CAROTENOID AND FRUIT DEVELOPMENT EFFECTS ON GERMINATION AND VIGOR OF
TOMATO (Lycopersicon esculentum Mill.) SEEDS.
DISSERTATION
Presented in Partial Fulfillment of the Requirements for the Degree
Doctor of Philosophy in the Graduate School of The Ohio State University
By
Gerardo Ramirez Rosales, BS, MS
The Ohio State University
2002
*****
Dissertation Committee
Dr. Mark A. Bennett, Adviser
Dr. Miller B. McDonald, Adviser
Dr. David M. Francis
Dr. Grady W. Chism
Approved by
______________________
Adviser
______________________
Co-Adviser
Graduate Program in
Horticulture and Crop Science
ABSTRACT
The tomato crop is an important source of carotenoids such as ßcarotene and lycopene. These carotenoids play important roles in human
health and nutrition. Consequently, humans benefit from the development
of tomatoes with enhanced carotenoid content.
High pigment genes, such
as dark green (dg) and high pigment (hp) that result in higher levels
of total carotenoids are available. However, the deleterious effects on
plant
development
homozygotes
in
caused
by
commercial
these
genes
varieties.
have
The
limited
effects
of
their
these
use
as
genes
on
plant development, including slow seed germination and plant growth,
are not well understood. In addition, the effects of these genes in
other traits such as seed longevity and total antioxidant capacity have
not
been
evaluated.
development
on
seed
This
study
quality
evaluated
of
tomato
the
effect
varieties
with
of
fruit
different
concentrations of carotenoids under field and greenhouse conditions.
Gibberellin
effects
and
on
norflurazon
speed
of
(an
inhibitor
germination
of
of
carotenoid
varieties
with
synthesis)
different
concentrations of carotenoids were also evaluated. Fruits and seeds of
these varieties were assayed for total antioxidant capacity using the
Photo-induced
Chemiluminescense
(PCL)
ii
and
the
Total
Equivalent
Antioxidant Capacity (TEAC) methods. Results indicated that the effect
of fruit development on seed germination depends on the genotype and
that the low speed of germination characteristic of the high lycopene
line is independent of the gradual accumulation of lycopene. Seeds of
the
high
pigment
line
treated
with
norflurazon
plus
gibberellin
germinated faster than the control indicating that ABA is involved in
the low speed of germination of high lycopene genotypes with the dg
gene
as
an
expression
of
dormancy.
Fruits
of
the
dg
line
had
significantly greater antioxidant capacity than fruits of the recurrent
parent. However, seeds of the recurrent parent had higher antioxidant
capacity than seeds of the high pigment line as assayed by both PCL and
TEAC methods, suggesting that antioxidants in the fruit may compete
with antioxidants in the seed.
In conclusion, genotypes carrying the
dg gene result in a high content of lycopene and antioxidant capacity
and have delayed seed germination possibly caused by higher levels of
ABA. Fruits with high content of lycopene may be desirable for human
health perspective but
result in lower quality planting material. The
application of ABA inhibitors can minimize the effect of high pigment
genes on speed of seed germination.
iii
DEDICATION
To my wife Guadalupe and my children Gerardo, Germaín and Gerson
for their love, patience, and moral support. I hope this document
will be a part of the reward they deserve for their sacrifice. To
my parents for their love, courage and the moral values that they
provided me with.
iv
ACKNOWLEDGMENTS
I want to express my gratitude to Drs. Mark A. Bennett and Miller
B.
McDonald
for
providing
me
with
excellent
suggestions,
ideas
and
invaluable intellectual contribution to my research and for helping me
in my writing. I also thank Dr. David Francis for his valuable support
on my field experiments and for introducing me to the problem of low
quality seed of high pigment tomato genotypes. I am grateful to Dr.
Grady Chism for his guidance and advice in the evaluation of total
antioxidant capacity of tomato seeds.
I give special thanks to Andy Evans for all his help on the
acquisition
of
Thanks
Soja
to
chemicals
and
Sekharan,
equipment
Wenting
Li,
for
seed
and
quality
Elaine
evaluation.
Grassbaugh
for
different kinds of support they provided me and for their friendship
during my stay at The Ohio State University.
I thank Dr. Steven Schwartz, Puspitasary-Nieben Liu and staff of
the Food Science and Technology Laboratory of The Ohio State University
for
use
of
his
laboratory
facilities
and
technological
advice
for
lycopene and antioxidant extractions. Thanks too to Dr. Jim Metzger and
T.J Doong for their advice and use of his laboratory facilities for
antioxidant quantifications.
v
Finally, I want to express my gratitude to the National Council
for
Science
and
Technology
(CONACyT)
of
Mexico
financial support through this dissertation process.
vi
for
providing
me
VITA
October 25, 1965..............
Born, Coahuila, México
1987 B.Sc.....................
Agrarian University “Antonio
Narro” Coahuila, México
1993 M.S......................
Agrarian University “Antonio
Narro” Coahuila, México
1993-1998.....................
Maize Seed Production
Coordinator Cargill Seeds
Guadalajara, México
1998-Present..................
Graduate Student at The Ohio
State University
PUBLICATIONS
Cano-Rios, P., Ramirez-Rosales, G., Ortegon-Perez, J., EsparzaMartinez-J.H. and Rodríguez-Herrera, S. 2000.Diallel Analisis of
Seed Vigor in Muskmelon. Agrociencia 34:337-342.
FIELDS OF STUDY
Major Field: Horticulture and Crop Science
vii
TABLE OF CONTENTS
Page
ABSTRACT............................................................. ii
DEDICATION........................................................... iv
ACKNOWLEDGMENTS....................................................... v
VITA................................................................ vii
LIST OF TABLES........................................................ x
LIST OF FIGURES.................................................... xiii
Chapters:
1. INTRODUCTION....................................................... 1
Overview........................................................... 1
Carotenoids and Seed Germination................................... 4
Seed Development and Quality...................................... 11
Seed aging and antioxidants....................................... 17
Literature Cited.................................................. 25
2. Effect of Fruit Development on the Germination and Vigor of High
Lycopene Tomato (Lycopersicon esculentum Mill.) Seeds................ 34
Introduction...................................................... 35
Materials and methods............................................. 38
Results and discussion............................................ 40
Literature Cited.................................................. 48
3. Environment and Fruit Development Effects on Seed Germination and
Vigor of Four Tomato (Lycopersicon esculentum Mill.)Genotypes........ 55
Introduction...................................................... 56
Materials and methods............................................. 57
Results and discussion............................................ 59
Literature Cited.................................................. 62
4. Effects of Overripe Fruits on Germination of Tomato (Lycopersicon
esculentum Mill.) Seeds.............................................. 66
Introduction...................................................... 67
Materials and methods............................................. 68
Results and Discussion............................................ 70
Literature Cited.................................................. 74
viii
5. Total Antioxidant Capacity of Seeds from Normal and Enhanced
Lycopene Tomato (Lycopersicon esculentum Mill.) Genotypes............ 77
Introduction...................................................... 78
Materials and methods............................................. 80
Results and Discussion............................................ 85
Literature Cited.................................................. 90
6. Gibberellin plus Norflurazon Enhance the Germination of Dark Green
Tomato (Lycopersicon esculentum Mill.) Genotypes.................... 100
Introduction..................................................... 101
Materials and Methods............................................ 104
Results and Discussion........................................... 106
Literature Cited................................................. 112
7. CONCLUDING REMARKS AND FUTURE STUDIES............................ 121
BIBLIOGRAPHY........................................................ 124
Appendices:
A. P-Values for Winter 2000 Study................................... 133
B. Effect of Cluster Position....................................... 134
C. Weight of One-hundred Seeds...................................... 135
ix
LIST OF TABLES
Table
page
2.1. Anova table for genotype, maturity and the interaction Genotype x
maturity of germination index (GI), germination percentage (GP) and
SSAA of the tomato genotypes ‘OH8245’ and ‘T4099’ harvested at five
maturities: mature green (MG), breaker, (BR), pink breaker (PB),
red mature (RM), and overripe (OR) . Winter 2000................. 51
2.2.
Germination
Index
(GI),
germination
percentage
(GP)
and
germination percentage after SSAA of ‘OH8245’ and ‘T4099’ tomato
seeds harvested at five different fruit maturities: mature green
(MG), breaker (BR), pink breaker (PB) red mature (RM) and overripe
(OR)............................................................. 52
2.3. Five-day count germination (radicle protrusion) of fresh tomato
seeds of ‘Flora-Dade’ and ‘T4099’ harvested at five maturities:
mature green (MG), breaker (BR), pink breaker (PB) red mature (RM)
and overripe (OR)................................................ 53
2.4. Five-day count germination (normal seedlings) of fresh seeds of
‘Flora-Dade’ and ‘T4099’ harvested at five maturities: mature green
(MG), breaker (BR), pink breaker (PB) red mature (RM) and overripe
(OR)). Summer 2001............................................... 53
2.5.
Five-day count germination (radicle protrusion) of dry seeds
‘Flora-Dade’ and ‘T4099’ harvested at five maturities (mature green
(MG), breaker (BR), pink breaker (PB) red mature (RM) and overripe
(OR)). Summer 2001............................................... 54
2.6. Five-day count germination (normal seedlings) of dry seeds ‘FloraDade’ and ‘T4099’ harvested at five maturities: mature green (MG),
breaker (BR), pink breaker (PB) red mature (RM) and overripe (OR)).
Summer 2001...................................................... 54
3.1.
Probability values for tomato germination index (GI) and
germination percentage (GP) and percentage of variance explained by
different sources of variation of four tomato genotypes grown in
two locations and harvested at four fruit maturities: mature green,
breaker, red mature. Fall 2000................................... 64
3.2.
Germination index (GI) and Germination percentage (GP) of four
tomato genotypes grown in two locations and harvested at four fruit
maturities: mature green (MG), breaker (BR), red mature (RM), and
overripe (OR).................................................... 65
x
4.1. Lycopene content and tomato fruit characteristics of ‘Flora-Dade’
harvested at different days after pollination (DAP). Winter 2002. 75
4.2.
Germination (GP) and Saturated Salt Accelerated Aging (SSAA)
percentages of tomato ‘Flora-Dade’ at different fruit ages. Fall
2001............................................................. 75
4.3. Germination percentage of 90 day-old fruit of tomato ‘Flora-Dade’
after different periods of fermentation.......................... 76
5.1.
Analysis of variance for lycopene content of two tomato
(Lycopersicon
esculentum
Mill.)
genotypes,
‘Flora-Dade’
and
‘T4099’.......................................................... 93
5.2. Fruit tissue lycopene content and Trolox Equivalent Antioxidant
Capacity (TEAC) of a wild type (‘Flora-Dade’) and a high lycopene
tomato line (‘T4099’............................................. 93
5.3.
Analysis of variance of total antioxidant capacity for lipidsoluble antioxidants of tomato (Lycopersicon esculentum Mill.)
fruits from two genotypes ‘Flora-Dade’ and ‘T4099’. Antioxidant
capacity was determined by the Trolox Equivalent Antioxidant
Capacity (TEAC) method........................................... 94
5.4.
Analysis of variance for lipid-soluble antioxidants (ACL)) of
tomato (Lycopersicon esculentum Mill.)
seeds from two genotypes
‘Flora-Dade’ and ‘T4099’. Total antioxidant capacity was determined
by the Trolox Equivalent Antioxidant Capacity (TEAC) method...... 94
5.5.
Analysis of variance for water-soluble antioxidants (ACW)) of
tomato seeds from two genotypes. Total antioxidant capacity was
determined by the Trolox Equivalent Antioxidant Capacity (TEAC)
method........................................................... 95
5.6.
Analysis of variance for water-soluble antioxidants (ACW) of
tomato seeds from two different genotypes. Total antioxidant
capacity was determined by the Photo-induced Chemiluminescence
method (PCL)..................................................... 95
5.7.
Analysis of variance for lipid soluble antioxidants (ACL) of
tomato seeds from two different genotypes. Total antioxidant
capacity was determined by the Photo-induced Chemiluminescence
(PCL) method..................................................... 96
xi
5.8
Total antioxidant capacity in water (ACW) and lipid (ACL)
fractions of tomato seeds (nmol/g) of ‘Flora-Dade’ and ‘T4099’
using the Trolox Equivalent Antioxidant Capacity (TEAC) and the
Photo-induced Chemiluminescence (PCL) methods for ‘Flora-Dade’ and
‘T4099’ tomato genotypes......................................... 96
6.1.
Percentage
germination
(radicle
protrusion)
of
two
tomato
genotypes ‘Flora-Dade’ and ‘T4099’ treated with solutions of
gibberellin, (GA3), norflurazon, and gibberellin plus norflurazon 115
6.2.
Means squares and significance for time to fifty percent
germination (T50), germination index (GI) and hypocotyl length (HL)
of two tomato genotypes ‘Flora-Dade’ and ‘T4099’. Seeds were
treated with gibberellin (GA3), norflurazon, or gibberellin plus
norflurazon. Germination was recorded daily when seeds showed
radicle protrusion.............................................. 116
6.3.
Time to fifty percent germination (T50), germination index (GI)
and hypocotyl length (HL) of two tomato genotypes (‘Flora-Dade’ and
‘T4099’) treated with solutions of gibberellin (GA3), norflurazon
(Nor), or gibberellin plus norflurazon.......................... 117
6.4.
Means squares and significance for time to fifty percent
germination (T50) and germination Index (GI) of two tomato
genotypes ‘Flora-Dade’ and ‘T4099’. Seeds were germinated in
darkness or under 8/16 h light/dark cycles (Experiment 1) and 16/8
h light/dark cycles (Experiment 2). Germination was recorded daily
when seeds showed radicle protrusion............................ 118
6.5. Time to fifty percent germination (T50), germination index (GI) of
two
tomato
genotypes:
‘Flora-Dade’ and ‘T4099’. Seeds were
germinated under darkness or under 8/16 h light/dark cycles
(Experiment 1) and 16/8 h light/dark cycles (Experiment 2).
Germination was recorded daily when seeds showed radicle protrusion
................................................................ 119
xii
LIST OF FIGURES
Figure
Page
1.1. The carotenoid pathway.......................................... 32
1.2. Recurrent parent ‘Flora-Dade’ and high lycopene line ‘T4099’ dg
ogc.............................................................. 33
5.1.
Total antioxidant capacity of tomato seeds determined by the
photo-induced
chemiluminescence
method
(PCL)
and
the
Trolox
Equivalent Antioxidant Capacity (TEAC). Each value represents the
average of five replications..................................... 97
5.2. Calibration curves developed with 0.125, 0.025, 0.5 and 0.1 mmol/L
of 6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid (Trolox).
(A) Standard curve used to calculate Trolox Equivalent Antioxidant
Capacity (TEAC) of lipid-soluble antioxidants of tomato fruits. (B)
Standard curve used to calculate TEAC values of water-soluble
antioxidants of tomato seeds and (C) Standard curve used to
calculate TEAC values of lipid-soluble antioxidants of tomato
seeds.
The chemical reagent 2,2’-Azinobis(3-ethylbenzothiazoline6-sulfocin acid) diammonium salt (ABTS) was incubated with Trolox
and
the
reduction
in
absorbance
was
determined
spectrophotometrically.................................................. 98
5.3.
Calibration curves used to calculate the total antioxidant
capacity of tomato seeds using the photo-chemiluminescence method.
(A) Standard curve used to calculate the total antioxidant capacity
of lipid-soluble antioxidants using 0.5, 1,2, and 2.5 nmol/L of 6hydroxy-2,5,7,8-tetramethylchroman2-carboxylic
acid
(Trolox).(B)
Standard curve used to calculate the total antioxidant capacity of
water-soluble antioxidants using 0,1,2, and 3 nmol/L of ascorbic
acid. TAC is determined based on the percentage of inhibition of
the chemiluminiscence due to presence of trolox or lipid-soluble
antioxidant (A) or the delay in seconds of chemiluminsence signal
due to ascorbic acid or water soluble antioxidant (B)............ 99
6.1. Percentage germination (radicle protrusion) of two tomato
genotypes ‘Flora-Dade’ (A) and ‘T4099’ (B). Seeds were germinated
under darkness or under 8/16 h light/dark cycles and 16/8 h
light/dark cycles............................................... 120
xiii
CHAPTER 1
. INTRODUCTION
Overview
Carotenoids are C40 isopropenoid compounds that participate in
a number of physiological processes in plants and other organisms
(Ronen et al., 1999; Shewmaker et al., 1999; Fraser et al., 2001).
These compounds are essential in photosynthesis where they function
as energy carriers and photo-oxidation protectors (Van Den Berg et
al., 2000). They are also important for the pigmentation of flowers
and fruits. In flowers, carotenoids are important for pollination
meadiated by insects, while in fruits they serve as indicators of
maturity that make fruits attractive for human consumption (Arias et
al., 2000).
Carotenoids also have antioxidant qualities that make
them important for human nutrition and disease prevention (Abushita
et al., 1997). Tomato (Lycopersicon esculentum Mill.) varieties that
differ
in
structures
fruit color (i.e. yellow, orange, and red) due to different
or
concentrations
of
carotenoids
exist.
In
addition,
plant
scientists are modifying the carotenoid pathway in different plant
species such as rice (Oryza sativa L.) and canola (Brassica napus
L.) to enhance their nutritional quality. However, the ability to
affect human diets with nutrition through genetic modification has
been limited by negative effects of increased carotenoid content on
plant processes.
1
Because
carotenoids
are
free
radical
scavengers,
tomato
breeders are now developing materials with high carotenoid content,
especially
lycopene.
traditional
metabolic
and
reduced
molecular
pathways
physiological
plant
However,
growth
biology
and
processes.
increasing
cause
For
have
approaches
delayed
reported
in
content
might
abnormalities
example,
been
lycopene
alter
in
other
important
germination
the
high
by
and
pigment
genotypes (see figure 1.1) in which lycopene content is much higher
(2-3X) compared to normal tomato genotypes (Jarret et al., 1984;
Wann et al., 1985).
2
Although
modify
the
there
is
synthesis
evidence
of
that
essential
high
levels
germination
of
carotenoids
promoters
such
as
gibberellins (Fray, 1995), the cause(s) of low speed of germination
in high lycopene lines remains unclear. Carotenoids are precursors
of abscisic acid (ABA) via an indirect pathway (Zeevaart, 2000);
therefore, the elevated levels of carotenoids may over-produce ABA
resulting
in
varying
intensities
of
dormancy.
However,
this
mechanism has not been demonstrated in high pigment genotypes. In
addition, more research is needed to determine whether there is a
direct
effect
of
tomato
fruit
development,
maturation,
and
the
concomitant carotenoid and lycopene synthesis on the expression of
seed
germination
and
longevity
in
tomato.
synthesis of lycopene characteristic of
Thus,
if
the
elevated
high pigment varieties is
closely related to reduced germination in tomato, then it might be
possible that harvesting at earlier stages of fruit maturation may
enhance
seed
germination
because
lycopene
accumulates
from
the
breaker to the overripe fruit maturity stages.
