0.96; p < 0.0001) between total flavonoid content quantified by
spectrophotometry and 3,4'-Qdg + 4'-Qmg quantified by HPLC analysis. Price and
Rhodes (1997) also found that over 80% of total flavonoid content was accounted for by
3,4'-Qdg and 4'-Qmg. Patil and Pike (1995) found total quercetin content to be as high
as 3066.83 mg/100 g dry weight in one red colored variety 'Redbone' and k ranged from
9.50 mg/100 g in a white colored onion 'Contessa' variety to the high in the 'Redbone'.
In 'Purplette' onions the leaves were on average, 45.4% and 73.3% more concentrated
than the bulbs and roots. The 'Redbone' variety the leaves on average were 31.6% and
88.9% more concentrated than the bulbs and roots. The plants had relatively the same
concentration of TF in the leaves but differed in thek bulb and root concentrations.
One of the major functions of quercetki ki plants is to act as a protective barrier
against deleterious effects from insect and fungal damage. Bohm (1998) observed an
increase in quercetin in European beech (Fagus sylvatica) after an attack of beech bark
disease. Quercetin also functions within microorganisms and insects. In lycaenid
butterflies and zebra swallowtail quercetin is a part of the wkig pigmentation, which is
formed durkig larval feeding on plants (Bohm 1998; Harbome 1999).
70
Antknicrobial and antibacterial properties of quercetki have been found agamst
species such as Bacillus cereus and Cladosporium cucumerinum (Bohm 1998).
The flavonol content in the leaves and bulbs is part of the plant's defense mechanism.
The bulbs and leaves were ki constant contact with light. Skice the leaves were the major
portion of the onions at 45 d of age, k stands to reason that they would contam the
highest amount or partkionkig of flavonols. Quercetm absorbs at different wavelengths
but maxknum absorption is ki the range of 362-375 nm has been established (Lombard
2000; Jones and others 1998; Hertog and others 1992a; Patil and Pike 1995). A
particularly knportant role ki this regard has been attributed to phenylpropanoids,
kicluding hydroxycmnamic acid derivatives and flavonoids with effective absorption ki
the UV-B spectral region (Reuber and others 1996; Sheahan 1996; Hoque and Remus
1999). In addition to UV-screenkig, other knportant UV-B-protective properties ascribed
to flavonoids include antioxidant activities (Dawar and others 1998), and energy
dissipation via intramolecular proton transfer (Smith and Markham 1998). Specific
quercetin accumulation has been observed as a response to a number of other forms of
stress, ranging from heavy metal pollution (Loponen and others 1998), N deficiency
(Bongue-Bartelsman and Phillips 1995), to electron donating paraquat application
(Steger-Hartmann and others 1994). Plants synthesize many phenolic compounds in
roots during normal growth and development. The products are the buildkig blocks of
plant pigments and protect the plant from UV light, pathogens, might be used to modify
hormones and can be induced by wounding (Peters and Verma 1990). Increases in light
concentration increase flavonol content in plants (Barabas and others 1998; Britt 1999).
71
To see a complete nutrkional data of the 'Purplette" and 'Redbone' onion plants and
different concentrations ki the plant parts refer to Appendix D (Table D.3-4).
Individual Growth Chamber Experiment
Crop responses to mcreased CO2 include an kicrease in biomass accumulation,
increase yield, and knprove the water use efficiency of the plants (Monje and Bugbee
1998). Plant weight of 'Purplette' onions significantly increased (Figure 3.28) (p < 0.05)
when CO2 levels mcreased to 2000 ppm but 'Purplette' had fewer leaves at 2000 ppm
compared to the plants gassed at 1,000 ppm and 370 ppm (Figure 3.29) Plant height was
not significantly (p > 0.05) affected by CO2 level (data not shovm). Plants exposed to
2000 ppm CO2 weighed on average 7.43 ± 0.44 g (mean ± SEM), to 5.34 ± 0.44 g (mean
± SEM) at 1000 ppm, and to 3.76 ± 0.44 cm (mean ± SEM) at ambient CO2 (370 ppm).
Several studies found that increased CO2 levels affect makitenance respiration by
changkig carbon-partkionkig rates. Baker and coworkers (1992) m long-term CO2
experiments with rice found that canopy dark respkation increased (per unit ground area)
as CO2 concentrations increased to 500 nmol/mol from 160 nmol/mol. However,
specific canopy respkation decreased (per unit dry mass). Higher specific respiration
rates ki subambient CO2 treatments were attributed to higher makitenance respkation. In
a study conducted by Thomas and Griffm (1994) on C02-enriched soybeans maintenance
respkation increased by 34%, while the growth respkation did not change. All these
reports showed higher biomass production rates at elevated CO2 supporting the data
72
reported here. For complete biomass data refer to Appendix E (Table E.l). These
physiological changes resulted ki different growth rates and edible biomass production.
The DM content of the 'Purplette' onions gassed at 2000 ppm for 96 h was
significantly (p < 0.05) (Figure 3.30) higher than those at 1000 and 370 ppm CO2.
Lippert and coworkers (1996) found that responses of plant to elevated CO2 are
substantially kifluenced by water and N supply. Skice the only factor different ki this
experiment was CO2 concentration the DM matter content could be due to specific
genetic responses to the kicrease ki CO2.
Plants grown at ambient (370 ppm) CO2 contained 0.36 ± 0.02% (mean ± SEM)
N, while plants gassed at 1000 ppm had only 0.25 ± 0.02% (mean ± SEM) and the 2000
ppm level resulted ki N levels of 0.30 ± 0.02% (mean ± SEM) (Figure 3.31). McKeehen
and coworkers (1996) found N levels ki radish (Raphanus sativus L. cv Giant White
Globe) leaves and roots mcreased ki plants grown at 1000 ppm CO2 and then decreased
as the concentration was mcreased to 10,000 ppm. Onions might morphologically act
different than radishes to elevated CO2 and thus N levels might not kicrease with
kicreases in concentration of CO2.
Overall C content ki the onions was significantly (p < 0.05) different between the
3 gassing treatments (Figure 3.32). Plants gassed at 2000 ppm contamed 3.62 ± 0.11%
(mean ± SEM) C while the 1000 ppm treatment created plant with the lowest percent at
2.81 ± 0.11% (mean ± SEM) with plants grovm at ambient levels averaging 3.53 ± 0.13%
(mean ± SEM). In wheat (Triticum aestivum L. cv. Veery-10) plants subjected to
elevated CO2 at 1200 ppm altered the plant partkionkig of C and increased root biomass
73
and increased root respkation. This kicrease ki CO2 kicreases skik strength, which
kicreases photosynthetic capacity of the plant (Monje and Bugbee 1998).
Percent ash content did not significantly (p > 0.05) vary among plants gassed at
different CO2 concentrations. McKeehen and coworkers (1996) found significant
kicreases ki ash content ki radish leaves exposed to elevated CO2. However, radishes
were grovm to maturity (36 d) for the entire production period. Onions in this experiment
were only grovm at elevated CO2 for 96 h. Significant differences (p < 0.05) ki Ca
(Figure 3.33) Mg (Figure 3.34), and K (Figure 3.35) concentrations were observed ki this
experiment. Gasskig at 2000 ppm CO2 caused a decrease in Ca, Mg, and K
concentration. However, the K concentration was sknilar to the reported value. Onions
contain on average 18.8 mg/100 g of Ca and 148.2 mg/100 g of K (National Onion
Association 2001) and Mg concentrations of 11 mg/100 g (Ensminger and others 1994).
Randle (2000) has shown that N content of the hydroponic solutions causes variations in
Ca, Mg, and K content, which could account for differences in the data reported here
compared to values reported ki the literature. McKeehen and coworkers (1996) observed
decreases ki K, Mg, and Ca content in radish roots of plants grown at elevated CO2
concentration, but an increase in all three mineral in the leaves. Super-elevated CO2 can
be detrknental to plants and some enviromnents have CO2 levels as high as 6000
nmol/mol such as the space shuttle cabki while ki orbk (Wheeler and others 1999).
There was no significant (p > 0.05) change ki S mg/100 g content due to increases
in CO2. The means ranged from 122.8 ± 4.6 mg/100 g (mean ± SEM) to 125.8 ± 9.3
mg/100 g (mean ± SEM) as the CO2 level decreased 2000 ppm to 370 ppm (ambient).