Fruit
development
effects
on
tomato
seed
quality
have
been
studied (Valdes and Gray, 1998; Demir and Samit, 2001). However,
studies conducted to date have included traditional genotypes with a
similar pattern of carotenoid synthesis and accumulation. Therefore,
little information exists on genotypes that over-produce carotenoids
including lycopene. This information is essential because genotypes
with
enhanced
nutrition
prostate
due
lycopene
to
the
may
be
desirable
correlation
between
for
human
lycopene
health
and
and
reduced
cancer (Clinton, 1998). As a result, enhancing the seed
quality of these increased carotenoid genotypes is physiologically
3
challenging and requires additional study. In addition, carotenoids
are natural antioxidants and it is important to determine whether
tomato
fruits
antioxidant
rich
levels
in
antioxidants
that
increase
produce
the
seeds
ability
to
with
enhanced
stored
seed
(McDonald, 1999).
Carotenoids and Seed Germination
One of the most significant values of carotenoids is their
essential
role
in
human
nutrition
and
the
prevention
of
many
diseases (Ye et al., 2000). Unfortunately, humans do not synthesize
carotenoids. Therefore, they must consume vegetables and vegetable
products to meet this nutritional requirement. That is one reason
that breeders have for enhancing carotenoid content in vegetables
and fruits (Ye et al., 2000). The carotenoids most studied include
β-carotene and lycopene, although other carotenoids such as lutein
and zeaxanthin have also received considerable attention because of
their antioxidant properties (Clinton et al., 1998; Volker et al.,
2002). In addition, violaxanthin and neoxanthin have been studied
with respect to their role in synthesis of ABA (Zeevaart, 2000).
Lycopene’s antioxidant properties and nutritive value make it
the
subject
of
many
studies,
although
most
of
these
have
been
oriented toward human health (Clinton et al., 1996; Clinton, 1998).
These reports propose that a diet rich in lycopene is beneficial for
human health and resultantly, tomato varieties with high lycopene
content are desirable.
In
tomato,
lycopene
has
received
considerable
attention
because it is responsible for the characteristic deep-red color of
4
ripe tomato fruits and tomato products (Shi et al., 1999). Lycopene
and β-carotene inhibit reactive oxygen species-mediated reactions,
which have been associated with many diseases (Giovanelli et al.,
1999).
Lycopene is also involved in the prevention of heart attacks
and different types of cancer (Shi et al., 1999; Giovanelli et al.,
1999).
The high pigment genes dark green (dg) and hp (variants hp-1
and hp-2) produce elevated lycopene content (2-3X) (Jarret, et al.,
1984; Wann et al., 1985; Berry and Uddin, 1991). These genes have
received considerable attention due to their effect on fruit color
(Stevens and Rick, 1986). One of the
high pigment variants (hp-2)
has been already cloned and the DNA sequence has high homology with
the
De-etiolated1
gene
in
Arabidopsis. Plants carrying this gene
show a higher sensitivity to light (i.e. hypocotyl is shorter under
light than under darkness) (Chiara et al., 1999). In tomato, the hp
genes
increase
carotenoid
content
by
increasing
the
amount
of
plastids. Other two methods by which carotenoids can be manipulated
include
increasing
the
biochemical
flux
through
the
pathway
and
manipulating the flux within the pathway using the crimson genes
(ogc).
5
The
high
pigment
genes
pose
pleiotropic
effects
which
have
been characterized (Jarret et al., 1984; Wann et al., 1985; Wann,
1996). In addition to elevated levels of carotenoids, high pigment
genotypes
produce
high
levels
of
chlorophyll,
ascorbic
acid
and
higher fruit firmness compared to wild types. However, the effect of
these
genes
on
other
carotenoids
and
seed
maturation,
total
antioxidant capacity and germination physiology has not been studied
to the same extent.
The slow seed germination and reduced plant height that these
genes
cause
on
plant
development
have
slowed
their
use
as
homozygotes in commercial cultivars (Sacks and Francis, 2001). One
reason
is
that
carotenoids;
by
other
increasing
metabolic
important
physiological
instance,
Fray
et
al.
levels
pathway
processes
(1995)
of
lycopene
abnormalities
(Croteau
reported
et
that
and
are
al.,
other
caused
in
2000).
elevated
For
levels
of
carotenoids resulted in shorter tomato plants because metabolites
from
the
gibberellin
pathway.
reported
pathway
Consistent
higher
with
content
were
this
of
re-directed
hypothesis,
gibberellins
to
the
Fraser
in
et
plants
carotenoid
al.
(1995)
deficient
in
carotenoid synthesis.
Wann
(1995)
gibberellins
to
observed
high
lycopene
that
exogenous
genotypes
applications
restored
plant
of
growth
to
levels close to those of the wild type. He claimed that high pigment
lines
were
deficient
observation,
however,
in
the
synthesis
provided
no
of
gibberellins.
conclusive
evidence
This
that
gibberellins were also responsible for the low speed of germination
associated
with
high
pigment
genotypes.
6
Recently,
Fraser
et
al.
(2001) noted that fruit specific expression of the phytoene synthase
gene from bacteria inserted into a tomato plant resulted in nondeleterious effects in other related pathways including tocopherols,
vitamin K, ubiquinones and plastiquinones. However, the study did not
examin effects specific to plant development such as plant height and
seed germination.
The human population will benefit from the generation of plant
varieties with enhanced carotenoid content, especially in developing
countries where the dietary consumption of vitamin A is low (Ye et
al.,
2000).
Studies
manipulating
carotenoid
content
have
been
successful in rice (Ye et al., 2000) in which the rice endosperm
synthesized
higher
levels
of
carotenoids
that
resulted
in
a
noticeable orange color compared to the wild type. Similar results
have been reported in canola (Brassica napus L.). The embryo of this
plant showed an intense orange color and a considerable difference in
carotenoid content in relation to the wild type. However, transformed
seeds had a two-day delay in germination (Shewmaker et al., 1999).
The delay in seed germination of transformed canola seeds might
be due to lower levels of gibberellins and higher ABA content which
results in delay in synthesis of hydrolytic enzymes necessary for
reserve
movilization
and
endosperm
weakening.
The
endosperm,
in
addition to being a reserve tissue, functions as a mechanical barrier
that impedes radicle protrusion (Leviatov, et al., 1994) and must be
weakened
for
germination
to
occur.
In
that
respect,
Liu
et
al.
(1996b) and Bradford et al. (2000) emphasized that gibberellins were
essential germination promoters for tomato endosperm weakening while
Bewley and Black (1994) and Raghavan (2000) stated that this hormone
7
was essential for the synthesis of hydrolytic enzymes. Liu et al.
(1996b)
noted
that
the
endosperm
also
serves
as
a
barrier
that
restricts water movement into the seed during development and that
the degradation of the endosperm must happen for germination to occur
regardless of the water potential gradient between the seed and fruit
tissue.
For
endosperm
degradation,
hydrolytic
enzymes
depend
on
gibberellins for their synthesis (Bradford et al., 2000).
The hormone ABA may moderate this activity and the synthesis of
essential enzymes (Liu et al., 1996b). Based on this information, a
deficiency in gibberellin synthesis and/or the over expression and
sensitivity
of
ABA
may
reduce
the
speed
of
germination
in
high
lycopene tomato genotypes.
The plant hormone ABA is a ubiquitous hormone that plays a
number
of
closure,
roles
seed
in
plant
reserve
development
accumulation
and
(e.g.,
seed
stimulates
dormancy)
stomatal
(Taiz
and
Zeiger, 1998). Alterations affecting loci that code proteins involved
in
ABA
synthesis
result
in
disruption
of
embryo
maturation,
accumulation of reserves, desiccation tolerance, and embryo dormancy
(Holdsworth et al., 1999). If high lycopene content results from
alterations
in
the
carotenoid
synthesized
indirectly
via
biosynthesis
carotenoids,
then
pathway
and
increasing
ABA
is
lycopene
content might alter the synthesis of ABA. Higher ABA levels could
lead to seed maturation and germination abnormalities such as greater
seed dormancy (Thomson et al., 2000).
Carotenoids are precursors of ABA via an indirect biochemical
pathway
2001).
(see
As
a
figure
result,
1.2)
it
(Cutler
might
be
8
and
Krochko,
possible
1999;
that
the
Milborrow,
levels
of
germination
genotypes
inhibitors
with
such
elevated
as
ABA
levels
of
are
over-produced
carotenoids.
in
Whether
tomato
elevated
levels of carotenoids consistently result in the over-expression of
ABA needs to be evaluated in other species.
In
maize
(Zea
mays
L.),
for
instance,
precocious
seed
germination has been observed in mutants defective in carotenoid
biosynthesis (such as the
Crozier
levels
et
of
al.,
ABA
2000).
being
vp5 mutant) (Cutler and Krochko, 1999;
This
observation
necessary
in
the
is
consistent
early
with
stages
low
of
seed
have
been
maturation.
Alterations
in
carotenoid
byiosynthesis
pathway
reported in some plant species. Bartley and Scolnik (1994) presented
a review of studies regarding carotenoid biosynthesis in Arabidopsis
thaliana, maize, and tomato. In Arabidopsis, the first mutants shown
to be arrested in some step of the carotenoid pathways were isolated
as ABA deficient mutants. In tomato, alterations on loci affecting
carotenoid content are
reported to modify pigmentation in flowers
and fruits, but little information exists about the effect on seeds.
Recently,
Thompson
et
al.
(2000)
reported
that
the
ectopic
expression of the 9-cis epoxy carotenoid dioxygenase, an enzyme that
catalyzes the formation of neoxanthin, resulted in over-production
of ABA in tomato causing a higher degree of dormancy compared to the
wild type. Dormancy was released with fluridone, an inhibitor of
carotenoid synthesis.
In fact, fluridone has been successfully used
to break dormancy in lettuce (Lactuca sativa L.) (Volmaro et al.,
1998; Yoshioka et al., 1998), tomato (Thomson et al., 2000), and
wheat (Triticum aestivum L.) (Garello and Le Page-Degivry, 1999).
9
These findings suggest that germination problems associated with a
modified carotenoid biosynthesis pathway (as in the case of high
lycopene
tomatoes)
might
be
related
to
elevated
rates
of
ABA
synthesis.
Constance et al. (2001) evaluated the effect of suppressing
the synthesis of gibberellins and preventing precocious germination
of
the
maize
mutants
vp5 and
vp1. Reducing gibberellin synthesis
prevented precocious germination of vp5 suggesting that gibberellins
and
ABA
act
antagonistically
in
the
germination
process
during
development. ABA prevents precious germination when gibberellins are
present.
However,
low
ABA
and
proper
gibberellins lead to precocious germination.
10
endogenous
levels
of
It
is
interesting
to
note
that
lycopene
and
β-carotene
accumulate late in fruit maturation, and continue accumulating even
after ripening (Abushita et al., 1996; Giovanelli et al., 1999). If
high lycopene results in altered ABA synthesis, and lycopene content
is greater in red fruits, then fruits harvested at the breaker or
pink
stages
might
have
improved
seed
germination.
In
contrast,
elevated levels of carotenoids may result in greater seed dormancy.
This
concept
suggests
that
seed
scientists
need
to
evaluate
the
synthesis and metabolism of carotenoids in seeds and how they affect
ABA and germination physiology. Little information exists at present
on
tomato
seed
information
currently
is
quality
important
developing
and
fruit
because
genotypes
carotenoid
the
tomato
differing
in
content.
seed
fruit
This
industry
color
due
is
to
changes in the structure and content of carotenoids. These genotypes
differ in total content of carotenoids, lycopene and ß-carotene, and
these
biochemical
alterations
might
have
a
detrimental
effect
of
seed quality.
Seed Development and Quality
Pioneer
studies
aimed
at
answering
seed
quality
and
physiological maturity considered days after pollination (dap) as an
indicator
of
information
demonstrated
seed
and
development.
concepts.
that
30-day
For
old
Those
studies
example,
Berry
tomato
seeds
provided
and
had
valuable
Bewley
the
(1991)
ability
to
germinate once separated from the fruit tissue. They also reported
that fresh and dry weight declined after 60 dap, a maximum content of
protein correlated with the capacity to tolerate desiccation and was
11
observed at 40-45 dap. They further suggested that water potential
and ABA were responsible for preventing precocious germination.
In
a
information
subsequent
on
development
on
germination
was
the
study,
effect
the
Berry
of
ABA
germination
prevented
by
and
and
of
water
Bewley
(1992)
osmotic
tomato
potential
presented
potential
seeds.
more
during
Precocious
than
by
the
presence of ABA. They also observed that the sensitivity of seeds to
water potential remains across all stages of seed development while
the sensitivity to ABA declined at maturity. Demir and Ellis (1992)
observed
that
seed
quality
was
constant
from
42
to
95
dap.
Importantly, they reported no decrease in seed quality of overripe
fruits. Maximum seed quality was observed after mass maturity, which
contradicts the accepted notion that seeds reach maximum quality at
maximum dry weight.
Liu et al. (1996a) evaluated the effect of osmo-priming on the
germination
of
tomato
seeds
extracted
from
fruits
of
different
maturities. Seeds had maximum germination capacity at 50-55 dap and
germination declined thereafter. Liu et al. (1996a) also observed
greater dormancy as seeds matured suggesting that these seeds better
tolerated desiccation and could be successfully stored for the next
growing season. However, after storage, more dormancy was observed
for immature seeds.
In a different study, Liu et al. (1996b) found that precocious
germination was prevented by reduced water potential and ABA content
on the fruit tissue that surrounds the embryo contrary to findings in
a report by Demir and Ellis (1992). They proposed that these two
factors prevented endosperm weakening and the subsequent penetration
12
of
the
embryo
through
the
seed
coat.
Thus,
degradation
of
the
endosperm in tomato is essential to initiate the germination process
and both gibberellins and ABA are involved (Bradford et al., 2000).
The
studies
information,
but
mentioned
because
they
previously
used
dap
generated
as
an
important
indicator
of
seed
development, they provided little information on the physiological
relationship between the plant and
fruit. That information is more
relevant in terms of what changes are taking place in the whole
plant, including fruits and seeds and how they interact. In addition,
it is easier to understand the effect of seed development on seed
quality regardless of the interacting environmental conditions during
production and the genotype.
The
use
of
dap
as
an
indicator
of
seed
development
poses
several disadvantages. For example, Valdes and Gray (1998), working
with several tomato genotypes, observed that fruits from the same age
had
different
quality
maturation
levels.
As
a
stages
result,
that
they
resulted
decided
in
to
different
seed
characterize
seed
development using fruit maturation stages (immature green, mature
green,
breaker,
pink
breaker,
red
mature
and
overripe)
that
correlated with seed quality parameters (seed weight, seed moisture
content and seed germination).
The use of plant physiological stages and the environmental
effects including water and nutrition on yield have been reported in
maize
Merr.)
(Iowa
State
production
precendence
for
University,
(Iowa
using
State
1996)
and
University,
physiological
soybean
1993).
criteria
in
(Glycine
Thus,
max
L.
there
is
assessing
seed
quality. In tomato, studies that evaluate physiological stages of
13
fruit and seed development and the effects of environmental stresses
on seed quality are lacking. In addition, it is not known whether the
environment is sensed by the mother plant or by the developing seed.
Therefore, studies that relate plant development with seed physiology
in differing environments need to be conducted. These studies should
use
physiological
parameters
to
indicate
development
instead
of
simple dap, which provides little information about the physiological
status of the plant and fruit. Thus, even though tomato is a good
model
to
study
seed
development
(Hilhorst
et
al.,
1998),
little
information exists that evaluates fruit development and seed quality
simultaneously.
In maize, TeKrony and Hunter (1995) evaluated a physiological
parameter
that
defines
quality.
Maximum
maturity
(Harrington,
the
seed
time
when
quality,
1972;
seeds
also
Delouche,
known
1980;
acquire
as
a
Powell
maximum
seed
physiological
and
Matthews,
1984), coincided with the formation of the black layer (abscission
layer)
regardless
of
genotype
and
production
environment.
The
formation of the black layer occurs at approximately 30-35% seed
moisture content. This information has been of great benefit to the
maize seed industry. Most seed companies inspect seed fields and
harvest when the black layer appears to obtain high quality seed.
In
tomato
there
is
no
clear
morphological
indicator
of
physiological maturity, although some have reported that maximum seed
quality is reached at the red mature fruit stage (Valdes and Gray,
1998). However, little is known whether high lycopene genotypes also
produce maximum seed quality at the red mature stage.
14
Valdes and Gray (1998) also reported that after seed quality
declines after the red mature stage. This observation was consistent
with that reported by Demir and Samit (2001). This decrease in seed
quality associated with overripe fruits has been also reported in
muskmelon
(Cucumis
melo
L.)
fruits
(Welbaum,
1999).
However,
the
cause(s) of this phenomenon is not completely understood and still
requires study.
Early studies argued that seed quality remains constant during
seed development until 95 dap (Demir and Ellis, 1992). In contrast,
recent reports (Liu et al., 1996a; Liu et al., 1997; Valdes and Gray,
1998; Demir and Samit, 2001) indicate that seeds from old fruits show
lower seed quality. The reasons for these discrepancies are likely
due to the use of dap as an indicator of seed development and the
different
tests
to
assess
seed
quality
as
well
as
the
genotypes
evaluated in each study. Another factor that has not been included is
the effect of the external environment. Stages of plant and fruit
development in which seeds are more sensitive to environmental stress
and the physiological effects that result from such stresses should
be studied.
Little
information
is
deterioration
associated
possible
the
that
provided
with
elevated
about
overripe
accumulation
the
type
fruits;
of
and
cause
however,
lycopene
in
it
of
is
overripe
fruits will result in an over production of ABA that results in
delayed
seed
synthesis
of
germination.
lycopene
It
that
is
also
results
possible
from
high
that
the
pigment
elevated
genes
may
redirect the synthesis of gibberellins as reported for genotypes with
elevated content of carotenoids which results in short plants (Fray
15
et
al., 1995). Interestingly, gibberellin deficient genotypes are
less
affected
Although
this
by
overripe
may
be
a
fruit
result
maturity
of
(Liu
altered
et
fruit
al.,
and
1996a).
seed
water
potential, the lack of synthesis of gibberellins in deficient mutants
during fruit development may cause these mutants to be less sensitive
to
the
overripe
fruit
condition
as
compared
to
wild
types
where
synthesis of gibberellins is changing during seed development. If
gibberellin synthesis is altered by elevated lycopene synthesis, then
overripe fruits, which have high levels of lycopene, might result in
low seed quality. Thus, the correlation between lycopene content and
seed germination would provide valuable information for understanding
whether these two processes are involved.
It
is
also
possible
that
advances
in
germination
occur
in
overripe tomato fruits. This observation was suggested by Liu et al.
(1997) who found greater 4c/2c DNA ratio in seeds of fruits harvested
75 dap indicating more advanced germination that resulted in lower
seed quality. However, it is not known whether this is a common
phenomenon and whether it is independent of the genotype.
Studies aimed at answering seed quality have also considered
fruit and seed position effects.
For example, in cucumber (Cucumis
sativus L.), Jing et al. (2000) found that the effect of fruit and
seed position on the expression of seed quality was more important in
fruits harvested before 42 dap. After that period, seeds had the same
level
of
determined
germination
seed
and
germination
vigor.
under
Nevertheless,
laboratory
these
and
authors
greenhouse
conditions providing little information about speed of germination
and vigor compared to other seed quality tests. In addition, little
16
information
was
provided
regarding
changes
in
cucumber
fruit
development. Thus, how fruit development affects seed quality remains
largely unknown in some species.
Tomato fruit color is affected by the environment and genetics
(Sacks and Francis, 2001), and color is defined by the type and
accumulation
of
different
carotenoids.