74
Randle (1992) reported S contents rangmg from 42.9 to 52.6 mg/g of dry weight ki
onions grown hydroponically and onions can accumulate as much as 100 mg of S/g of
dry weight. The S content in the data here is reported on a wet matter basis but converted
to a DM the values are below Randle's reported values. This could be due to the kicrease
ki the CO2 or differences due to variety and plant age.
Total flavonol content did not significantly (p > 0.05) differ due to changes ki
CO2 concentrations. Plants grown at ambient CO2 contamed 531.7 mg/100 g of TF.
Plants gassed at 2000 ppm CO2 averaged 473.8 mg/100 g of TF, while plants gassed at
1000 ppm contained 324.0 mg/100 g. Even though the numbers seem different the SD
was rather large at 69.4 mg/100 g and the sample size was small (n = 12) so no
significant differences were noted. For complete data refer to Appendix E (Table E.2).
Skice the plants were the same age and the flavonol levels were not different the variation
could be attributed to genetic variation and not to elevated CO2 levels.
Lombard (2000)
and Price and Rhodes (1997) have demonstrated that the majority of the TF in onions is
quercetin. Specific quercetin accumulation has been observed as a response to a number
of other forms of stress, ranging from heavy metal pollution (Loponen and others 1998),
N deficiency (Bongue-Bartelsman and Phillips 1995), to electron donating paraquat
application (Steger-Hartmann and others 1994). So by creatkig another stress by
increasing CO2 concentration, TF content could be elevated.
75
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79
Table 3.1. Mean ± SEM for ash percent and magnesium (Mg) concentration of 45 d
old 'Redbone' and 'Purplette' onions grown hydroponically ki two different
envkonmental growth chambers, plants placed ki chamber at 24 d of age, one
with long-day (16 h) (1) and the other a short-day ( l i b ) (2) photoperiod.
(n = 45). No significant differences were noted (p > 0.05).
EGC
Variety
Redbone
1
Purplette
Redbone
2
Purplette
Plant Part
Leaf
Bulb
Root
Leaf
Bulb
Root
Leaf
Bulb
Root
Leaf
Bulb
Root
n
0
1
0
4
4
2
3
2
0
4
2
4
Ash (%)
n/a'
1.22 ±0.00
n/a
1.14 ±0.04
0.91 ± 0.06
0.81 ±0.06
1.11±0.13
1.06 ±0.08
n/a
1.16 ±0.04
1.20 ±0.06
1.14 ±0.06
n/a- not enough sample available to complete analysis.
80
Mg (mg/100 g)
n/a
10.1 ±0.00
n/a
24.7 ±5.4
10.1 ±2.4
5.6 ±1.9
28.2 ±1.2
8.3 ± 0.9
n/a
26.0 ±3.7
12.9 ±2.3
28.0 ±3.0
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CHAPTER IV
CONCLUSIONS AND IMPLICATIONS
Greenhouse Experiments
Experiment 1
From these results it can be concluded that age does play a significant role in
variation in edible biomass production as well as plant composition. Edible biomass
production in onion grown hydroponically followed the typical biological trend of linear
growth until about 70-75 d of age and then leveled off as bulbing begins. Significant
interaction between planting date and plant age was present in height, weight and net
number of leaves. The DM and S contents were not significantly different among plant
ages. The Ca, Mg, and K levels in the plants were extremely high but at 98 d of age
decreased to amounts close to previously reported values. Ash content and TF content
increased as the plants matured. Since quercetin is the major flavonol in onions it is
likely that quercetin increased as TF increased. Individually onion plants vary but do
show trends related to environmental conditions, but genetic variation is still apparent
given the variation m compostion within samples from the same planting date and age.
Experiment 2
Plant weight and net number of leaves are significantly different across age and
growing media with an interaction between the two. The height of the plants was also
different across age and between growing media. From these results it can be concluded
that plant maturity does not play a significant role in DM, C, Ca, K, and S content.
116
Growing medium does have a significant affect on ash content with onion grown in
potting soil having lower percent ash. The cation exchange system could be the cause by
tying up free, absorbable mineral decreasing the concentration available for plants to use.