Thus,
those
factors
that
govern carotenoid synthesis may affect other processes or pathways
related
to
them
as
well.
For
instance,
Gnayfeed
et
al.
(2001)
reported changes in carotenoid and other bioactive compounds because
of genotypic differences and maturity stage in pepper (Capsicum annum
L.). Carotenoid content was highly correlated with genotype and the
stage of fruit maturity. Similar studies in addition to seed quality
evaluation are necessary in tomato and other fleshy-fruited species.
This is especially important in high lycopene tomato genotypes, in
which
changes
in
color
intensity
may
follow
different
patterns
compared to normal lycopene genotypes. It is probable that seeds from
high
lycopene
genotypes
accumulate
different
levels
of
stored
reserves compared to wild types. For instance, Liu et al. (1996b)
suggested that gibberellin and ABA deficient mutants have impaired
reserve accumulations.
Seed aging and antioxidants
Seed
aging
and
deterioration
may
be defined as the gradual
loss of seed quality as measured by loss in germination capability,
delayed speed of germination and low vigor. Better understanding of
the causes of seed deterioration will benefit seed companies and
seed producers and seed consumers. As a consequence, considerable
17
research has been conducted on the factors leading to rapid seed
aging
and
deterioration.
genotypes
(Doijode,
For
2001),
instance,
genetic
environmental
and
differences
biological
among
factors
during seed production and storage (Copeland and McDonald, 2001),
desiccation
tolerance
(Pammenter
and
Berjak,
1999)
and
maturity
level (Ajayi and Fakorede, 2000) are all involved in seed aging and
deterioration. However, defining a precise cause of seed aging and
deterioration is difficult. Most of the information suggests that
membrane damage and degradation are at least partly responsible for
seed deterioration. This degradation generates electrolyte leakage,
reduction
in
ATP
synthesis,
chromosome
damage,
reduction
of
seed
vigor and eventually seed death (Bewley and Black, 1994; Priestley,
1986; Smith and Berjak, 1995, Pukacka, 1998; McDonald, 1999).
Membrane damage is primarily caused by lipid peroxidation that
results
from
free
radicals
that,
once
generated,
create
a
chain
reaction that terminates in a destructive process (McDonald, 1999).
Natural
enzymatic
and
non-enzymatic
antioxidants
or
free
radical
scavengers can minimize the effect of free radical attack (Balz,
1994; McDonald 1999; Noctor and Foyer, 1998).
18
The effect of free radicals on the deterioration of biological
systems is well documented. For example, Balz (1994) presented a
comprehensive
organs
and
literature
resultant
review
human
of
free
diseases.
radical
He
damage
also
to
reported
human
studies
related to the use of antioxidants as a means of prevention of human
diseases and aging. It is interesting to note that the effects of
oxidants
on
humans,
such
as
damage
to
membranes,
aging,
and
reduction of mitochondrial activity, among others, are similar to
those reported for seeds.
There is abundant and increasing information on dietary levels
of antioxidants or free radical scavengers in vegetables and fruits.
For
instance,
antioxidants
cabbage
Kurilich
such
(Brassica
as
et
al.
(1998)
carotenoid,
oleracea
evaluated
var.
tocopherol,
capitata),
the
and
levels
of
ascorbate
in
cauliflower
(Brassica
oleracea var. botrytis), broccoli (Brassica oleracea var. italica),
Kale
(Brassica
oleracea
L.
var.
acephala)
and
Brussels
sprouts
(Brassica oleracea var. gemmifera Zeak.). Their study was conducted
for
breeding
food.
They
varieties.
similar
varieties
found
Kale
study,
that
enhance
substantial
had
the
Conner
Ng
antioxidant
variability
highest
and
the
levels
(1998)
of
within
these
evaluated
potential
and
between
compounds.
changes
of
in
In
a
lipid
peroxidation and antioxidant status in ripening muskmelon fruits.
Their objectives were to correlate the levels of antioxidants with
the
developmental
information
increasing
that
fruit
stage
can
be
storage
of
used
muskmelon.
for
potential.
They
breeding
Thus,
found
programs
studies
valuable
aimed
are
at
being
conducted on the potential of antioxidants as a means to increase
19
storage
life
However,
or
to
little
enhance
research
the
has
antioxidant
examined
potential
antioxidants
of
in
food.
seeds,
particularly as a means of reducing seed deterioration in genotypes
contrasting in carotenoid content to assess the relationship between
the total antioxidant capacity and seed longevity between species
and genotypes.
The literature on antioxidants available in seeds is still
scarce
and
contradictory.
Some
researchers
have
found
that
free
radicals are at least partly responsible for the reduction in seed
longevity
(Thapliyal
and
Connor,
1997;
Trawatha
et
al.,
1993).
Others argue that the reduction in seed longevity is accompanied by
a decrease in the level of free-radical scavengers (Chui et al.,
1995; Hsu and Sung, 1997; Bailly et al., 1997; De Vos, et al., 1994,
Bernal-Lugo, 1999). Other researchers have found no evidence of the
action
of
free
deterioration
radicals
(Powell,
and
1986;
free
radical
Powell
and
scavengers
Harman,
on
seed
1985).
The
discrepancies between these reports may be a result of the methods
of
measuring
addition,
the
deterioration
free
radical
evaluation
has
attack
of
included
and
antioxidant
antioxidants
individual
on
mechanisms.
seed
antioxidants
aging
such
In
and
as
glutathione. Therefore, little information is available about the
total antioxidant capacity, the capacity to delay or inhibit the
oxidation of molecules by inhibiting their initiation or propagation
(Zheng and Wang, 2001).
Plant genetic background among other factors is implicated in
seed longevity. For instance, Doijode (1990) reported differences in
the longevity of tomato genotypes. In watermelon (Citrullus lanatus
20
L.),
triploid
seeds
were
more
susceptible
to
accelerated
aging
compared to diploid seeds (Chiu et al., 1995). In maize, the response
to the accelerated aging test is different among hybrids (Santipracha
et
al.,
1997).
However,
the
fundamental
cause(s)
of
genetic
differences in seed longevity among species and varieties has not
been evaluated.
It
is
unknown
whether
tomatoes
or
other
plant
species
that
differ in carotenoid content also differ in antioxidant capacity, and
whether
this
may
cause
differences
in
seed
longevity.
Certainly,
tomato fruits with different carotenoid contents are different in
nutritional
value
and
probably
possess
different
antioxidant
capacities. How these differences relate to seed longevity remains
unknown. Do the levels of antioxidants in the tomato fruit have any
effect on the total antioxidant capacity of tomato seeds? How does
this relationship affect seed longevity and deterioration rate? Do
enhanced-antioxidant
tomato
fruits
produce
seeds
with
increased
longevity? These and other questions regarding antioxidant capacity
of tomato fruits and seeds need to be answered. It is interesting to
speculate whether differences in antioxidant capacity correlate with
seed longevity or seed vigor so plant breeders could select genotypes
with enhanced total antioxidant capacity and enhanced seed longevity.
This achievement would clearly benefit the seed industry and seed
consumers.
The
role
of
enzymatic
has
been
studied.
longevity
observed
a
reduction
in
factors
For
activity
on
seed
instance,
levels
of
Sung
deterioration
and
Jeng
superoxide
and
(1994)
dismutase,
peroxidase, and ascorbate peroxidase (enzymatic antioxidants) in the
21
axis and cotyledons of aged seed of two peanut (Arachis hypogaea L.)
cultivars. Chiu et al. (1995) reported a reduction in the peroxide
scavenging enzymes because of accelerated aging of watermelon seeds.
The
enzymatic
contributions
free
of
radical
previous
protection
researchers
mechanisms
may
suggest
that
increase
seed
longevity and decrease seed deterioration. However, total antioxidant
capacity involves mechanisms and compounds other than enzymes (i.e.,
carotenes, ascorbic acid, tocopherol, and phenols). Therefore, it is
important
to
include
antioxidant
capacity
evaluation
in
seed
longevity studies.
Seed longevity is also determined by the stage at which seeds
are harvested. Therefore, it is possible that the stage at which
seeds
express
maximum
antioxidant
capacity
coincide
with
maximum
seed longevity. However, little information exists about changes in
total antioxidant capacity during seed development and maturation in
plant species. In cocoa (Theobroma cacao L.), for example, Li and
Sun
(1999)
mentioned
that
the
level
of
antioxidant
protection
increases as seeds mature, and is highly dependent on water content
in recalcitrant seeds. Whether the levels of free radical scavengers
are changing during seed development and maturation, and how these
changes affect seed longevity in tomato seeds are questions that
have not been answered.
In pearl millet (Pennisetum glaucum L. R.
Br.), seed longevity was greater when seeds were harvested one week
after
maximum
dry
weight
was
attained
(Kameswara
et
al.,
1991).
However, why differences in physiological maturity are related to
seed longevity remains poorly understood.
22
Another
aspect
related
to
seed
longevity
is
desiccation
tolerance (Pammenter and Berjak, 1999). Desiccation tolerance can be
defined
as
the
capacity
of
certain
seeds
to
tolerate
drying
to
certain levels without detrimental effects on seed quality. If seed
longevity is influenced by free radical protection mechanisms, then
desiccation tolerance and free radical protection mechanisms may be
closely related as well. Thus, the presence and efficient operation
of antioxidant systems in an intracellular environment may be an
important
aspect
of
a
physiological
mechanism
implicated
in
desiccation tolerance (Pammenter and Berjak, 1999). Holdsworth et
al. (1999) suggested that decreased water content was crucial for
acquisition of desiccation tolerance. However, genotypes harvested
at
the
same
water
content
might
differ
in
their
desiccation
tolerance, which reflects differences in seed longevity. Desiccation
tolerance varies for a given species, depending on the stage of seed
development and maturation. Therefore, there may be a developmental
stage
where
the
levels
of
protection
mechanisms
are
maximum desiccation tolerance and increased seed longevity.
23
ideal
for
The objectives of this research are to:
(1) Determine the effects of fruit development on the germination of
seed from high lycopene and related tomato varieties with normal
concentrations of lycopene,
(2) determine whether harvesting tomato
fruits in the early stages of fruit maturation might improve seed
germination
and
vigor
in
high
lycopene
genotypes;
(3)
determine
whether slow germination in high lycopene genotypes is caused by
higher
levels
germination
produce
seeds
endogenous
process,
fruits
with
of
and
with
(4)
levels
total
ABA
determine
super-elevated
higher
of
whether
levels
antioxidant
during
of
the
genotypes
lycopene
capacity
and
early
that
result
how
in
that
antioxidant capacity relates to seed longevity.
The hypotheses to be tested include:
1. Seed germination and vigor of high lycopene genotypes at different
fruit maturation stages follows a different pattern compared to normal
lycopene genotypes. Therefore, maximum seed germination and vigor of
high lycopene genotypes might be obtained before fruits reach red
color (i.e., mature green and breaker stages).
2. The reduced speed of germination of the tomato genotype dark green
is caused by elevated ABA content during imbibition, which results
from higher levels of carotenoids.
3.
Dormancy
breaking
treatments,
such
as
GA3
and
Norflurazon
(an
inhibitor of carotenoid synthesis), improve the germination of high
lycopene tomato seeds.
4.
High
lycopene
varieties
produce
seed
with
a
higher
total
antioxidant capacity of fruits and seeds, which results in enhanced
seed longevity and storability.
24
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31
geranylgeranyl
diphosphate
synthase
(Ggps)
IPP
Tocopherols
Chlorophyll
Gibberellins
GGPP
Phytoene synthase
(Psy )
Phytoene
Phytoene
desaturase (Pds)
?-Carotene
Carotene
desaturase
(Zds)
Lycopene
epsilon
cyclase
(crtL-e)
Lycopene
d-Carotene
Lycopene
beta
cyclase
(crtL-b)
?-Carotene
Lycopene
beta
cyclase
(crtL-b)
Lycopene
beta
cyclase
(crtL-b)
a-Carotene
ß-Carotene
Ring
hydroxylases
beta and epsilon
(crt-R-b)
(crt-R-e)
Ring
hydroxylase
beta
(crtR-B-2)
Lutein
Zeaxanthin
Zeaxanthin
epoxidase (ZEP)
Violaxanthin
Antheraxanthin
9-cis-epoxycarotenoid
dioxygenase (NCED)
Neoxanthin
Xanthin
Figure 1.1. The carotenoid pathway
32
ABA
Figure 1.2. Recurrent parent ‘Flora-Dade’ and high lycopene line
‘T4099’ dg ogc
33
CHAPTER 2
Effect of Fruit Development on the Germination and Vigor of High
Lycopene Tomato (Lycopersicon esculentum Mill.) Seeds
Summary
Lycopene is a carotenoid compound correlated with reduced risk
of human diseases such as prostate cancer; consequently, tomatoes
with elevated levels of lycopene are desirable in the human diet.
Tomato genes such as dark green (dg) and high pigment (hp-1 and hp2) that increase carotenoid content are available. However, their
use in commercial cultivars is limited because of the undesirable
pleiotropic effects such as reduced plant growth and low speed of
seed
germination.
during
fruit
This
and
study
seed
evaluated
maturation
whether
would
harvesting
result
in
early
improved
germination of a high lycopene genotype. Plants of an experimental
line ‘T4099’ (dg ogC), its recurrent parent ‘Flora-Dade’ (+ +), and
the variety ‘OH8245’ (+ +) were greenhouse cultured and harvested at
different
stages
of
fruit
maturity
(mature
green,
breaker,
pink
breaker, red mature and overripe) in winter 2000 and summer 2001.
Seed
quality
was
evaluated
by
germination
index,
saturated
salt
accelerated aging (winter 2000) and standard germination (5 and 14
d) tests of fresh and dry seeds (summer 2001). Germination
of the
high lycopene line was slower than both ‘OH8245’ and ‘Flora-Dade’
lines regardless of fruit maturity. Differences between ‘Flora-Dade’
34
and ‘T4099’ were more marked for normal seedlings than for radicle
protrusion in both fresh and dry seeds indicating that the dg gene
affects
seedling
development
more
than
germination.
In
general,
overripe and mature green fruits showed the lowest seed quality.
These results suggest that the cause of delayed speed of germination
is
independent
tissues
and
of
the
that
gradual
speed
of
accumulation
germination
of
lycopene
dg
genotypes
of
in
fruit
is
not
improved by harvesting during early fruit maturation. In addition,
the use of dg genes in commercial cultivars, although desirable, is
still
dependent
on
the
ability
to
overcome
negative
production
effects such as low seed quality
Introduction
Tomato fruits are important sources of antioxidants such as
lycopene that are considered beneficial nutrients. (Van Den Berg et
al., 2000). Because antioxidants function as free radical scavengers
and
have
been
implicated
in
a
reduced
risk
of
certain
cancers,
tomato breeders have developed plant materials with high lycopene
content. However,
traditional
also
breeding
alter
abnormalities
example,
increasing carotenoid and lycopene content
other
in
delayed
techniques
metabolic
plant
and
pathways
development
germination
molecular
and
and
(Croteau
reduced
manipulation
cause
et
plant
using
might
undesirable
al.,
2000).
growth
have
For
been
reported in tomatoes carrying the high pigment (hp-1 and hp-2) and
dark green (dg) genes in which carotenoid content is much higher (23X) than normal tomato genotypes (Jarret et al., 1984; Wann et al.,
1985;
Berry
and
Uddin,
1991).
35
In
addition,
high
levels
of
carotenoids
might
decrease
the
synthesis
of essential germination
promoters such as gibberellins (Fray et al., 1995; Croteau et al.,
2000) leading to a reduced speed of germination in high carotenoid
tomato genotypes. Because lycopene synthesis increases as the fruit
matures (Giovanelli et al., 1999), elevated levels of lycopene may
be responsible for the reduced speed of germination of seeds from dg
and
hp
varieties.
determine
whether
As
a
a
result,
direct
additional
relationship
research
exists
is
between
needed
to
increased
lycopene synthesis during fruit development and seed germination and
longevity.
36
Most studies examining the influence of seed development on
seed
quality
in
tomato
have
used
days
after
pollination
as
an
indicator of seed maturity (Berry and Bewley, 1991; Demir and Ellis
1992,
Liu
et
al.,
1996).
However,
influenced
by
environmental
fruit
conditions
development
making
it
can
be
difficult
to
identify specific physiological stages of development of the seed.
More
recent
studies
have
evaluated
changes
in
fruit
color
as an
improved measure of seed development (Valdes and Gray, 1998; Demir
and Samit, 2001). Seeds extracted from red tomato fruits possessed
maximum seed germination compared to seeds extracted from earlier
stages of fruit maturity. While these studies evaluated changes in
fruit development based on color intensity and fruit firmness, they
provide little information on other fruit quality aspects such as
the accumulation of total carotenoids and lycopene. This information
is valuable since the tomato seed industry is currently developing
genotypes
with
different
levels
of
lycopene
in
which
fruit
development effects on seed quality may differ from those found in
traditional varieties.
37
Another aspect of tomato seed quality that has received little
attention is the combined effect of environment, fruit maturity and
genotype. Therefore, little is known about which factor (genotype,
environment or fruit maturity) is more critical for the expression of
optimum tomato seed quality. The specific objective of this study was
to
evaluate
fruit
development
and
environmental
effects
on
seed
quality of normal and dark green tomato genotypes. Such information
will
help
to
improve
the
speed
of
germination
and
vigor
in
high
lycopene tomato genotypes and enable a better understanding of the
combined
effects
of
fruit
maturity
and
genotype
on
tomato
seed
quality.
Materials and methods
Seed Production Cycles.
Two seed production cycles were used. The first was in winter
2000 and the second in summer 2001. In winter 2000,
seeds of two
unrelated
and
tomato
genotypes
differing
in
seed
vigor
lycopene
synthesis and accumulation were planted in a greenhouse. The first
was the open pollinated variety ‘OH8245’ developed at The Ohio State
University (Berry et al., 1991) and the second was the high lycopene
line ‘T4099’ (dg ogC) developed at The United States Department of
Agriculture (USDA) by backcrossing with the open pollinated variety
‘Flora-Dade’
followed
by
several
generations
of
self-pollination
(Wann, 1996). The variety ‘OH8245’ produces lycopene contents of 810 mg/100 g DW and the line ‘T4099’ produces about 25 mg/100 g DW.
Fruits
were
harvested
at
five
maturity
stages.
Seeds
were
extracted from the mature green, breaker, pink-breaker, red fruit
38
and overripe stages following the criteria described by Valdes and
Gray
(1998).
The
extraction
procedure
was
completed
by
hand
and
0
seeds were fermented in a beaker at room temperature (~24
C) for 48
h. Seeds were then rinsed and surface dried at room temperature to
approximately
8
%
moisture
content
(dry
weight
basis).
Once
extracted, seeds from all fruits were placed in coin envelopes and
stored at 4
In
O
C until evaluation.
summer
experimental
2001,
line
the
‘T4099’
recurrent
parent
ogc were
dg
‘Flora-Dade’
planted
in
and
the
the greenhouse.
Seeds were sown in 200 cell trays 4 cm deep. Seedlings transplanted
to
21
L
(35
cm
diameter
and
42
cm
depth)
pots
filled
with
the
cultivation media Metromix 360TM. Plants were maintained at 24 °C
during
the
growing
season
and
fruits
harvested
at
five
maturity
stages.
Seed Quality
For
standard
the
winter
2000
germination,
study,
germination
accelerated
aging
(SSAA)
germination
index
(GI)
tests
was
seed
quality
index
(Jianhua
calculated
and
and
for
was
the
evaluated
saturated
McDonald,
each
1996).
treatment
by
by
salt
The
the
algebraic sum of the ratio of normal germinated seedlings and the
day after planting at which time the count was made. The standard
germination and the GI were evaluated up to 14 days (ISTA, 1999).
Fifty seeds were planted in each of four Petri plates containing two
layers of blotter paper saturated with 10 ml dd water. The plates
were then placed in a germination chamber at 25 °C with a 16/8 h
light
and
dark
cycles.
In
all
39
cases,
seeds
were
recorded
as
germinated
when
the
essential
structures
to
be
considered
normal
seedlings were present. For SSAA, seeds were aged in a chamber at 41
o
C and 75% RH for 96 h. After this treatment, seeds were evaluated
for standard germination.
For the summer 2001 study, seed quality was evaluated by the
standard
seeds
germination
(seeds
test,
extracted
which
after
was
the
conducted
on
fermentation
dry
and
process
fresh
without
desiccation). Germination conditions were as described previously.
Percentage of germinated seeds (radicle protrusion) and percentage
of normal seedlings were determined after 5 and 14 d, respectively.
Seedlings with a radicle and shoot greater than 2.0 and 1.5 cm,
respectively, were considered normal.
Experimental Design
The experimental design was a complete randomized design with
four (winter 2000) and two (summer 2001) replications. The analysis
was conducted using the analysis of repeated measures to determine
statistical
maturity
differences
(Hinkelmann
between
and
genotypes
Kempthorne,
at
1994).
each
stage
Data
of
fruit
expressed
in
percentage were transformed using arcsine square root (Steel et al.,
1997), but the original data are shown. Data were analyzed with the
Statistical Analysis System (SAS) software (SAS institute, Cary NC
2001).
Results and discussion
Germination Index (GI)
Highly
genotype
by
significant
maturity
for
effects
GI
were
40
of
genotype
observed
and
(Table
interaction
2.1).
The
of
main
effect
of
between
genotype
‘OH8245’
indicated
and
a
clear
‘T4099’.
The
difference
genotype
in
x
seed
quality
maturity
effect
suggests the effect of the stage of fruit maturity on seed quality
is different for ‘OH8245’ compared to ‘T4099’. Similarly, there were
significant effects of maturity for GI (Table 2.1) indicating that
the stages of fruit maturity affected this seed quality attribute.
Genotype ‘T4099’ had a lower GI at all fruit maturities (p<0.01) than
‘OH8245’
(Table
2.2)
indicating
lower
speed
of
germination
of
‘T4099’ versus ‘OH8245’.
Germination Percentage
Significant
and
highly
significant
differences
were
also
observed for GP for genotype, maturity and the interaction maturity
x genotype respectively (Table 2.1). Genotype ‘T4099’ had a higher
final germination percentage than ‘OH8245’ at the mature green and
breaker
stages
(Table
2.2).
Genotype
‘OH8245’
showed
higher
germination at the red mature and the overripe fruit stages (p<0.01)
indicating differences in the effect of fruit maturity on GP between
these
two
genotypes.
Germination
percentage
of
‘T4099’
increased
from mature green to breaker stage and then declined resulting in
85%
germination
at
the
overripe
stage.
On
the
other
hand,
germination of ‘OH8245’ increased from mature green to red mature
fruit stages and then declined. This genotype followed the general
pattern reported in the literature that red mature fruits result in
maximum
seed
quality,
and
after
that
seeds
start
to
deteriorate
(Valdes and Gray, 1998; Demir and Samit, 2001). Apparently, ‘T4099’
followed a different pattern with the mature green and breaker fruit
stages producing high quality seed.
41
Saturated Salt Accelerated Aging (SSAA)
Highly significant differences of genotype and the interaction
genotype by maturity were observed for SSAA (Table 2.2) consistently
with the difference in seed quality between ‘OH8245’ and ‘T4099’
observed
for
germination
index
and
germination
percentage.
Differences between maturities within genotypes were observed after
accelerated aging. For ‘T4099’, red mature and overripe fruit stages
had
a
lower
final
germination
after
SSAA
than
seeds
from
mature
green and breaker stage fruit (p <0.05)(Table 2.2). In contrast,
seeds
from
red
mature
‘OH8245’
fruit
had
a
higher
germination
percentage than mature green and breaker stage fruit (p <0.05). The
SSSA
test
showed
artificial
that
‘T4099’
deterioration,
seed
especially
was
seeds
more
from
susceptible
overripe
to
fruits
(Table 2.4). Seeds from ‘T4099’ overripe fruit stage resulted in 62%
germination after SSAA, compared to 95% germination for ‘OH8245’. In
addition, the difference between germination percentage before and
after SSAA was greater for ‘T4099’ than for ‘OH8245’. For example,
seeds
from
before
and
overripe
after
‘T4099’
the
fruits
treatment,
had
85%
and
respectively,
63%
while
germination
seeds
from
overripe ‘OH8245’ had 96% and 95% germination respectively (Tables
2.2).
Because tomato fruit maturity is accompanied by an increase in
lycopene
synthesis
harvesting
‘T4099’
and
in
accumulation,
the
early
the
stages
of
question
fruit
of
whether
maturity
(i.e.
breaker stage) would improve the speed of germination was addressed.
The
GI
indicated
that
‘T4099’
had
lower
speed
of
germination
compared to ‘OH8245’ regardless of fruit maturity (Table 2.1). This
42
finding
indicates
that
the
speed
of
germination
of
this
high
lycopene line is not improved by harvesting at early stages of fruit
maturity, and suggests that the effect of the dg gene in germination
physiology is independent of the gradual accumulation of lycopene
that accompanies fruit development.
Germination of ‘T4099’ was less affected by seed harvest at
the mature green fruit stage than for ‘OH8245’. In addition, red
mature and overripe fruit stages resulted in lower seed quality in
‘T4099’ compared to ‘OH8245’ (Table 2.2).
that
‘T4099’
fruit
might
maturity
acquire
stages
and
maximum
that
This finding suggests
germination
red
mature
capacity
and
in
early
overripe
fruit
maturities might have a detrimental effect on the final germination
percentage of ‘T4099’. Low seed germination of ‘OH8245’ was observed
for
the
mature
green
stages
while
in
‘T4099’
the
overripe stage
resulted in lower seed quality. This observation also suggests that
fruit development effects differ among genotypes, particularly at
the
mature
green
and
overripe
stages
where
seeds
may
be
more
susceptible to environmental stresses. In addition, fruits of two
different genotypes might be classified using the same stage based
on color and firmness as criteria. However, seeds from such fruits
might have different maturity levels, and reserve accumulation that
produces different seed quality levels. Thus, classifications based
on
tomato
fruit
color
and
firmness,
although
practical,
are
no
assurance that they reflect similarities in seed maturity.
Seeds from the red mature fruit stage are reported to have the
highest quality (Valdes and Gray, 1998). In ‘T4099’, results of the
SSAA test indicated that seed quality was higher at the mature green
43
breaker and pink breaker compared to the red mature and overripe
stages
(Table
firmness
(Wann,
and
2.2).
since
1996),
initiated
Fruits
fruits
seeds
were
of
within
deterioration
‘T4099’
the
with
classified
no
late
based
possessed
red
visual
mature
symptoms
on
color
greater
stage
in
and
firmness
might
the
have
fruit
as
reported in previous studies for overripe fruits (Valdes and Gray,
1998; Demir and Samit, 2001).
In
replaced
the
summer
‘OH8245’
study,
to
the
minimize
recurrent
the
effect
parent
of
‘Flora-Dade’
different
genetic
background between ‘OH8245’ and ‘T4099’. We also evaluated whether
planting
seeds
germination
of
without
‘T4099’.
desiccation
In
would
addition,
affect
normal
the
seedling
speed
and
of
radicle
protrusion studies were evaluated to determine whether the effect of
slow
germination
was
more
marked
for
seedling
growth
than
for
germination physiology.
Radicle protrusion
Results
indicated
that
at
5
d,
‘Flora-Dade’
had
a
higher
germination for fresh seeds than ‘T4099’. However, this difference
was only significant at the overripe fruit stage (Table 2.3). For
dry seed comparisons, ‘Flora-Dade’ showed
‘T4099’
in
all
fruit
higher germination than
maturities except the overripe stage (Table
2.5). There were no significant differences between ‘Flora-Dade’ and
‘T4099’ at 14 d for fresh and dry seeds at any of the fruit maturity
stages (data not shown).
44
Percentage of normal seedlings
‘Flora-Dade’
seeds
produced
a
higher
percentage
of
normal
seedlings than ‘T4099’ seeds at all fruit maturities for fresh seeds
(Table
2.4).
In
‘Flora-Dade’
seed
lots,
percentage
germination
increased from mature green to red mature fruit stages and remained
constant from red mature to overripe fruit maturity. Germination was
greater
than
90%
in
seeds
from
red
mature
and
overripe
fruit
maturity stages. In ‘T4099’, germination increased from mature green
to pink breaker fruit stages; however, germination was never greater
than 25%. Similar results were observed for dry seeds, ‘Flora-Dade
seeds produced a higher percentage of normal seedlings than ‘T4099’
in all but the mature green fruit stage (Table 2.6). ‘Flora-Dade’
seeds showed overall higher germination than ‘T4099’ seeds at all
fruit
stages
and
this
difference
was
greater
for
red
mature
and
overripe fruit stages. Similarly, as for radicle protrusion, there
were no significant differences between ‘Flora-Dade’ and ‘T4099’ at
14 d for fresh and dry seeds at any of the fruit maturity stages.
The
difference
between
‘Flora-Dade’
and
‘T4099’
was
more
marked for seedling growth than for radicle protrusion, suggesting
the effect of the dg gene is more critical for seedling development
than for germination.
and
‘T4099’,
consistent
which
with
This difference at 5 d between ‘Flora-Dade’
is
the
more
low
GI
obvious
for
found
in
normal
the
seedlings,
winter
study
was
and
demonstrates the low seed vigor of ‘T4099’. Similarly, for fresh
seeds,
the
normal
seedlings
germination
difference
for
in
compared
fresh
and
percentage
germination
to
radicle
dry
seeds,
45
was
protrusion.
drying
greater
Based
affected
on
seeds
for
the
from
‘Flora-Dade’ more than ‘T4099’. This observation is possibly caused
by the fact that at five days after planting, fresh seeds of ‘T4099’
had a percentage germination ranging from o% to 21 % across fruit
maturities while ‘Flora-Dade’ had a germination percentage ranging
above 15 to 93% suggesting that germination reduction in ‘T4099’
seeds is independent of drying.
Although seedling length was not measured, it was observed that
‘Flora-Dade’ possessed taller seedlings than ‘T4099’. Differences were
found between ‘Flora-Dade’ and ‘T4099’ in percentage germination as
measured by radicle protrusion at the mature green (fresh seeds) and
overripe (dry seeds) fruit maturity stages, but the small number of
replications
failed
to
show
a
statistical
difference.
The
more
consistent difference between ‘Flora-Dade’ and ‘T4099’ was observed at
5 d for normal seedlings, indicating lower growth rate of ‘T4099’
although this also may indicate that radicle protrusion should be
evaluated
from
the
first
day
after
sowing
to
detect
differences
between ‘Flora-Dade’ and ‘T4099’ early in the germination process.
The
difference
between
‘Flora-Dade’
and
‘T4099’
was
less
pronounced for percentage of seeds with radicle protrusion compared to
percentage
of
difference
between
development
normal
rather
seedlings.
these
than
two
This
indicates
genotypes
germination
is
(radicle
that
the
greatest
limited
to
seedling
protrusion).
However,
‘Flora-Dade’ seeds germinated earlier than seeds of ‘T4099’ and, by
the fifth day, some seeds already produced normal seedlings. Genotype
‘T4099’, in contrast, germinated later, so by the fifth day, seedlings
were not sufficiently developed to be considered normal. However, they
continued to develop and eventually became normal seedlings.
46
A question that remains is why it was not possible to increase
the speed of germination of ‘T4099’ fresh seeds given that fruit and
locular
tissue
prevent
precocious
germination
and
tomato
generally
regain the capability to germinate once separated from these fruit
tissues (Berry and Bewley, 1992). It is probable that the low speed of
germination of ‘T4099’ is caused by a mechanism that is independent of
the gradual accumulation of fruit lycopene, maturity stage and drying.
Fruit development affected seed quality differently in ‘T4099’
compared to the other genotypes evaluated in this study. ‘OH8245’
and
‘Flora-Dade’
had
maximum
percentage
germination
at
the
red
mature fruit stage (Table 2.2). In contrast, ‘T4099’ had maximum
percentage germination at the breaker (Table 2.2) and pink breaker
(Table 2.2) fruit stages. Genotype ‘T4099’ had maximum seed quality
in the early fruit maturity stages so harvesting in the latest phase
of red mature when fruits are becoming soft might result in seeds
with advanced deterioration as indicated by the SSAA test. Thus, the
seed quality response to fruit maturity affects some genotypes more
than
others.
Therefore,
harvesting
tomato
fruits
from
certain
genotypes by machine in a single day would result in seeds with
different development and quality attributes.
Gibberellins might be responsible for both the reduced plant
growth
and
delayed
germination
responses
in
‘T4099’
(Wann
1995).
Gibberellins are essential for the synthesis of hydrolytic enzymes
necessary
for
endosperm
degradation
and
subsequent
radicle
protrusion (Bewley and Black, 1994; Bradford et al., 2000), as well
as for cell and stem elongation. However, genotypes carrying the dg
gene
result
in
high
levels
of
carotenoids
47
and
because
ABA
is
a
derivative from carotenoid precursors (Zeevaart et al., 2000); it is
also
possible
causing
and/or
that
delayed
increased
dg
genotypes
germination.
ABA
may
be
produce
Thus,
both
involved
in
greater
levels
decreased
the
slow
of
ABA
gibberellins
germination
of
‘T4099’.
Currently, the tomato seed industry is developing genotypes
with enhanced lycopene and fruit firmness. Based on the results of
the
SSSA
test,
some
of
these genotypes might produce seeds that
deteriorate faster even if fruits are harvested at the red mature
stage.
Genotypes
that
produce
higher
levels
of
lycopene
possibly
should be harvested at the breaker and pink breaker stages.
Acknowledgment
We thank the Ohio Agricultural Research and Development Center
Research
Enhancement
Competitive
Grant
Program
(OARDC-
RECG)
for
partial funding of this research. We also thank the National Council
for Science and Technology of Mexico (CONACyT) for financial support
of Gerardo Ramirez-Rosales’ doctoral program.
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Berry, T. and Bewley, D.J. (1991).
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the fruit switch from a developmental
a requirement for desiccation. Planta,
(1991).
‘Ohio
8245’
Seeds of tomato (Lycopersicon
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Berry, S.Z. and Uddin, M.R. (1991). Breeding tomato for quality and
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49
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50
Source
DG
GI
GP
SSAA
Genotype
1
25.938 **
31.686 NS
899.145
***
Maturity
4
4.2588 **
18422.83 *** 15235.385***
Genotype x maturity
4
5.8277 ***
55.6627 *** 278.4332 ***
Table 2.1. Anova table for genotype, maturity and the interaction
Genotype
x
maturity
of
germination
index
(GI),
germination
percentage (GP) and SSAA of the tomato genotypes ‘OH8245’ and
‘T4099’ harvested at five maturities: mature green (MG), breaker,
(BR), pink breaker (PB), red mature (RM), and overripe (OR) . Winter
2000.
*, **, *** = Significance level at a= 0.05, 0.01, 0.001 and
respectively.
NS not significant at a=0.05
51
Genotype
MG
GI
FG
BR
SSAA
GI
FG
PB
SSAA
GI
FG
SSAA
'OH8245' 15.80 86.70 80.20
18.40 90.70 84.50
18.20 94.70 95.00
'T4099' 12.20 92.50 84.00
13.10 95.70 82.50
12.50 91.50 82.50
Prob.
0.01
0.25
0.59
0.01
RM
GI
Prob.
0.59
0.01
0.19
0.07
OR
FG
SSAA
GI
FG
SSAA
99.2
97.5
18.9
96.2
95.0
12.2
90.5
75.0
11.0
85.2
63.0
0.01
0.01
0.01
0.01
0.01
0.01
'OH8245' 19.5
'T4099'
0.04
Table 2.2. Germination Index (GI), germination percentage (GP) and
germination percentage after SSAA of ‘OH8245’ and ‘T4099’ tomato
seeds harvested at five different fruit maturities: mature green
(MG), breaker (BR), pink breaker (PB) red mature (RM) and overripe
(OR) ). Winter 2000.
52
Genotypes
MG
BR
PB
RM
OR
----------------------------------%---------------------------------‘Flora-Dade’ 92.7 a
98.0 az
90.5 a
99.0 a
95.0 a
‘T4099’
93.0 a
76.0 a
97.0 a
59.0 b
73.2 a
Table 2.3. Five-day count germination (radicle protrusion) of fresh
tomato seeds of ‘Flora-Dade’ and ‘T4099’ harvested at five
maturities: mature green (MG), breaker (BR), pink breaker (PB) red
mature (RM) and overripe (OR) . Summer 2001.
Z
values within columns with the same letter are not significantly
different at a 0.05.
Genotype
MG
BR
PB
RM
OR
----------------------------------%--------------------------------‘Flora-Dade’
‘T4099’
15 a
0 b
35.5 az
75.5 a
3.75 b
21.2 b
92.0 a
10.0 b
92.7 a
17.7 b
Table 2.4. Five-day count germination (normal seedlings) of fresh
seeds of ‘Flora-Dade’ and ‘T4099’ harvested at five maturities:
mature green (MG), breaker (BR), pink breaker (PB) red mature (RM)
and overripe (OR)). Summer 2001.
Z
values within columns with the same letter are not significantly
different at a 0.05.
53
Genotypes
MG
BR
PB
RM
OR
----------------------------------%--------------------------------‘Flora-Dade’ 85.5 a
99.5 az
98.7 a
97.5 a
92.6 a
‘T4099’
61.2 b
83.0 b
83.0 b
85.3 a
55.2 b
Table 2.5. Five-day count germination (radicle protrusion) of dry
seeds ‘Flora-Dade’ and ‘T4099’ harvested at five maturities (mature
green (MG), breaker (BR), pink breaker (PB) red mature (RM) and
overripe (OR)). Summer 2001 .
Z
values within columns with the same letter are not significantly
different at a 0.05.
Genotypes
MG
BR
PB
RM
OR
---------------------------------%--------------------------------‘Flora-Dade’ 0
26.5 az
50.5 a
69.2 a
64.0 a
‘T4099’
1.0 b
13.7 b
4.9 b
24.0 b
0
Table 2.6. Five-day count germination (normal seedlings) of dry
seeds ‘Flora-Dade’ and ‘T4099’ harvested at five maturities: mature
green (MG), breaker (BR), pink breaker (PB) red mature (RM) and
overripe (OR)). Summer 2001.
Z
values within columns with the same letter are not significantly
different at a 0.05.
54
CHAPTER 3
Environment and Fruit Development Effects on Seed Germination and
Vigor of Four Tomato (Lycopersicon esculentum Mill.) Genotypes.
Summary
Considerable
information
exists
on
the
effect
of
seed
development on tomato (Lycopersicon esculentum Mill.) seed quality.