Nitrogen and Mg contents significantly decreased as plants matured, but TF level
increased. A lack of effect due to growmg medium (with the exception of the ash
content) could be due to the fact that both sets of plants were watered daily with HydroSol™, unlike field conditions which are just watered with water and m some cases
fertilizer is used.
Environmental Growth Chamber Experiment
Even though an apparent photoperiod affect was observed, the EGCs were
different in temperature and humidity. EGCl was 25-30% lower in relative humidity
than EGC2 and at night EGCl's mean temperature dropped as low as 15 °C. So effect of
photoperiod cannot be determined for certain from this data. Photoperiod effects plant
biomass production with long-day onions growing faster and heavier compared to shortday onions despite photoperiod length. Nutritional content of onions may vary due to
photoperiod. Different parts of the plant require and mamtain different levels of minerals
and phytonutrients to perform the required biological ftinctions.
Individual Growth Chamber Experiment
Elevated CO2 increases biomass production even if plants are exposed to
increased concentration of CO2 for only a short duration. Nutritional components were
117
different at elevated CO2 treatment effects and in some cases decreased below levels in
the plants not exposed to elevated levels and other components increased. However
increasmg CO2 levels did not effect the plant height, percent ash, S and TF level.
Increased CO2 levels m plant growth chambers could be an alternative to increasing plant
growth by using natiu*al means.
Overall Implications
Growuig plants hydroponically, in environmental growth chambers, and at
elevated CO2 affected plant growth and altered composition. Growing plants m EGCs
allows growing conditions to be optimized or changed to increase one or more individual
factors to alter growth rate of plants and alter nutritional composition. Elevated CO2 is
found in certain biological regions as well as in space. Plants grown at elevated CO2
have been shown to have extreme growth rates as well as an altered composition.
Overall onions do grow well in hydroponic conditions and maintain a relatively
constant nutritional profile. Biomass and some nutrients can be affected by changes in
photoperiod, soil type, and carbon dioxide level. Onions could be a reliable food crop
grown hydroponically m elevated CO2 levels, under a long photoperiod in a growth
chambers and controlled environments. This can be usefiil when growing space, time or
length is limited such as m the International Space Station or on long-term manned space
mission.
118
APPENDIX A
FLAVONOL STANDARD CURVE
A standard curve for TF content was established to determine flavonol
concentrations in onion samples reported. Patel and Pike (1995) have stated that
quercetin content in onions can range from 94.99-30668.31 mg/kg fwt. Hertog and
coworkers (1992) found onions to contain 284-486 mg/kg fwt of quercetm.
Concentration of TF (mg/ml) were calculated in a 2 g sample of onion extracted with 8
ml of 80% EtOH and ranged from 0.00625-0.125 mg Q/ml (Table A.l.).
Concentrations of unknowns were determined using linear regression. An
isoquercitrin (Extrasynthese, Geney, France) stock solution was made by dissolving 62.5
mg of isoquercitrin m 500 ml of 80% EtOH. Eight dilutions were prepared from the
stock solution. The absorbance of each of the 9 standards was determined 10 separate
times on the spectrophotometer to create an average absorbance for each dilution. The
linear working relationship between isoquercitrin concentration (mg/ml) and absorbance
was as follows: (spectrophotometer AU ± SEM for each dilution: 0.0163 ± 0.002; 0.0245
± 0.002; 0.0918 ± 0.003; 0.1587 ± 0.003; 0.1785 ± 0.003; 0.2586 ± 0.002; 0.3286 ±
0.004; 0.4478 ± 0.004; 0.5562 ± 0.009; r^ = 0.988; CV < 0.68%) (Figure A.l.)
The absorbance value was used to obtain flavonol concentration on a mg/ml basis.
Unknown values were then converted to mg flavonol/100 g fwt (Table A.2.).
119
Table A.l. Isoquercitrin (Q) calculations for standard curve.