However,
most
studies
have
evaluated
one
genotype
and
a
single
environment. As a consequence, little comparative information exists
on
which
factor(s)
(fruit
maturity,
production
environment
or
genotype) are most important for expression of optimum tomato seed
quality.
This
production
genotypes
study
evaluated
environment
varying
in
on
fruit
the
effect
germination
color
and
and
of
fruit
vigor
lycopene
of
maturity
four
content.
and
tomato
Genotypes
‘Flora-Dade’, ‘OH9242’ (ogc), ‘FG-218’ (dg ogC) and ‘T4099’ (dg ogc)
were grown in two locations, and harvested at four fruit maturity
stages. Seeds were extracted by fermentation at 24
quality
was
evaluated
as
percentage
germination
O
C for 48 h. Seed
and
Germination
Index (GI). Highly significant effects of genotype and maturity were
observed for GI. Highly significant effects for maturity and the
interaction genotype by maturity were also observed for germination
percentage.
55
The experimental line ‘T4099’ (dg ogc) showed lower GI values
than the other genotypes. Germination percentage of seeds from the
mature
green
fruit
(ogc)
‘OH9242’
indicated
and
that
stage
was
‘FG-218’
fruit
reduced
ogc).
(dg
maturity
in
genotypes
‘Flora-Dade’,
Variance component analysis
accounted
for
36
and
49%
of
the
variance for percentage germination and GI. Genotypes explained 25%
for GI and 6% for germination percentage. The effect of tomato fruit
maturity varied among genotypes, especially for the mature green and
overripe
fruit
harvesting
maturity
tomato
stages.
fruits
from
These
the
results
breaker
to
suggest
red
mature
that
stages
results in good seed germination.
Introduction
The effect of fruit maturity on tomato seed quality has been
studied extensively (Berry and Bewley, 1991; Berry and Bewley, 1992;
Demir
and
studies
Ellis,
on
1992;
fruit
Liu
et
development
al.,
have
1996).
However,
evaluated
one
most
genotype
of
the
and
a
single environment. Valdes and Gray (1998) evaluated the effect of
fruit maturity on seed quality of four tomato genotypes produced
under
different
genotypes
overripe
for
fruit
environments.
the
effect
maturity
of
They
fruit
stage.
observed
maturity,
However,
they
differences
among
especially
at
failed
provide
to
the
information about which factor(s) (genotype, environment or fruit
maturity)
was
most
important
for
the
expression
of
optimum
seed
germination. It is important to know when seeds are most susceptible
to environmental stresses at various fruit stages and whether this
response differs among genotypes.
56
The
with
fruits
seed
high
industry
carotenoid
with
higher
is
currently
content,
red
developing
including
color
intensity
tomato
lycopene,
compared
genotypes
that
to
produce
traditional
genotypes. How fruit maturity affects seed quality across different
production environments for these high pigment genotypes is unknown.
Recent information on the effect of environments and genotype on
tomato flesh color is available (Sacks and Francis, 2001). However,
such studies are lacking for seed related traits such as germination
and vigor in tomato.
Seed vigor can be defined as the sum of seed attributes that
ensure the successful development of a new plant under a range of
environmental
determined,
conditions.
it
is
also
Although
highly
seed
modified
vigor
by
is
the
genetically
environment
and
physiological processes. While it is well known that the genetic,
environment and maturity components are important to the expression
of tomato seed germination and vigor, the relative importance of
each
of
evaluated.
influence
these
The
of
factors
in
objective
fruit
seedling
of
this
maturity,
establishment
study
genetic
and
was
has
to
not
been
determine
production
the
environment
components on the expression of tomato seed germination and vigor as
measured by a Germination Index.
Materials and methods
Plant material
Genotypes
‘Flora-Dade’,
‘OH9242’,
‘FG-218’
and
‘T4099’
were
used in this study. The varieties were selected because they contain
significantly
different
amounts
of
57
lycopene,
but
are
paired
by
pedigree.
Variety
developed
by
background.
‘T4099’
crossing
The
two
was
the
described
ogc
dg
genotypes
share
by
genes
97%
of
Wann
(1996),
into
a
genes
and
was
‘Flora-Dade’
by
descendent.
‘OH9242’ is a crimson (ogc) breeding line with moderately elevated
levels
of
Lycopene
Experimental
line
(Francis,
‘FG-218’
was
in
progress)
developed
by
and
excellent
‘OH9242’
and
color.
‘T4099’
followed by two backcrosses with selection for dg. ‘OH9242’ and ‘FG218’ share 87.5% of their genes by descendent. Seeds were planted
for fruit and seed production in the Vegetable Crops Branch (VCB) of
The Ohio State University (Fremont, OH) and the Ohio Agricultural
Research and Development Center (OARDC) (Wooster OH). Fruits were
harvested in a single day in fall 2000 at four maturity stages:
mature green, breaker, red fruit and overripe stages based on the
criteria reported by Valdes and Gray (1998). Fruits were transported
to the Seed Biology Laboratory at Columbus, OH for seed extraction.
Seeds were extracted by hand, and fermented at room temperature (~
24 °C) for 48 h in sealed 0.250L bags. Seeds were rinsed and dried
at
room
temperature
(~24
O
C)
to
content (fresh weight basis).
58
approximately
8%
seed
moisture
Seed Quality
Seed
quality
was
evaluated
by
germination
percentage
and
germination Index (GI) for up to 14 days (ISTA, 1999). Fifty seeds
were planted in each of four Petri plates containing two layers of
blotter paper saturated with 10 ml dd water. The plates were then
placed in a germination
chamber at 25 °C with a 16/8 h light/dark
cycle. The GI was calculated for each treatment by the algebraic sum
of
the
ratio
of
normal
germinated
seedlings
and
the
days
after
planting at which time the count was made.
Statistical Analysis
Statistical
including
four
two
replications
genotypes.
replications
analysis was conducted as a combined experiment
Genotypes
and
using
repeated
measures
genotypes
at
the
a
SAS
to
given
location
were
locations
conducted
by
considered
were
random
procedure
determine
fruit
(Fremont
as
fixed
effects.
GLM
(SAS
Wooster)
effects
The
stage
2001)
differences
and
while
analysis
Institute,
statistical
maturity
and
was
as
among
(Hinkelmann
and
Kempthorne, 1994). To determine the variance explained by maturity,
genotype, and locations for germination percentage and GI, SAS PROC
VARCOMP
was
used.
Data
expressed
in
percentage
were
transformed
using arcsine square root (Steel et al., 1997) although the original
germination values are shown.
Results and discussion
There
were
significant
effects
of
genotype,
maturity,
and
maturity x location x genotype for GI (table 3.1) indicating that
the production environment (location), maturity and genotype affect
59
the
speed
of
germination
of
tomato
seeds.
For
percentage
germination, significant effects were found for location, maturity
and maturity x genotype indicating environment and maturity affect
germination percentage and the effect of maturity was dependent on
the genotype (Table 3.1). In all fruit maturities, except in the
mature
green
stage,
genotype
‘T4099’
showed
lower
GI
than
other
genotypes (Table 3.2). All genotypes had a lower GI at the mature
green stage compared to breaker, pink breaker and overripe stages;
however, this difference was larger for ‘Flora-Dade’, ‘OH9242’ ogc
and ‘FG-218’ dg ogc. Apparently, seed quality of these genotypes was
more
affected
by
harvest
at
the
mature
green
fruit
stage
than
‘T4099’. This effect was most evident for germination percentage,
where
‘T4099’
had
10%
higher
germination
‘OH9242’ogc, and ‘FG-218’ dg ogc (Table 3.2).
than
‘Flora-Dade’,
Significant differences
were also present between ‘T4099’ and ‘Flora-Dade’ genotypes at the
red mature and overripe fruit maturities, but these differences were
not greater than 5%. Overripe fruit maturity stage did not result in
significant
reductions
in
seed
germination
(Table
3.2).
Genotype
‘T4099’ showed low seed quality in red mature and overripe fruit
maturity stages in previous studies (Ramirez-Rosales et al., chapter
2).
However,
overripe
in
fruits
this
was
experiment,
not
germination
affected.
It
is
of
red
possible
mature
that
and
this
difference between winter and fall studies is because fruits from
the fall study were harvested in a single day and classified based
on color and firmness, which was estimated visually. In contrast, in
the winter study, plants were tagged at bloom to assure that fruits
at given maturity stages differed from other maturities in age in
60
addition
to
color
and
firmness.
It
is
also
possible
that
discrepancies in overripe effects are due to different production
conditions under greenhouse and field conditions. For example, in
the greenhouse, fruits were sometimes affected by the physiological
disorder known as blossom end rot which was not observed in the
field study.
In a previous study, percentage germination of line ‘T4099’
did not decrease in the mature green stage suggesting that this line
achieves maximum seed germination in the early fruit maturity stages
(Ramirez-Rosales
et
al.,
chapter
2
unpublished
data).
Genotypes
‘OH9242’ dg and ‘FG-218’ dg ogc showed a speed of germination similar
to ‘Flora-Dade’. The reason for this observation is unclear since
the
both
genotypes
germination
(Jarret
carry
et
the
al.,
dg
gene,
1984;
which
Berry
and
can
result
Uddin,
in
1991).
slow
These
results suggest that pleiotropic effects associated with dg can be
moderated by genetic background.
Variance component analysis indicated that maturity accounted
for 36 and 49% of the variance for percentage germination and GI.
Genotypes explained 25% for GI and 6% for germination percentage,
respectively.
germination
This
genotype
percentage
is
difference
possibly
a
effect
result
of
on
the
GI
low
versus
speed of
germination of ‘T4099’. Genotype x maturity explained 16% of the
variance for germination percentage, probably due to the effect of
the mature green stage. Genotype maturity, and genotype x maturity
explained nearly 60% of the variance of germination percentage, while
genotype,
location
approximately
86%
x
of
genotype
the
x
maturity
variance
61
for
and
GI
maturity
(Table
explained
3.1).
Thus,
production location environment did not have a central effect on the
expression of percentage germination and vigor measured as GI; these
two variables were more influenced by genotype and fruit maturity
stage.
The
mature
green
stage
may
be
more
affected
by
adverse
environmental conditions because this fruit maturity stage showed the
lowest overall GI and germination percentage.
Based on data from the four tomato genotypes evaluated, our
results show that tomato genotypes vary in their seed vigor response
to the effect of fruit maturity stage. These results suggest that
harvesting from breaker stage to red mature fruit stage results in
optimum seed of high pigment tomato genotypes.
Acknowledgment
We thank the Ohio Agricultural Research and Development Center
Research Enhancement Competitive Grant Program (OARDC- RECG) of The
Ohio State University for partial funding of this research. We also
thank
The
National
Council
for
Science
and
Technology
of
Mexico
(CONACyT) for financial support of Gerardo Ramirez-Rosales’ doctoral
program.
Literature Cited
Berry, T., and Bewley, J.D. (1991).
esculentum Mill.) which develop in a
the fruit switch from a developmental
a requirement for desiccation. Planta,
Seeds of tomato (Lycopersicon
fully hydrated environment in
to a germinative mode without
186, 27-34.
Berry, T. and Bewley, J.D. (1992) A role for the surrounding fruit
tissue in preventing the germination of tomato (Lycopersicon
esculentum) seeds. Plant Physiology, 100, 951-957.
Berry, S.Z. and Uddin, M.R. (1991). Breeding tomato for quality and
processing attributes. In: Genetic Improvement of Tomato. (Ed.)
Kaloo, G. pp. 197-206. Springer-Verlag, Inc., Berlin.
62
Demir, I. and Ellis, R.H. (1992). Changes in seed quality during
seed development and maturation in tomato. Seed Science Research, 2,
81-87.
Hinkelmann, K. and Kempthorne, O. (1994). Design and analysis of
experiments. V (1.) 495 P. John Wiley & Sons, Inc., New York.
Jarret, R.L., Sayama, H. and Tigchelaar, E.C. (1984). Pleiotropic
effects associated with the chlorophyll intensifier mutants high
pigment and dark green in tomato. Journal of the American Society
for Horticultural Science, 109, 873-878.
International Seed Testing Association. (1999). Rules
testing. Seed Science and Technology, 27, supplement.
for
seed
Liu, Y., Bino, R.J., Karssen, C.M. and Hilhorst, H.W.M. (1996).
Water relations of GA and ABA deficient tomato mutants during seed
development
and
their
influence
on
germination.
Physiologia
Plantarum, 96, 425-432.
Sacks, E.J. and Francis, D.M. (2001). Genetic and environmental
variation for tomato flesh color in a population of modern breeding
lines. Journal of the American Society for Horticultural Science 126,
221-226.
SAS Institute, Inc., 2001. Cary, NC.
Steel, R., Torrie, J.H. and Dickey, D.A. (1997). Principles
procedures of statistics. P. 666, McGraw-Hill, New York.
and
Valdes, V.M. and Gray, D.
(1998). The influence of stage of fruit
maturation on seed quality in tomato (Lycopersicon lycopersicum (L.)
Karsten) Seed Science and Technology, 26, 309-318.
63
GI
GP
P-Value
Var
(%)
Location
0.09
2.30
0.04
3.90
Replication
0.98
0.00
0.73
0.00
Genotype
0.00
25.40
0.19
5.50
Location x Genotype
0.18
0.00
0.84
0.00
Maturity
0.00
49.40
0.00
36.50
Maturity x Genotype
0.21
0.00
0.01
16.00
Maturity x Location
0.06
0.00
0.42
1.40
Maturity x Location x Genotype
0.02
12.10
0.00
0.30
Sources
P-Value Var (%)
Table 3.1. Probability values for tomato germination index (GI) and
germination percentage (GP) and percentage of variance explained by
different sources of variation of four tomato genotypes grown in two
locations and harvested at four fruit maturities: mature green,
breaker, red mature. Fall 2000.
64
MG
Genotypes
GI
BR
GP (%)
GI
RM
GP (%)
GI
OR
GP (%)
GI
GP (%)
‘Flora-Dade'
16.1 a 80.2 b 18.0 b 98.0 a 19.0 a 98.2 a 18.9 a 98.0 a
‘T4099'dg ogC
14.5 a 93.3 a 16.1 c 95.2 a 16.4 b 98.7 a 16.7 b 98.7 a
‘OH9242' ogC
16.0 a 84.7 b 19.2 a 98.7 a 18.9 a 97.7 ab 19.2 a 97.7 ab
‘FG-218’ dg ogc 15.1 a 79.4 b 19.0 a 98.2 a 19.1 a 93.3 b 18.4 a 93.3 b
Table 3.2. Germination index (GI) and Germination percentage (GP) of
four tomato genotypes grown in two locations and harvested at four
fruit maturities: mature green (MG), breaker (BR), red mature (RM),
and overripe (OR).
Z
Values with same letter within columns are not significantly
different at a 0.05.
65
CHAPTER 4
Effects of Overripe Fruits on Germination of Tomato (Lycopersicon
esculentum Mill.) Seeds.
Summary
Tomato seed quality is highly influenced by the stage of fruit
maturation. Maximum seed quality generally believed to occurs when
seeds
are
harvested
from
fruits
at
the
red
mature
stage.
Most
studies demonstrate that tomato seed deterioration occurs following
the red mature stage. However, the cause(s) of this deterioration
and
the
relationship(s)
understood.
This
study
with
examined
fruit
the
maturation
effect
of
remain
fruit
poorly
maturity
on
tomato seed quality after the red mature stage. The variety ‘FloraDade’
was
grown
in
the
greenhouse
during
fall
2001.
Fruits
were
harvested at 55, 60, 70, 80 and 90 days after pollination (dap).
Fruits were evaluated for maturity based on changes in red color,
lycopene
content
fermentation at 24
standard
and
fruit
firmness.
Seeds
were
extracted
by
O
germination
C for 48 h. Seed quality was assayed using the
and
saturated
salt
accelerated
aging
(SSAA)
tests. Decreases in germination percentage were observed at 60 dap
and coincided with a reduction in fruit firmness. Seeds from 90 dap
66
tomato fruits resulted in germination percentages approximately 40%
lower than that of seed from fruits at 55 dap. Seeds from fruits at
55 dap had lower germination following SSAA than seeds from 90 dap
fruits. The higher germination of seeds from overripe fruits after
the SSSA test may suggest that these seeds become dormant as fruit
deterioration
proceeds
in
a
fashion
similar
to
an
after-ripening
process.
Introduction
Seed
development
extensively
maximum
studied.
seed
effects
Seeds
on
tomato
extracted
seed
from
red
quality
tomato
have
been
fruits
have
germination (Valdes and Gray 1998; Demir and Samit,
2001). However, the effect of over ripe fruits is poorly understood.
Early
studies
reported
that
seed
quality
remains
constant
after
physiological maturity and until 95 dap (Demir and Ellis, 1992) In
contrast, more recent reports (Liu et al., 1996; Liu et al., 1997;
Valdes and Gray, 1998; Demir and Samit, 2001) indicate that seeds
from overripe tomato fruits show lower seed quality. The reasons for
these differences may be due to the use of different parameters to
determine seed development (e.g dap vs. fruit stages), genotype and
tests
used
to
evaluate
seed
quality.
Studies
have
shown
that
decreased germination can occur in overripe tomato fruits (Liu et
al., 1997). These authors reported greater 4c/2c DNA ratios in seeds
from
tomato
fruits
harvested
75
dap,
indicating
more
advanced
germination physiology. They speculate that this results in lower
seed
quality.
Seeds
from
overripe
fruits
may
also
be
more
susceptible to damage during seed fermentation. However, no direct
67
reports exist that have evaluated the effect of fermentation and
drying
on
unclear
quality
when
of
overripe
fruits
fruits.
initiate
In
addition
substantial
it
changes
is
still
in
the
deterioration process that may result in lower tomato seed quality.
The
objective
of
this
study
was
to
determine
seed
quality
of
overripe tomato fruits and to test whether overripe fruits yield
seeds
of
lower
quality
because
they
are
less
tolerant
to
fermentation and drying.
Materials and methods
Plant material
Thirty plants of the open pollinated tomato variety ‘FloraDade’ were grown in a greenhouse in Columbus, OH for fruit and seed
production
in
fall
2001.
The
experiment
consisted
of
three
replications of 10 plants each. Flowers were tagged to determine
fruit
age
pollination
visual
and
harvested
(dap).
changes
in
Fruit
fruit
at
55,
age
60,
was
firmness,
70,
80
determined
color,
and
based
lycopene
degree of fruit deterioration were also evaluated.
68
90
days
on
after
dap,
but
content,
and
Lycopene Extraction and Quantification
A sample of approximately 10 fruits from each replication and
each
maturity
stage
was
homogenized
in
a
blender.
A
portion
was
placed in 50 ml tubes at –20 °C until extraction. Lycopene extraction
was
conducted
using
a
method
similar
to
that
of
Volker
et
al.
(2002). Briefly, a 5 g sample was homogenized in 50 ml methanol plus
1 g calcium bicarbonate and 5 g celite. The sample was then filtered
through
Whatman
no.
1
and
no.
42
filter
papers.
Lycopene
was
extracted using a sample of hexane-acetone (1:1, v:v) and quantified
spectrophotometrically at 472 nm and expressed in mg/100 g FW.
Seed extraction and quality.
Seeds
were
extracted
from
fruit
tissue
by
fermentation
conducted in a sealed plastic bag placed in a closed container at
room
temperature
surface
dried
at
(~24
0
room
C)
for
48
h.
temperature
content (dry weight basis).