100 mgQkgfVvt
4feg4000g
= ImgQ
lOg
=
.2 mg O
2g
.2mgO
8ml EtOH'
= 0.025 mg O
1ml EtOH
X 5 stock solutions = 0.125 mg/ml
A. Concentration of isoquercitrin standards
Level
1
2
3
4
5
6
7
8
9
Dilution
Factor
0.05
0.10
0.20
0.30
0.40
0.50
0.70
0.80
1.00
X
X
X
X
X
X
X
X
X
Stock
Solution
mg/ml
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
0.125
=
=
=
=
=
=
=
=
=
mg standard/ml
0.00625
0.0125
0.025
0.0375
0.05
0.0625
0.0875
0.10
0.125
B. Preparation of isoquercitrin standards
To make standard curve of 100 ml for each concentration:
Dissolve 62.5 mg isoquercitrin in 500 ml 80% EtOH then diluted as below:
Level
1
2
3
4
5
6
7
8
9
Dilution
Factor
X
0.05
X
0.10
X
0.20
X
0.30
X
0.40
X
0.50
X
0.70
X
0.80
X
1.00
0.125 mg Q/ml
stock amt (ml)
5
10
20
30
40
50
70
80
100
in
in
in
in
in
in
in
m
m
^Calculations based on Lombard (2000).
120
EtOH (ml)
95
90
80
70
60
50
30
20
0
Table A.2. Calculation offlavonolconcentration using linear regression.
A. Linear regression calculations
Dilution Factor
b =
0.05
0.10
0.20
0.30
0.40
0.50
0.70
0.80
1.00
0.00625
0.01250
0.02500
0.03750
0.05000
0.06250
0.08750
0.10000
0.12500
0.0163
0.0245
0.0918
0.1587
0.1785
0.2586
0.3268
0.4478
0.5562
0.00004
0.00016
0.00063
0.00141
0.00250
0.00390
0.00766
0.01000
0.01563
xy
0.0001
0.0003
0.0023
0.0060
0.0089
0.0161
0.0287
0.0448
0.0695
n
sum
.avg_
9
0.50625
0.05625
9
2.061
0.229
0.04191
0.1768
m.iXi) - l(IxJ{gyi)/nl
Sxi' - [(Ix,)2/n]
b = 4.5297 =
0.06087
0.01343
Y = a + bX
a = y - ( bx)
-0.0258 = 0.229 - (4.5297*0.05625)
Y = -0.05265 +(4.808*X)
To obtain concentration rearrange formula to X = (Y - (-0.0258))/4.5297
B. Determination offlavonolconcentration
So at dilution level 9 (0.125 mg/ml) the average absorbance is 0.5562 ± 0.009
X = (0.5562 + 0.0258)/4.5297 = 0.1285 mg/ml
To determine concentration in mgflavonol/lOOgfwt:
0.1285 mg = 5 ml * 4 n J = 5.14 * 100 = 514 mgflavonol/100g fwt
ml
0.5 ml 1 g
121
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APPENDIX B
PLANT VARIABILITY
Tables B.l-2 presents phenotypic and composite characteristics of 'Purplette'
variety onions grown hydroponically in a greenhouse from March 2001 to July 2001.
123
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o Tt o Tt o
t^ o m m o\
CM f^ o o o
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126
APPENDIX C
SOIL VERSUS HYDROPONICS
Tables C.l-3 presents phenotypic and composite characteristics of'Purplette'
variety onions grown hydroponically and in potting soil in a greenhouse from June 2001
to August 2001.
127
Table C.l. Average phenotypic characteristics of 'Purplette' onions grov^n
hydroponically (H) and in potting soil (S) in a Texas Tech University
greenhouse from May to August 2001.
Soil Type Age (d)
n
LV number
SD
SEM
CV
CI 95%
Ht (cm)
SD
S
SEM
CV
CI 95%
Wt(g)
SD
SEM
CV
CI 95%
n
LV number
SD
SEM
CV
CI 95%
Ht (cm)
SD
H
SEM
CV
CI 95%
Wt(g)
SD
SEM
CV
CI 95%
14
40
2.00
0.00
0.00
0.00
21
40
2.00
0.00
0.00
0.00
7.74
0.31
0.05
4.06
0.10
0.08
0.00
0.00
3.90
0.00
40
2.00
0.00
0.00
0.00
9.65
0.53
0.08
5.48
0.16
0.09
0.01
0.00
6.57
0.00
40
2.00
0.00
0.00
0.00
7.54
0.35
0.06
4.68
0.11
0.07
0.00
0.00
3.79
0.00
9.67
0.31
0.05
3.19
0.10
0.09
0.00
0.00
3.39
0.00
28
40
3.80
0.82
0.13
21.65
0.26
24.46
3.82
0.60
15.62
1.18
1.39
0.56
0.09
40.37
0.17
40
3.05
0.75
0.12
24.57
0.23
23.93
4.30
0.68
17.96
1.33
0.99
0.52
0.08
52.73
0.16
128
35
40
4.05
0.68
0.11
16.73
0.21
27.94
2.61
0.41
9.36
0.81
1.67
0.31
0.05
18.23
0.09
40
3.93
0.57
0.09
14.58
0.18
26.50
2.55
0.40
9.61
0.79
1.20
0.28
0.04
23.42
0.09
42
40
4.25
0.67
0.11
15.76
0.21
29.68
3.49
0.55
11.75
1.08
2.01
0.37
0.06
18.53
0.12
40
4.08
0.66
0.10
16.09
0.20
28.46
3.13
0.49
10.98
0.97
1.85
0.32
0.05
17.34
0.10
49
40
3.93
0.76
0.12
19.47
0.24
26.53
3.75
0.59
14.13
1.16
2.31
1.03
0.16
44.52
0.32
40
3.08
0.73
0.12
23.74
0.23
25.72
3.56
0.56
13.85
1.10
1.43
0.50
0.08
34.64
0.15
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129
d d --^ d
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d r^ in CM
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in
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O
APPENDIX D
PHOTOPERIOD LENGTH IN ENVIRONMENTAL GROWTH CHAMBERS
Tables D.l-4 presents phenotypic and composite characteristics of 'Purplette' and
'Redbone' varieties of onions grown hydroponically in envirorunental growth chambers
with two different photoperiods. One short-day photoperiod of 1 Ih/of light and 16 h/of
light for the long-day period.
131
Table D.l. Phenotypic characteristics of 'Redbone' (RB) variety grown hydroponically
in two different environmental growth chambers (EGC)with two
photoperiods.
EGC
1
2
Daylight (h)
16
11
Block
n
LV number
SD
SEM
CV
CI 95%
Ht (cm)
SD
SEM
CV
CI 95%
Wt(g)
SD
SEM
CV
CI 95%
n
LV number
SD
SEM
CV
CI 95%
Ht (cm)
SD
SEM
CV
CI 95%
Wt(g)
SD
SEM
CV
CI 95%
132
1
3
4.67
0.58
0.33
12.37
0.65
18.92
2.70
1.56
14.25
3.05
2.17
1.20
0.69
55.31
1.36
3
4.00
1.00
0.58
25.00
1.13
24.92
1.66
0.96
6.68
1.88
2.50
0.68
0.39
27.25
0.77
2
3
4.33
1.15
0.67
26.65
1.31
21.75
6.06
3.50
27.87
6.86
2.64
1.20
0.69
45.23
1.35
3
4.00
0.00
0.00
0.00
24.58
5.26
3.04
21.39
5.95
5.37
2.12
1.22
39.38
2.39
3
3
4.33
0.58
0.33
13.32
0.65
16.00
3.54
2.05
22.15
4.01
2.33
0.15
0.09
6.61
0.17
3
3.67
0.58
0.33
15.75
0.65
20.83
8.80
5.08
42.26
9.96
3.14
0.97
0.56
30.95
1.10
4
3
3.67
1.53
0.88
41.66
1.73
14.17
5.71
3.30
40.33
6.47
1.84
0.95
0.55
51.58
1.07
3
3.33
1.53
0.88
45.83
1.73
15.17
8.65
4.99
57.01
9.78
7.44
2.75
1.59
37.00
3.12
Table D.2. Phenotypic characteristics of 'Purplette' (P) variety grown hydroponically in
two different environmental growth chambers (EGC) with two different
photoperiods.