Seeds
to
were
then
approximately
rinsed
8%
and
moisture
Seed quality was evaluated using the
standard germination (ISTA, 1999) and the saturated salt accelerated
aging (SSAA) (Jianhua and McDonald, 1996) tests. For the standard
germination test, 50 seeds were sown in Petri dishes filled with 10
ml dd water and placed in a germination chamber at 24 °C. For the
SSAA test, seeds were subjected to 41°C and 75% RH for 96 h using
sodium
desired
chloride
relative
as
the
saturated
humidity.
After
salt
the
solution
SSAA
to
treatment,
evaluated for standard germination as described above.
69
achieve
seeds
the
were
To evaluate whether overripe fruits produce seed less tolerant
to the fermentation process, three replications of 50 seeds each
from
90
day-old
fruits
were
germinated
after
0,
24
and
48
h
fermentation.
Statistical Analysis
Data
were
analyzed
using
a
one-way
ANOVA
with
three
replications (blocks within greenhouse). Means were compared using
LSD (P=0.05) and data transformed using arcsine square root although
the original values are shown (SAS Institute, 2001).
Results and Discussion
Fifty-five
while
60-day-old
day
old
fruits,
tomato
fruits
although
were
still
red
firm,
mature
were
and
firm
starting
to
soften. Fruits 90 dap showed advanced levels of deterioration as
indicated by the presence of fungi and considerable reduction in
fruit firmness. Fruits 70 and 80 dap showed reduced firmness, but
did
not
exhibit
a
high
degree
of
deterioration
as
observed
for
fruits 90 dap. There were no visual changes in color from 55 to 90
dap
which
was
verified
by
lycopene
content.
Lycopene
content
remained constant from 55 to 90 dap giving values between 6-8 mg/100
FW (Table 4.1). Ninety-day-old fruits produced no more than 3% seeds
with radicle protrusion (e.g. precocious germination).
70
A highly significant difference among seedlots from different
fruit
ages
was
observed
for
standard
germination.
Germination
declined from 100 to 60% for seeds from fruits harvested 55 to 90
dap (Table 4.2). There was no significant difference for SSAA values
across
fruit
ages,
fruit
ages
with
germination
following
SSAA
remaining constant within a range of 87-94% (Table 4.2).
After fruits reached the
red mature stage, they initiated a
process of deterioration characterized by reduction of firmness. For
this
particular
genotype,
reduction
in
tomato
fruit
firmness
was
observed approximately 5 days after the initial red mature stage and
was accompanied by a reduction in standard germination percentage.
This
observation
suggests
there
is
a
direct
relationship
between
tomato fruit deterioration expressed as a marked change in fruit
firmness and reduction in seed quality. These results confirm those
reported by Valdes and Gray (1998) and Damir and Samit (2001). The
implications of these findings suggest that harvesting tomato fruits
from
different
degrees
of
maturation
after
the
red
mature
stage
compromises seed quality because fruits from differing degrees of
firmness, and consequently seed quality, are pooled to produce a
single seed lot. Although it is possible to sort fruits based on
color,
which
red
was
firmness,
color
remained
verified
especially
by
unchanged
lycopene
after
after
content
fruits
reach
the
(Table
color,
red
mature
4.1).
Thus,
appears
a
stage,
fruit
better
measure of when tomato seeds begin to deteriorate than fruit color.
71
The seed industry has developed tomato genotypes with extra
fruit firmness for extended storage in the marketing and processing
channels. It is not known whether these extended storage genotypes
will show a reduction in seed quality before a reduction in fruit
firmness occurs.
Results
of
the
SSAA
test
suggest
that
high
quality
tomato
seeds (i.e. high germination percentage) are more affected by this
test as observed by the reduction in germination percentage of 55
dap fruits (Table 4.2). Seeds from overripe fruits that already have
a degree of deterioration were unaffected and had higher germination
values (Table 4.2). Although it is not known why the germination of
seeds from 90 dap fruits was improved by SSAA, it is possible that
repair occurred similar to that reported in seed priming (McDonald,
2000). It is also possible that seeds from overripe fruits show a
dormancy mechanism that enables them to withstand adverse conditions
encountered after they are released from the
seeds
do
not
germinate
in
the
mother
mother plant. Tomato
plant
because
of
the
concomitant effect of reduced osmotic potential and inhibitors in
the locular tissue (Berry and Bewley, 1992). Seeds germinate once
they are separated from that medium. However, as fruits deteriorate,
seeds may develop endogenous dormancy mechanisms that allow them to
withstand adverse conditions. If tomato seeds develop dormancy as
the fruit deteriorates, then the reduced seed quality reported in
seeds
from
overripe
fruits
might
not
be
an
accurate
conclusion.
However, this study does not present any data that overripe fruits
result in dormant seeds. More studies are needed with more genotypes
and
under
different
environments
72
to
test
the
dormancy
model
in
overripe
tomato
fruits.
This
study
overripe
tomato
fruits
result
in
although
this
effect
may
be
an
demonstrates,
seeds
with
indication
of
however,
low
that
germination,
dormancy more than
reduced seed quality.
Germination after 24 h of fermentation resulted in a higher
germination percentage of seeds with radicle protrusion compared to
48
and
0
h
(Table
4.3).
However,
no
significant
difference
was
observed for percentage normal seedlings. Fermentation is a method
of tomato seed extraction; this study suggests that seeds coming
from
overripe
than
24
h
fruits
and
might
are
more
have
susceptible
lower
to
germination
fermentation
percentage
longer
(radicle
protrusion). As the fruit advances in maturation, the fermentation
process should be conducted in periods no longer than 24 h.
Acknowledgment
We thank the Ohio Agricultural Research and Development Center
Research Enhancement Competitive Grant Program (OARDC- RECGP) of The
Ohio State University for partial funding of this research. We also
thank
the
National
Council
for
Science
and
Technology
of
Mexico
(CONACyT) for financial support of Gerardo Ramirez-Rosales’ doctoral
program.
Finally, we thank Drs. Steve Schwartz and Puspitasari-
Nienaber Liu, and the personnel of the Food Science and Technology
Department of The Ohio State University for use of their facilities
for lycopene quantification.
73
Literature Cited
Demir, I. and Ellis, R.H. (1992). Changes in seed quality during
seed development and maturation in tomato. Seed Science Research, 2,
81-87.
Demir, I. and Samit, Y. (2001). Seed quality in relation to fruit
maturation and seed dry weight during development in tomato. Seed
Science and Technology, 29,453-462.
Jianhua,
Z.
and
McDonald,
M.B.
(1996).
The
saturated
salt
accelerated aging test for small-seeded crops. Seed Science and
Technology, 25, 123-131.
International Seed Testing Association. (1999). Rules
testing. Seed Science and Technology, 27, supplement.
for
seed
Liu, Y., Bino, R.J., Karssen, C. M. and Hilhorst, H.W.M. (1996).
Water relations of GA and ABA deficient tomato mutants during seed
development
and
their
influence
on
germination.
Physiologia
Plantarum, 96, 425-432.
Liu, Y., Hilhorst, H.W.M., Groot, S.P.C. and Bino, R.J. (1997).
Effects of nuclear DNA and internal morphology of gibberellin- and
abscisic acid deficient tomato (Lycopersicon esculentum Mill.) seeds
during maturation, imbibition, and germination. Annals of Botany 96,
161-168.
McDonald, M.B. (2000). Seed Priming. In: Seed technology and its
biological basis. (Eds.) Black, M. and Bewley, J.D. pp. 287-325.
Sheffield Academic Press, England.
SAS Institute, Inc., 2001. Cary, NC
Valdes, V.M. and Gray, D.
(1998). The influence of stage of fruit
maturation on seed quality in tomato (Lycopersicon lycopersicum (L.)
Karsten). Seed Science and Technology, 26, 309-318.
Volker, B., Puspitasari-Nienaber, N.L., Ferruzzi, M. and Schartz,
S.J. (2002). Trolox equivalent antioxidant capacity of different
geometrical
isomers
of
∝-carotene,
Β-carotene,
lycopene
and
Zeaxanthin. Journal of Food and Agricultural Food Chemistry 50, 221226.
74
Genotype
DAP
Lycopene
Fruit characteristics
(mg/100 g FW)
‘Flora-Dade’
55
7.9 az
Red mature firm
60
6.0 a
Red mature soft
70
7.0 a
Red mature soft
80
7.7 a
Overripe
90
7.0 a
Advanced deterioration
Table 4.1. Lycopene content and tomato fruit characteristics of
‘Flora-Dade’ harvested at different days after pollination (DAP).
Winter 2002
Z
values within columns with the same letter are not significantly
different at a 0.05.
Fruit age (DAP)
GP
SSAA
55
100 az
91 a
60
83
b
87 a
80
81
b
93 a
90
60
c
94 a
Table 4.2. Germination (GP) and Saturated Salt Accelerated Aging
(SSAA) percentages of tomato ‘Flora-Dade’ at different fruit
ages. Fall 2001
Z
values within columns with the same letter are not significantly
different at a 0.05.
75
Fermentation period
Radicle Protrusion (%)
Normal Seedlings (%)
0 h
44 c
34 a
24
74 a
45 a
48
60 b
44 a
Table 4.3. Germination percentage of 90 day-old fruit of tomato
‘Flora-Dade’ after different periods of fermentation.
Z
values within columns with the same letter are not significantly
different at a 0.05.
76
CHAPTER 5
Total Antioxidant Capacity of Seeds from Normal and Enhanced
Lycopene Tomato (Lycopersicon esculentum Mill.) Genotypes.
Summary
Elevated antioxidant content in seeds may be a desirable trait
for increased seed storability and slower deterioration rates. This
study was conducted to test whether tomato fruits from a genotype
with elevated levels of natural antioxidants produce seeds with a
functionally greater total antioxidant capacity. The tomato genotype
‘T4099’,
acid,
which
and
the
produces
elevated
recurrent
levels
parent
of
lycopene
‘Flora-Dade’
were
and
ascorbic
field
and
greenhouse produced under standard agronomic practices. Fruits and
seeds were evaluated for antioxidant capacity and lycopene content.
Total antioxidant capacity of the water and lipid soluble fractions
of
seeds
were
evaluated
using
the
Trolox
Equivalent
Antioxidant
Capacity (TEAC) and Photo-induced Chemiluminescence (PCL) methods.
The
high pigment line ‘T4099’ resulted in a higher fruit tissue
lycopene content and total antioxidant capacity than ‘Flora-Dade’.
However, both TEAC and PCL methods indicated that seeds of ‘T4099’
had lower antioxidant capacity and that the difference was greater
for water-soluble antioxidants.
77
Based on these results it is hypothesized that tomato fruits
and
seeds
may
compete
for
antioxidants.
Fruits
with
enhanced
lycopene content are desirable for human consumption, yet this may
produce seeds with lower antioxidant levels and reduced longevity in
storage.
Introduction
Tomato genes that produce elevated lycopene such as dark green
(dg) and high pigment (hp-1 and hp-2) are available (Stevens and
Rick, 1986). Genotypes carrying these genes have high chlorophyll
content due to their increased chloroplast number (Jarret et al.,
1984; Sacks and Francis, 2001). The conversion of chloroplasts into
chromoplasts results in higher carotenoid levels compared to normal
tomato
genotypes.
In
addition
to
high
lycopene
content,
these
genotypes also produce elevated levels of ascorbic acid (Jarret et
al., 1984). Even though the dg genotype has higher carotenoids and
ascorbic acid, direct information on the total antioxidant capacity
of the dg genotype compared to wild type tomatoes is unavailable.
The beneficial effects of the production and consumption of
tomato varieties rich in carotenoids including lycopene for human
health and nutrition are well established (Giovanelli et al., 1999;
Van Den Berg et al., 2000). But, the effect that such varieties have
on other traits is unknown. For example, do tomatoes with superelevated levels of lycopene produce seeds with increased antioxidant
levels? Even though tomato seeds do not have a major nutritive value
per se, their level of antioxidants might be an important trait for
seed longevity, storability and stress tolerance (McDonald, 1999).
78
Studies
least
have
partly
(Thapliyal
and
reported
responsible
Connor
that
levels
of
for
differences
free
radicals
in
seed
are
at
longevity
1997; Trawatha et al., 1993). Others argue
that reductions in seed longevity are accompanied by a decrease in
the levels of free-radical scavengers (Chui et al., 1995; Hsu and
Sung, 1997; Bailly et al., 1997; De Vos et al., 1994). The decreased
levels
of
free
deterioration.
radical
Thus,
scavengers
seeds
with
therefore
enhanced
may
levels
increase
of
seed
protective
compounds such as antioxidants might increase their longevity and
storability.
One indirect method of measuring antioxidants is through an
assessment of total antioxidant capacity. Although several methods
exist
to
calculate
the
total
antioxidant
capacity
of
fruits,
vegetables and vegetable products (Packer, 1999), little information
is available as to their application in seeds as planting material.
79
The
evaluation
of
antioxidants
and
their
influence
on
seed
quality aspects such as increased longevity has included assessments
of individual enzyme activity such as superoxide dismutase, catalase
and ascorbate peroxidase (Sung and Jeng, 1994; Chiu et al., 1995).
However,
these
studies
failed
to
report
information
on
total
antioxidant capacity as it relates to seed quality. It is important
to evaluate whether methodologies to estimate the total antioxidant
capacity used in vegetables and food products, such as the Trolox
Equivalent Antioxidant Capacity (TEAC) (Miller et al., 1996) and the
Photo-induced Chemiluminescence (PCL) (Popov and Lewin, 1999) assays
can
be
used
to
evaluate
the
antioxidant
capacity
of
seeds.
The
objective of this study was to determine whether a tomato genotype
rich in carotenoids also results in a higher antioxidant capacity of
fruits and seeds as measured by the TEAC and PCL methods.
Materials and methods
Plant material
Seeds
recurrent
of
the
parent,
high
the
lycopene
open
line
pollinated
‘T4099’
variety
ogc
dg
and
‘Flora-Dade’,
its
were
produced under standard agronomic practices in Wooster, OH in summer,
2000. Fruits were harvested at the red mature stage and transported
to the Seed Biology Laboratory (Columbus, OH) for seed extraction
which was conducted by fermentation at 24
0
C for 48 h. Seeds were
then washed several times and dried at room temperature (~24 h) to
approximately 8% seed moisture content (fresh weight basis). Seeds
were then placed in coin envelopes and stored at 4
until analysis for antioxidant capacity.
80
o
C and 70% RH
To determine lycopene content and total antioxidant capacity of
tomato fruits, plants of the variety “Flora-Dade’ and ‘T4099’ were
greenhouse grown in winter, 2002. At harvest, a sample of about 10
fruits per replication was homogenized with a commercial blender, and
part of the homogenate placed in a 50 ml centrifuge tube and stored
at –20
0
C until used.
Fruit lycopene extraction
Fruit lycopene extraction was conducted as described by Volker
et al. (2002). A sample (5 g) of fruit tissue was homogenized in 50
ml methanol plus 1 g calcium bicarbonate and 5 g celite. The sample
was filtered through Whatman no. 1 and no. 42 papers. Lycopene was
extracted using a sample of hexane-acetone (1:1, v:v). Acetone and
methanol
were
removed
by
washing
with
distilled
water
and
the
remaining volume was adjusted to 100 ml with pure hexane. Lycopene
was quantified spectro-photometrically at 472 nm and expressed in
mg/100 g FW.
Three fruit tissue samples (3 ml per replication) were dried
under nitrogen and stored at
–20 °C under darkness. These samples
were used for the antioxidant capacity assays. In the case of fruit
tissues, the total antioxidant capacity was assayed only for those
compounds soluble in acetone-hexane (lipid soluble antioxidants).
Antioxidant extraction of seeds
Tomato
seeds
were
ground
with
mortar
and
pestle
in
the
presence of liquid nitrogen. The total antioxidant capacity of seeds
was
evaluated
for
the
water
and
lipid-soluble
fractions.
The
extraction method was similar to that reported by Pellegrini et al.
81
(1999) with some modifications. Hexane was used to extract lipidsoluble antioxidants instead of dichloromethane, and nitrogen was
used to dry samples instead of rotary evaporation. For water-soluble
antioxidant extraction, samples were used for antioxidant capacity
without drying.
A ground sample (200 mg) was placed in a 50 ml centrifuge tube
filled with 10 ml hexane and 10 ml water. Samples were vortexed for
2
min
and
centrifuged
at
1000
g
for
15
min.
The
upper
phase
consisting of lipid soluble compounds was transferred to a new tube.
For maximizing lipid extraction, this step was repeated twice. Five
ml of the lipid extraction solution were dried under nitrogen and
stored
in
the
dark
at
–20
ο
C
until
used.
For
water-soluble
antioxidant extraction, 20 ml of pure methanol were added to the
aqueous phase. Samples were then shaken at 30
ο
C for 30 min and
filtered using a vacuum with a square funnel and Whatman paper no.
1. The filtrate was then stored at –20
ο
C until used.
Determination of total antioxidant capacity
Total
antioxidant
capacity
was
assayed
by
both
the
Trolox
Equivalent Antioxidant Capacity (TEAC) assay (Miller et al., 1996)
and
the
photo-induced
Chemiluminescence
method
(Popop
and
Lewin,
1999).
The TEAC assay consisted of the generation of a free radical.
The chemical reagent 2,2’-Azinobis(3-ethylbenzothiazoline-6-sulfocin
acid) diammonium salt (ABTS) (Sigma Co., St. Louis, MO) was passed
through a funnel with MnO2 (Merck, Dasmstadl, Germany) and a Whatman
filter paper no. 1. Free radical (ABTS.+) was dissolved in phosphate
buffer saline (PBS pH 7.4) to an absorbance of 0.7 at 734 nm and
82
stored at room temperature for 2h for stabilization. A standard curve
was generated incubating 1 ml ABTS.+ with 100 µl 6-hydroxy-2,5,7,8tetramethylchroman 2-carboxylic acid (Trolox) at concentrations of
0.0125, 0.025, 0.05, 0.1 mmol/L for 2 min. The absorbance at 734 nm
was then determined after 2 min using PBS as a blank and correcting
for hexane.
Lipid-soluble antioxidants
Samples dried under nitrogen were reconstituted in 1 ml and 3
ml
hexane
for
seeds
and
fruits,
respectively.
The
reconstituted
sample (100 µl) was incubated with 1 ml of ABTS.+. The antioxidant
capacity was determined based on the standard curve and expressed as
Trolox
Equivalent
Antioxidant
Capacity
(TEAC
value)
in
mmol/g
of
seed.
Water-soluble antioxidants
One hundred µl of the methanol (66%) solution containing watersoluble antioxidants was used for the assay that
was performed as
described for the lipid soluble antioxidant, but corrected for the
decay in absorbance caused by methanol.
83
The photo-induced Chemiluminescence method (PCL) consists of
the excitation of a photo-sensitizer (luminol) which results in the
generation of the superoxide radical O2.- (Popov and Lewin, 1999).
During the assay, the irradiated solution is transferred to a cell
where chemiluminescence is determined. The chemiluminescence signal
is
reduced
in
the
quantification.
compound
such
The
as
presence
of
antioxidants,
quantification
trolox
(lipid
is
which
performed
soluble
permits
using
antioxidants)
their
a
standard
or
ascorbic
acid (water soluble antioxidants). In this study, the antioxidant
capacity
was
evaluated
(antioxidant
capacity
using
of
Photochem®
the
lipid
soluble)
instrument
and
ACW
and
ACL
(antioxidant
capacity of water-soluble) kits (Analytik-Jena USA, Inc. Delaware,
OH). The Photochem® instrument determines the antioxidant capacity of
samples based
on the appropriate trolox or ascorbic acid standard
curves.