EGC
Daylight (h) Block
16
11
n
LV numbei• 5.33
SD
0.58
SEM
0.33
CV
10.83
CI 95%
0.65
Ht (cm)
20.67
SD
0.63
SEM
0.36
CV
3.04
CI 95%
0.71
Wt(g)
6.12
SD
0.78
SEM
0.45
CV
12.70
CI 95%
0.88
n
3
LV numbei• 5.00
SD
1.00
SEM
0.58
CV
20.00
1.13
CI 95%
31.08
Ht (cm)
6.21
SD
3.58
SEM
19.97
CV
7.02
CI 95%
3.09
Wt(g)
0.36
SD
0.21
SEM
11.71
CV
0.41
CI 95%
133
4.33
1.15
0.67
26.65
1.31
25.17
2.32
1.34
9.23
2.63
6.53
1.41
0.82
21.65
1.60
3
5.00
0.00
0.00
0.00
35.17
2.32
1.34
6.61
2.63
5.74
0.82
0.47
14.26
0.93
5.00
0.00
0.00
0.00
26.17
2.52
1.45
9.62
2.85
9.39
1.71
0.99
18.21
1.93
3
5.33
0.58
0.33
10.83
0.65
35.08
3.13
1.80
8.91
3.54
8.31
0.67
0.39
8.05
0.76
5.67
0.58
0.33
10.19
0.65
24.83
2.18
1.26
8.80
2.47
8.38
1.47
0.85
17.53
1.66
3
4.67
0.58
0.33
12.37
0.65
29.25
4.83
2.79
16.51
5.46
4.31
0.90
0.52
20.93
1.02
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APPENDIX E
ELEVATED CO2 GROWING CONDITIONS
Table E.l-2 presents phenotypic and composite characteristics o f Purplette'
onions grown hydroponically in individual growth chambers and treated with elevated
CO^ levels.
138
Table E.l. Phenotypic characteristics of 36 d old 'Purplette' onions grown at ambient
CO2 (370 ppm), acclimated for 48 h in individual growth chambers and then
exposed to elevated CO2 levels (1000 and 2000 ppm) for 96 h.
CO2
LV number
SD
SEM
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CI 95%
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SD
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CI 95%
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CI 95%
370
1000
2000
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APPENDIX F
CHEMICAL COMPOSITION
Tables F.l-2 present the chemical composition of Hydro-Sol™ (Scotts-Sierra
Horticultural Product Co, Marysville, OH, USA) and Ball Growing Mix 2™ (Ball Seed;
West Chicago, IL, USA) potting soil.
141
Table F.l. Chemical composition of Hydro-Sol™ 5-11 -26 (Scotts-Sierra Horticultural
Product Co, Marysville, OH, USA).
Chemical constituent
Nitrogen (total) - nitrate nitrogen
Available phosphate - P2O5
Soluble potash - K2O
Magnesium (total) water soluble - Mg
Sulfur combined - S
Boron - B
Copper chelated - Cu
Iron chelated - Fe
Manganese chelated - Mn
Molybdenum - Mo
Zmc potentially chelated - Zn
Concentration (%)
5.0
11.0
26.0
3.11
4.04
0.05
0.015
0.30
0.05
0.05
0.015
142
Table F.2. Chemical composition of Ball Growmg Mix 2™ (Ball Seed; West Chicago,
IL, USA) potting soil.
Component
Composite pine bark
Vermiculite
Canadian sphagnum peat
Perlite
Other
Percent of total (%)
45-55
20-30
15-25
5-15
0-3
143
APPENDIX G
EXPERIMENTAL LAYOUT AND PLANT LOCATION
Figures G.1-2 present the experimental layout and the plant location of short-day
onion plants (Allium cepa L. 'Redbone') and long-day (Allium cepa L. 'Purplette') onion
plants grown hydroponically in EGCs with different photoperiods.
144
© © © ©
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Block 1
Block 2
Block 3
Block 4
Figure G.l. Experimental diagram of'Redbone' and 'Purplette' onions grown m EGCl
with a photoperiod of 16 h. S = 'Redbone' and P = ' Purplette' with bold *
letters indicating plants sampled and partitioned.
145
Block 1
Block 2
Block 3
Block 4
Figure G.2. Experimental diagram of 'Redbone' and 'Purplette' onions grown in EGC2
with a photoperiod of 11 h. S = 'Redbone' and P = ' Purplette' with bold *
letters indicating plants sampled and partitioned.
146
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