Antioxidant capacity of lipid soluble (ACL) materials
Blank, standard curve and experimental samples were prepared
with
reagents
of
the
ACL-Kit.
Previously
dried
samples
were
reconstituted in 1 ml of ACL-1. This solution (100 µl) was mixed with
2.3 ml of ACL-1, 200 µl of ACL-2 and 25 µl of ACL-3. A standard curve
was prepared using 0.5, 1.0, 2.0 and 2.5 nmol of Trolox. Samples and
standard were injected into the Photochem® instrument to obtain the
antioxidant
capacity,
which
was
concentration.
84
later
adjusted
for
seed
Antioxidant capacity of water-soluble (ACW) materials
Similarly, as with lipid soluble antioxidants, blank, standard
curve
and
experimental
samples
were
prepared
using
an
ACW-kit
containing 1.5 ml ACW-1, 1.0 ml of ACW-2 and 25 ml of ACW-3. The
standard curve was prepared using 0.0, 1.0 and 3.0 nmol ascorbic
acid.
Samples
instrument
to
and
standard
obtain
the
were
injected
antioxidant
into
capacity,
Photochem®
the
which
was
later
adjusted for seed concentration.
Statistical analysis
Differences
between
‘Flora-Dade’
and
‘T4099’
for
lycopene
content were evaluated by ANOVA with three replications of 10 plants
each
and
three
antioxidant
sub-samples
capacity
for
of
10
fruit
fruits
tissue
per
was
replication.
evaluated
with
Total
two
replications of 10 plants each. Each replication was further divided
into
three
samples,
which
were
assayed
in
duplicate.
Differences
between ‘Flora-Dade’ and ‘T4099’ for antioxidant capacity of seeds
were computed using the SAS procedure GLM. The TEAC value for watersoluble antioxidants was analyzed twice (two experiments) and five
replications
per
experiment.
The
TEAC
value
for
lipid
soluble
antioxidants was evaluated using five replications.
Total
antioxidant
capacity
by
the
PCL method for water and
lipid soluble antioxidants was evaluated with five replications and
three samples per replication.
Results and Discussion
A highly significant difference (P<0.0001) for fruit lycopene
content between ‘Flora-Dade’ and ‘T4099’ was observed (Table 5.1).
85
Fruits from genotype ‘T4099’ contained lycopene levels approximately
70%
greater
than
fruits
from
‘Flora-Dade’
(Table
5.2).
These
differences in lycopene content between the ‘Flora-Dade’ and ‘T4099’
fruits are consistent with those reported by Wann et al. (1985).
A
significant
difference
between
genotypes
for
total
antioxidant capacity was observed (Table 5.3). This difference in
total antioxidant capacity between these two genotypes was even more
marked than the difference in lycopene content. Total antioxidant
capacity of tomato fruits could not be measured with the PCL method
possibly because of the high content of carotenoids, particularly
lycopene
and
ß-carotene.
These
compounds
were
extracted
with
acetone-hexane then reconstituted in reagent (ACL) kit reagent 1 for
the assay.
It is possible that the failure of the PCL method to
read results were incompatible between high carotenoid content and
ACL-reagent 1.
Genotype ‘T4099’ produced fruits with 135% greater antioxidant
capacity
than
Flora-Dade’
fruits
(Table
5.2).
However,
seeds
of
‘Flora-Dade’ and ‘T4099’ failed to show a significant difference in
TEAC values for lipid soluble antioxidants (Tables 5.4 and 5.8).
There
was
a
highly
significant
difference
between
genotypes
for
water-soluble antioxidants (P< 0.0001) as measured by TEAC and PCL
methods (Tables 5.5 and 5.6). Similarly, there was a significant
difference
for
lipid-soluble antioxidants (P=0.02) as measured by
the PCL method (Table 5.7).
Water-soluble
and
lipid
soluble
antioxidants for seeds from
‘Flora-Dade’ resulted in greater TEAC and PCL values than seeds from
‘T4099’ (Table 5.8). ‘Flora-Dade’ had a value 8 to 65% greater than
86
seeds
from
‘T4099’
respectively
(Table
for
lipid
5.8).
and
The
water-soluble
PCL
method
antioxidants,
distinguishes
the
antioxidant capacity of seeds of these two genotypes in both waterand lipid- soluble fractions while the TEAC method only identified
the difference in the water-soluble fraction.
This
study
demonstrates
that
higher
fruit
tissue
lycopene
content of a high lycopene genotype resulted in higher antioxidant
capacity.
It
antioxidants
is
important
were
not
to
assayed
emphasize
in
this
that
study.
fruit
The
water-soluble
reason
is
that
samples used for lycopene extraction were also used for antioxidant
capacity assays and the extraction procedure is accomplished using
acetone and hexane. However, it is likely that the ‘T4099’ fruit
will also have higher water soluble antioxidants since the dg gene
also causes increased levels of ascorbic acid (Jarret et al., 1984)
and
a
high
water
correlation
soluble
exists
antioxidant
between
capacity
ascorbic
(Popov
acid
and
content
Lewin,
and
1999).
Therefore, the introduction of this gene into commercial varieties
will
improve
the
nutritional
quality
of
tomato
fruits
and
their
products.
Both methodologies to determine total antioxidant capacity of
tomato seeds for the two genotypes evaluated in this study yielded
similar
results.
The
difference
in
antioxidants
between
seeds
of
these two genotypes was more marked for the water-soluble fraction
compared
to
the
lipid-soluble
fraction.
Although
both
methods
resulted in similar information in terms of differentiating between
total antioxidant capacity of both genotypes, a difference between
the values obtained for TEAC compared to the PCL method was found
87
(Table 5.2). This may be because the PCL method uses a standard
curve based on very low concentrations of trolox (nmol) while TEAC
generates
a
standard
curve
based
on
higher
trolox
concentration
(mmol) (Figures 5.2 A, B, C and 5.3 A). Further, the water-soluble
TEAC method uses trolox as a standard while PCL uses ascorbic acid
as
a
standard.
However,
statistical
analysis
showed
a
positive
correlation (r= 0.92) (Figure 5.1) between PCL and TEAC results for
ACW.
Both
methodologies
therefore
can
be
used
for
assaying
the
antioxidant capacity of tomato seeds for lipid and water fractions.
However, the PCL method may be more sensitive for lipid fraction
antioxidants
because
it
minimizes
the
possibility
of
error
since
experimental samples are prepared with reagents kits that do not
interfere with the accuracy of readings. In contrast, lipid fraction
samples using the TEAC method are reconstituted in hexane and the
hexane must be separated via centrifugation prior to the assay. If
some hexane remains during the spectrophotometric reading, it will
cause inaccurate results.
Another advantage of the PCL method is that it generates O2.free radicals that are more related to natural oxidation processes
(Hauptmann and Cadenas, 1997).
The
higher
TEAC
levels
results
of
demonstrate
antioxidants
that
while
‘T4099’
seeds
from
fruit
tissue
had
these
fruits
had
lower levels of antioxidants. Wild type ‘Flora-Dade’ fruit tissue
had
lower
levels
of
antioxidants
but
higher
seed
antioxidants
levels. These results suggest that higher levels of antioxidants in
the tomato fruit may compete with antioxidants found in the seed.
This observation may explain why ‘T4099’ seeds may be more prone to
88
deterioration:
the
lower
levels
of
antioxidants
being
unable
to
provide protection against free radical attack. Although this study
does not show direct evidence that lower levels of antioxidants are
associated with more rapid seed deterioration, studies do exist that
support this relationship. For example, maize (Zea mays L.) seeds
with lower levels of enzymic antioxidants deteriorate faster during
storage
(Bernal-Lugo
enzymatic
antioxidant
et
al.,
2000).
protection
In
has
addition,
been
a
decrease
associated
with
in
lipid
peroxidation in cocoa (Theobroma cacao L.) seeds (Li and Sun, 1999)
and
lipid
peroxidation
deterioration
(Chiu
et
is
considered
al.,
1995;
as
a
McDonald,
cause
of
1999).
In
seed
wheat
(Triticum aestivum L.), Pinzino et al. (1999) reported a decrease on
lutein
(a
potent
carotenoid
antioxidant)
as
wheat
seeds
age.
If
antioxidant contents decrease as seeds age, seeds containing lower
initial levels of antioxidants will likely deteriorate faster.
It
elevated
is
hypothesized
lycopene
content,
that
the
although
selection
of
tomatoes
with
desirable for human nutrition
and health, may result in seeds with lower antioxidant levels. This
may
result
genotypes
in
as
lower
quality
previously
planting
reported
seed
compared
to
(Ramirez-Rosales,
wild
2002
type
in
preparation).
Acknowledgment
We thank the Ohio Agricultural Research and Development Center
Research Enhancement Competitive Grant Program (OARDC- RECG) of The
Ohio State University for partial funding of this research.
89
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91
J.,
The
the
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Volker, B., Puspitasari-Nienaber, N.L., Ferruzzi, M. and Schwartz,
S.J. (2002). Trolox equivalent antioxidant capacity of different
geometrical
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92
Source
DF
MS
F CALC
Genotype
1
146.66
99.80
Replications
2
2.9213
2.64
Sample
2
2.1944
Error
16
23.51
P
< 0.0001
0.112
1.98
0.1803
Table 5.1. Analysis of variance for lycopene content of two tomato
(Lycopersicon esculentum Mill.) genotypes, ‘Flora-Dade’ and ‘T4099’.
Genotype
Lycopene (mg/100 g FW)
TEAC (mmol/g)
(mg/100 g FW)
(mmol/g)
‘Flora-Dade’
7.05 + 1.20
232.7 + 54.0
‘T4099’
12.7 + 12.7
548.1 + 59.1
1.20
44.8
LSD (0.05)
Table 5.2. Fruit tissue lycopene content and Trolox Equivalent
Antioxidant Capacity (TEAC) of a wild type (‘Flora-Dade’) and a high
lycopene tomato line (‘T4099’).
93
Source
DF
MS
FCALC
Genotype
1
599545.7
Replication
2
6496.74
2.38
0.12
Sample
2
8049.23
2.95
0.103
Reading
1
361.87
0.13
0.121
Error
18
2732.38
219.42
P
< 0.0001
Table 5.3. Analysis of variance of total antioxidant capacity for
lipid-soluble antioxidants of tomato (Lycopersicon esculentum Mill.)
fruits from two genotypes ‘Flora-Dade’ and ‘T4099’. Antioxidant
capacity was determined by the Trolox Equivalent Antioxidant
Capacity (TEAC) method.
Source
DF
MS
F CALC
P
Genotype
1
0.0065
0.13
0.738
Rep
4
0.048
0.96
0.516
Error
4
0.051
Table 5.4. Analysis of variance for lipid-soluble antioxidants
(ACL)) of tomato (Lycopersicon esculentum Mill.)
seeds from two
genotypes ‘Flora-Dade’ and ‘T4099’. Total antioxidant capacity was
determined by the Trolox Equivalent Antioxidant Capacity (TEAC)
method.
94
Source
DF
MS
F CALC
P
Genotype
1
16528131
75.51
< 0.0001
Experiment
1
150430
0.69
0.428
Replications (Exp)
8
145117.78
0.66
0.7132
Error
9
218875.46
Table 5.5. Analysis of variance for water-soluble antioxidants
(ACW)) of tomato seeds from two genotypes. Total antioxidant
capacity was determined by the Trolox Equivalent Antioxidant
Capacity (TEAC) method.
SOURCE
DF
MS
F CALC
P
Genotypes
1
39969.611
253.9
< 0.0001
Replications
4
1077.90
6.85
0.0010
Sample
2
238.06
1.51
0.2425
Error
22
157.42
Table 5.6. Analysis of variance for water-soluble antioxidants (ACW)
of tomato seeds from two different genotypes. Total antioxidant
capacity was determined by the Photo-induced Chemiluminescence
method (PCL).
95
SOURCE
DF
MS
F CALC
P
Genotypes
1
43.05
6.06
0.022
Replications
4
109.96
15.47
0.0001
Sample
2
6.60
1.51
0.409
Error
22
7.10
Table 5.7. Analysis of variance for lipid soluble antioxidants (ACL)
of tomato seeds from two different genotypes. Total antioxidant
capacity was determined by the Photo-induced Chemiluminescence (PCL)
method.
TEAC
Genotype
ACW
PCL
ACL
ACW
ACL
--------------------------(nmol/g)-------------------------------‘Flora-Dade’
5815
874
184
41.2
‘T4099’
3997
834
111
38.3
LSD (0.05)
473
NS
9.5
2.0
Table 5.8. Total antioxidant capacity in water (ACW) and lipid
(ACL) fractions of tomato seeds (nmol/g) of ‘Flora-Dade’ and
‘T4099’ using the Trolox Equivalent Antioxidant Capacity (TEAC)
and the Photo-induced Chemiluminescence (PCL) methods for ‘FloraDade’ and ‘T4099’ tomato genotypes.
96
250
y = 0.0381x - 39.148
R 2 = 0.8532
TEAC
200
150
100
50
0
3000
3500
4000
4500
5000
5500
6000
6500
PCL
Figure 5.1. Total antioxidant capacity of tomato seeds determined by
the Photo-induced Chemiluminescence method (PCL) and the Trolox
Equivalent Antioxidant Capacity (TEAC). Each value represents the
average of five replications.
97
Absorbance
(ABTS- Trolox)
A
y = 2.4779x + 0.0108
2
R = 0.9921
0.30
0.20
0.10
0.00
0
0.025
0.05
0.075
0.1
Trolox concentration( mm/L)
Absorbance
(ABTS-Trolox)
B
y = 2.6643x + 0.0666
R 2 = 0.9997
0.30
0.20
0.10
0.00
0
0.025
0.05
0.075
0.1
Absorbance (ABTS
- Trolox)
Trolox concentration (mmol/L)
y = 2.2024x + 0.1913
R2 = 0.9918
C
0.4
0.3
0.2
0.1
0
0
0.025
0.05
0.075
0.1
Trolox Concentration (mmol/L)
Figure 5.2. Calibration curves developed with 0.125, 0.025, 0.5 and
0.1 mmol/L of 6-hydroxy-2,5,7,8-tetramethylchroman2-carboxylic acid
(Trolox). (A) Standard curve used to calculate Trolox Equivalent
Antioxidant Capacity (TEAC) of lipid-soluble antioxidants of tomato
fruits. (B) Standard curve used to calculate TEAC values of watersoluble antioxidants of tomato seeds and (C) Standard curve used to
calculate TEAC values of lipid-soluble antioxidants of tomato seeds.
The chemical reagent 2,2’-Azinobis(3-ethylbenzothiazoline-6-sulfocin
acid) diammonium salt (ABTS) was incubated with Trolox and the
reduction in absorbance was determined spectro-photometrically.
98
Inhibition (%)
A
y = 0.2098x + 0.3772
R 2 = 0.9828
1.00
0.80
0.60
0.40
0.20
0.00
0
0.5
1
1.5
2
2.5
Trolox (nmol/L)
Lag phase (seconds)
y = 50.131x + 5.0745
2
R = 0.9758
B
200
150
100
50
0
0
0.5
1
1.5
2
2.5
3
Asc Acid (nmol/L)
Figure 5.3. Calibration curves used to calculate the total
antioxidant
capacity
of
tomato
seeds
using
the
photochemiluminescence method. (A) Standard curve used to calculate the
total antioxidant capacity of lipid-soluble antioxidants using 0.5,
1,2,
and
2.5
nmol/L
of
6-hydroxy-2,5,7,8-tetramethylchroman2carboxylic acid (Trolox).(B) Standard curve used to calculate the
total antioxidant capacity of water-soluble antioxidants using
0,1,2, and 3 nmol/L of ascorbic acid. TAC is determined based on the
percentage of inhibition of the chemiluminiscence due to presence of
trolox or lipid-soluble antioxidant (A) or the delay in seconds of
chemiluminsence signal due to ascorbic acid or water soluble
antioxidant (B).
99
CHAPTER 6
Gibberellin plus Norflurazon Enhance the Germination of Dark Green
Tomato (Lycopersicon esculentum Mill.) Genotypes
Summary
Tomato
lycopene
(Lycopersicon
are
desirable
esculentum
for
human
Mill.)
nutrition
varieties
because
rich
of
in
their
associated reduced risk of various cancers. Tomato genes that cause
elevated lycopene content such as dark green (dg) are available.
However, several genes that result in elevated lycopene result in
negative pleiotropic effects including slow germination and reduced
plant height. It is uncertain whether low gibberellin levels, high
ABA content or high light sensitivity account for the low speed of
germination of dg tomato genotypes. This study evaluated gibberellin
(GA3), norflurazon (inhibitor of carotenoid and ABA synthesis), and
light effects on the speed of germination of the high pigment line
line
‘T4099’
and
its
recurrent
parent
‘Flora-Dade’.
These
tomato
genotypes were greenhouse produced in summer 2001 for fruit and seed
production.
Seeds
from
red
fruits
were
sown
in
solutions
of
gibberellin, norflurazon and norflurazon plus gibberellin (GA3). In
another
light
experiment,
conditions.
protrusion.
Speed
tomato
seeds
Germination
of
were
was
germination
was
germinated
recorded
evaluated
under
daily
as
as
time
different
radicle
to
reach
fifty percent germination (T50) and germination index. Norflurazon
alone and gibberellin plus norflurazon resulted in higher speed of
100
germination of ‘T4099’ compared to the control but not at the same
level as ‘Flora-Dade’. Light reduced the speed of germination and
the
effect
was
more
marked
for
‘T4099’.
Darkness
alone
and
gibberellin plus norflurazon resulted in similar values for T50 and
germination
index,
suggesting
that
both
treatments
have
the
same
site of action. These data suggest that the high lycopene tomato
genotype produce greater amounts
of ABA during imbibition and this
process is possibly regulated by light.
Introduction
The
red
color
associated
with
tomatoes
is
determined
by
lycopene, a carotenoid compound. Because lycopene has been correlated
with reduced prostate and other cancers, tomato genotypes rich in
lycopene are thought to be desirable in the human diet. Therefore, it
is important to incorporate tomato germplasm that enhances lycopene
content into commercial varieties.
Several major genes, such as dark green (dg) crimson (ogc) and
high pigment (hp-1 and hp-2) types result in super elevated lycopene
content,
and
are
these
genes
plant
growth,
available
cause
in
undesirable
slow
improved
tomato
pleiotropic
germination
and
germplasm.
effects
brittle
stems
such
However,
as
(Jarret
reduced
et
al.,
1984). These negative effects on plant development have slowed the use
of the dg and hp genes as homozygotes in commercial tomato varieties
(Sacks and Francis, 2001).
The
associated
reduced
with
plant
low
growth
endogenous
of
dg
and
gibberellin
hp
genotypes
content
has
(Wann,
been
1995).
Gibberellins are required for the synthesis of hydrolytic enzymes and
101
endosperm
weakening,
and
both
processes
are
required
for
radicle
protrusion in tomato seeds (Bradford et al., 2000). Unfortunately,
there is no direct evidence that the low speed of germination of high
lycopene
tomato
genotypes
is
caused
by
low
endogenous
gibberellin
content. Wann (1995) evaluated the effect of gibberellin on plant
height of dg and hp tomato genotypes but did not consider the effect
of seed germination. Since carotenoids are precursors of ABA via an
indirect pathway (Zeevaart, 2000), it is possible that this delay in
seed germination is caused by higher levels of abscisic acid (ABA).
For example, increasing carotenoid content in canola (Brassica napus
L.) resulted in a 1 to 2 day delay in seed germination (Shewmaker et
al., 1999).
It is possible that elevated levels of carotenoids result in
over-production
of
ABA
causing
a
delay
in
germination.
Ectopic
expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene caused
over-production
of
ABA
(Thomson
delayed seed germination.
relationship
between
et
al.,
2000a)
that
resulted
in
Even though these are examples of the
high
carotenoid
lines
and
germination
abnormalities, information about how genetic changes resulting in
different fruit carotenoid levels may affect tomato seed quality is
limited.
Because
dg
and
hp
genotypes
produce
higher
fruit
carotenoid
content compared to wild types, these tomato varieties might also
produce higher carotenoid content in their seeds resulting in higher
levels of ABA and consequently slower germination. Recent reports also
demonstrate
that
an
increase
in
ABA
synthesis
during
imbibition
results in delayed seed germination in some species. This phenomenon
102
has been reported in Arabidopsis thaliana (Debeaujon and Koornneeef,
2000),
Nicotiana
(Triticum
plumbaginifolia
aestivum
L.)
(Garello
(Grappin
and
Le
et
al.,
Page-Degivry,
2000),
wheat
1999)
yellow
cedar (Chamaecyparis nootkatensis) (Schmitz et al., 2000), and tomato
(Thomson et al., 2000b). Since dg and hp genotypes produce elevated
tomato
fruit
decreased
carotenoid
gibberellins
content,
but
it
also
is
possible
increased
that
ABA
not
levels
only
acting
antagonistically are responsible for the low speed of germination of
these genotypes.
Norflurazon is a bleaching herbicide that interferes with the
carotenoid biosynthetic pathway. This herbicide acts directly on the
enzyme
phytoene
disaturase,
preventing
the
formation
of
cyclic
carotenoids and resulting in accumulation of phytoene (Breitenbach et
al., 2001). Because carotenoids are precursors of ABA, norflurazon and
other
carotenoid
inhibitors
such
as
fluridone
have
been
used
to
prevent formation of ABA and release seed dormancy (Garello and Le
Page-Degivry, 1999; Schmitz et al., 2000; Thompson et al., 2000b).
Release
of
hypothesis
that
imbibition.
tomato
dormancy
ABA
with
is
Norflurazon
lines
resulting
carotenoid
also
inhibitors
synthesized
has
been
in
lower
de
demonstrates
novo
in
successfully
used
ABA
and
content
seeds
in
the
during
transgenic
decreased
seed
dormancy (Thompson et al., 2000b). But, it has not been used to date
in tomato genotypes with increased lycopene.
The
caused
by
delayed
higher
speed
of
germination
sensitivity
to
of
light
an
hp
mediated
genotype
by
is
also
phytochrome
responses (Shichijo et al., 2001). The germination of this genotype is
favored by darkness and delayed by far red light. However, little
103
information exists about whether the slow germination of dg tomato
seeds also results from decreased gibberellin, increased ABA content
or light sensitivity. This study was designed to evaluate the effects
of
gibberellin
(GA3), norflurazon (inhibitor of carotenoid and ABA
synthesis), and light treatments on the speed of germination of seeds
from the high lycopene genotype ‘T4099’ (dg ogc) compared to ‘FloraDade’.
Materials and Methods
Plant material
Plants
of
genotype
‘T4099’
dg
ogc and
its
recurrent
parent
‘Flora-Dade’ were grown under greenhouse conditions for fruit and
seed at The Ohio State University (Columbus, OH) in summer 2001.
Gibberellin and norflurazon studies
Four
replications
(50
seeds
each)
from
the
same
batch
were
planted in Petri dishes containing either a solution of dd water
(control), 10-4 M gibberellin (GA3) (Aldrich, Milwaukee, WI), 20 mg/L
of
norflurazon
norflurazon.
(Supelco,
Bellefonte,
PA)
or
gibberellin
plus
Petri dishes were placed in a germination chamber at 24
º C and 16/8 dark/light cycle.
104
Seed Quality
The effect of treatments on germination was measured as the
germination
index,
hypocotyl
length
and
time
for
fifty
percent
germination (T50). Germination index was calculated as the algebraic
sum
of
the
ratio
obtained
dividing
the
number
of
seeds
showing
radicle protrusion and the days after sowing. Hypocotyl length (cm)
was measured 8 days after sowing using 10 seedlings per replication.
Time to fifty percent of visible germination (T50) was calculated
using probit analysis on time (SAS Institute, 2001).
Experimental design
Data
were
analyzed
as
a
completely
randomized
design
in
a
factorial arrangement with four replications. The main factors were
genotypes,
levels:
gibberellin
‘Flora-Dade’
and
and
norflurazon.
‘T4099’
for
All
main
genotype,
factors
and
had
presence
two
or
absence of gibberellin and norflurazon. Data were analyzed with the
SAS procedure GLM and Least Squares Means (SAS Institute, 2001).
Light and darkness studies
Two
experiments
were
conducted.
In
experiment
1,
seeds
of
‘Flora-Dade’ and ‘T4099’ were germinated in Petri dishes filled with
10 ml of distilled water and covered with aluminum foil for 24, 48,
72, 96, and 120 h. Three replications of 50 seeds each were sown for
each period and visible germination (radicle protrusion) was recorded
at the end of each period. The control consisted of germination under
8/16 h light/dark cycles. The effect of treatments on germination was
measured
as
the
germination
index,
and
time
to
fifty
percent
germination (T50); this value was obtained using SAS proc probit.
105
In experiment 2, seeds of both genotypes were germinated in
darkness as
in experiment 1, but the light treatment consisted of
germination in 16/8 light/dark cycle.
Experimental design
The
experimental
design
was
a
completely
randomized
design
with factorial arrangement. Data were analyzed using proc GLM and
Least Squares Means. Main effects were genotypes (‘Flora-Dade’ and
‘T4099’), light treatment (light and dark), and experiments (8/16
light/dark experiment 1, 16/8 light/dark experiment 2).
Results and Discussion
Gibberellin and Norflurazon Effects
Genotype
‘Flora-Dade’
initiated
germination
earlier
than
‘T4099’ (Table 6.1). ‘Flora-Dade’ had 60-75% germination two days
after sowing, while ‘T4099’ showed less than 10% at this time (Table
6.1). Germination of ‘T4099’ was improved by the gibberellin plus
norflurazon treatment (Table 6.1). Three days after sowing, seeds
treated
with
gibberellin
plus
norflurazon
had
approximately
35%
greater germination than the control while norflurazon had only 20%
germination
greater
than
the
control.
None
of
the
treatments
(norflurazon, gibberellin, or norflurazon plus gibberellin) improved
early germination percentage (3d) of ‘T4099’ to the same extent as
‘Flora-Dade’. However, there was a positive response of ‘T4099’ to
gibberellin and norflurazon, especially when applied simultaneously.
There were no significant effects of gibberellin treatment for
T50 (p<0.05) (Table 6.2). Significant effects for germination index
106
(p<0.05) and highly significant effects on hypocotyl length (HL),
however, were observed suggesting that exogenous gibberellin was more
important in determining hypocotyl elongation expression than speed
of germination. Significant effects for genotype and norflurazon for
the three variables (T50, germination index and HL) were observed
(Table 6.2), indicating that genotypes differ in speed of germination
and plant growth (as measured by hypocotyl length), and that these
variables are influenced by the presence or absence of norflurazon.
Similarly,
the
interaction
of
genotype
by
gibberellin
was
highly
significant for T50 and hypocotyl length indicating that the effect
of gibberellin on T50 and hypocotyl length was different for ‘FloraDade’ compared to ‘T4099’.
107
‘Flora-Dade’
values
than
showed
‘T4099’
lower
T50
regardless
and
of
higher
germination
gibberellin
and
index
norflurazon
treatments (P<0.05) (Table 6.3), indicating faster germination of
‘Flora-Dade’
independent
of
the
presence
or
absence
of
exogenous
gibberellin and/or norflurazon. None of the treatments (gibberellin,
norflurazon or gibberellin plus norflurazon) had an effect on T50 or
germination index. In contrast, ‘T4099’ showed different speed of
germination
for
some
treatments.
For
example,
T50
was
lower
for
‘T4099’ seeds sown in solutions containing gibberellin, norflurazon
and
gibberellin
greatest
plus
reduction
norflurazon
compared
(approximately
0.5
to
days)
the
in
control.
The
between
any
T50
treatment and control was observed for gibberellin plus norflurazon.
For
germination
highest
value
index,
(41.8)
Germination
index
gibberellin
compared
gibberellin
(P<0.05)
of
plus
followed
‘T4099’
was
to
control
the
norflurazon
not
by
showed
norflurazon
significantly
(P<0.05).
(38.4).
different
Thus,
the
for
gibberellin
alone influenced speed of germination as measured by T50, but not
germination index (Table 6.3)
Hypocotyl
gibberellin
control
compared
and
to
in
length
both
was
genotypes,
gibberellin
‘T4099’.
greater
in
and
treatment
the
the
Norflurazon
was
presence
difference
greater
resulted
in
for
of
exogenous
between
the
‘Flora-Dade’
seedlings
with
the
shortest hypocotyls in both genotypes (Table 6.3)
Gibberellin plus norflurazon enhanced the speed of germination
measured by T50 and germination index (Table 6.3). Norflurazon is an
inhibitor
of
carotenoid
synthesis
108
(Breitenbach
et
al.,
2001)
and
consequently ABA synthesis. Thus, the effect of this compound on the
germination of ‘T4099’ suggests that this line synthesizes higher
levels of ABA during imbibition. Gibberellin alone had little or no
effect on speed of germination of ‘T4099’. However, when combined
with
norflurazon,
the
germination
of
‘T4099’
was
enhanced.
This
finding suggests that gibberellins may be more effective breaking
dormancy
once
the
synthesis
of
post-imbibition
ABA
is
prevented.
Tomato seed germination involves the rupture of endosperm cap (Dahal
et al., 1997) a process prevented by ABA (Toorop et al., 2000). This
is
likely
the
norflurazon
reason
resulted
values
compared
action
is
to
in
that
norflurazon
lower
T50
gibberellin
prevented
by
and
greater
alone.
norflurazon,
or
Once
gibberellin
plus
germination
index
post-imbibition
gibberellin
ABA
activates
the
expression of hydrolytic enzymes that result in degradation of the
endosperm followed by subsequent radicle protrusion.
Hypocotyl length was greater in both genotypes when treated
with gibberellin (Table 6.3), and these results are in agreement with
those
of
Wann
(1995)
who
reported
gibberellin
resulted
in
similar
the
type.
to
observed
for
wild
plant
germination,
that
growth
However,
suggesting
exogenous
of
a
high
pigment
similar
that
application
response
germination
of
genotypes
was
not
not
only
involves gibberellins, but also ABA (Bewley and Black, 1994; Bewley,
1997).
Since
(Breitenbach
norflurazon
et
al.,
is
2001),
an
inhibitor
and
of
consequently
carotenoid
ABA,
the
synthesis
different
norflurazon effect between ‘Flora-Dade’ and ‘T4099’ suggests that ABA
produced after imbibition is greater in ‘T4099’ than in ‘Flora-Dade’,
which
culminates
in
delayed
seed
109
germination.
Tomato
fruits
with
enhanced levels of carotenoids including lycopene may produce seeds
with greater ABA levels. The modification of the carotenoid pathway
may
also
result
in
higher
ABA
synthesis
during
imbibition
as
documented for Arabidopsis seed dormancy (Debeaujon et al., 2000).
The fact that the application of norflurazon, or gibberellin plus
norflurazon did not improve speed of germination of ‘T4099’ to the
same level as ‘Flora-Dade’ seeds suggests the possibility of another
mechanism
such
as
ABA
being
produced
during
seed
development
(Debeaujon et al., 2000).
Light Treatment Effects
‘Flora-Dade’ had higher germination values than ‘T4099’ during
the first 2 to 3 days after sowing regardless of light treatment.
However, in both genotypes, germination under darkness was higher
than
under
light.
Germination
under
8/16
h
light/dark
cycles
resulted in higher germination than 16/8 h light/dark cycles, and
this effect was more marked for ‘T4099’ (Figure 6.1).
There
(genotype,
were
highly
experiment
significant
and
light)
differences
for
T50
and
for
main
germination
effects
index.
Similarly, there were significant differences for the interactions
between
genotype
by
experiment,
genotype
by
light,
light
by
experiment, and genotype by light by experiment for T50 (Table 6.4).
However, for germination index, there were significant effects only
for the interaction between light by treatment. The significance for
interactive effects on T50 indicates that the effect of light vs.
darkness, and light/dark cycles (experiment 1 vs. experiment 2) on
speed
of
‘T4099’.
germination
For
were
example,
different
the
for
difference
110
‘Flora-Dade’
between
compared
‘T4099’
to
between
darkness and 8/16 h light/dark cycle was 0.32 days and that for
‘Flora-Dade’ was 0.20 days (Table 6.5). In addition, the difference
between
darkness
and
16/8
h
light/dark
cycle
was
0.85
days
for
‘T4099’ and 0.37 for ‘Flora-Dade’.
In both genotypes, germination was higher under darkness than
under light. In addition, more light hours resulted in the lowest
speed
of
germination.
For
‘Flora-Dade’,
16/8
h
light/dark
cycles
resulted in a T50 value of 1.95 versus 1.58 for darkness and lowest
germination index 61.5 versus 70.3. For ‘T4099’, the 16/8 light/dark
cycle resulted in a T50 of 3.82 versus 2.97, and a germination index
of 28.4 versus 40.3 for darkness (Table 6.5). These results clearly
suggest
that
the
germination
of
these
genotypes
is
darkness and that this effect is higher in ‘T4099’.
also
suggest
that
‘Flora-Dade’,
and
phytochrome
A
as
‘T4099’
this
shows
a
higher
hypersensitivity
previously
observed
is
for
light
improved
These results
sensitivity
probably
an
hp
by
than
mediated
tomato
by
variety
(Shichijo et al., 2001). However, the fact that both ‘Flora-Dade’
and ‘T4099’ respond to dark treatment while only ‘T4099’ responds to
norflurazon suggests that ABA presence is more important than light
treatments in explaining the low speed of germination for seeds of
‘T4099’. Interestingly, the gibberellin plus norflurazon treatment
resulted in similar values for T50 and germination index (2.86 and
41.8,
respectively)
as
respectively)
(Tables
darkness
gibberellin
and
6.3
those
and
plus
for
6.5).
darkness
(2.97
and
This
finding
suggests
norflurazon
improve
the
40.3,
that
speed
of
germination of ‘T4099’ by a common mechanism, the regulation of ABA
biosynthesis. Regulation of ABA biosynthesis by light may be related
111
to
the
activity
of
the
enzymes
9
cis-epoxycarotenoid
dyoxygenase
(NCED) and zeaxanthin epoxidase (ZEP) mRNAs (Thomson et al., 2000b).
These
authors
different
evaluated
light/dark
the
cycles
mRNAs
and
levels
concluded
of
that
NCED
and
ZEP
at
up-regulation
of
these two enzymes may be necessary to maintain high rates of ABA
biosynthesis. On the other hand, norflurazon acting directly on the
enzyme
phytoene
resulting
in
desaturase
a
higher
inhibits
the
accumulation
desaturation
of
of
phytoene
phytoene
and
lower
accumulation of carotenoids and consequently ABA.
Acknowledgment
We thank the Ohio Agricultural Research and Development Center
Research Enhancement Competitive Grant Program (OARDC- RECG) of The
Ohio State University for partial funding of this research. We also
thank
The
National
Council
for
Science
and
Technology
of
Mexico
(CONACyT) for financial support of Gerardo Ramirez-Rosales’ doctoral
program.
Literature Cited
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9, 1055-1066.
Bewley, J.D. and Black, M. (1994). Seeds: Physiology of development
and germination. Springer-Verlag, Inc., Berlin.
Bradford, K.J., Chen, F., Cooley, M.B., Dahal, P., Downie, B.,
Fukunaga, K.K., Gee, O.H., Gurushinge, R.A., Mellia, H., Nonogaki,
H., Wu, C-T., Yang, H. and Yim, K.O. (2000). Gene expression prior
to radicle emergence in imbibed tomato seeds. In: Seed Biology:
Advances and Applications (Eds.) Black, M. Bradford, K.J. and
Vázquez-Ramos J. pp. 231-251. CABI Publishing, New York.
Breitenbach, J., Zhu, C. and Sandmann, G. (2001). Bleaching herbicide
norflurazon inhibits phytoene desaturase by competition with the
cofactors. Journal of Agriculture and Food Chemistry, 49, 5270-5272.
112
Dahal, P., Nevins, D. and Bradford, K.J. (1997). Relationship of endoß-mannanase activity and cell wall hydrolysis in tomato endosperm to
germination rates. Plant Physiology, 113, 1243-1252.
Debeaujon, I. and Koornneef, M. (2000). Gibberellin requirement for
Arabidopsis
seed
germination
is
determined
both
by
testa
characteristics and embryonic abscisic acid. Plant Physiology, 122,
415-424.
Garello, G. and Le Page-Degivry, M. T. (1999). Evidence for the role
of abscisic acid in the genetic and environmental control of dormancy
in wheat (Triticum aestivum L.). Seed Science Research, 9, 219-226.
Grappin, P., Bouinot. D., Sotta, B., Miginiac, E. and Julien, M.
(2000). Control of seed dormancy in Nicotiana plumbaginifolia: postimbibition abscisic acid synthesis imposes dormancy maintenance.
Planta, 210, 279-285.
Jarret, R.L., Sayama, H. and Tigchelaar, E.C. (1984). Pleiotropic
effects associated with the chlorophyll intensifier mutants hp and dg
in tomato. Journal of the American Society for Horticultural Science,
109, 873-878.
Sacks, E. and Francis, D. (2001). Genetic and environmental
variation for tomato flesh color in a population of modern breeding
lines. Journal of the American Society for Horticultural Science,
126, 221-226.
SAS Institute, Inc., (2001). Cary, NC
Schmitz, N., Abrams, R.S. and Kermode, A.R. (2000). Changes in
abscisic acid content and embryo sensitivity to (+)-abscisic acid
during the termination of dormancy of yellow cedar seeds. Journal of
Experimental Botany, 51, 1159-1162.
Shewmaker, C. K., Shelley, J.A., Daley, M. Colburn, S. Yank, K.
(1999). Seed-specific overexpression of phytoene synthase: increase
in carotenoids and other metabolic effects. The Plant Journal, 20,
401-402.
Shichijo, C., Katada, K., Tanaka, O. and Hashimoto,
Phytochrome A-mediated inhibition of seed germination
Planta, 213, 764-769.
T.
in
(2001).
tomato.
Toorop, P.E., Aelst, A.C.V. and Hilhorst, H.W.M. (2000). The second
step of the biphasic endosperm cap weakening that mediates tomato
(Lycopersicon esculentum) seed germination is under control of ABA.
Journal of Experimental Botany, 51, 1371-1379.
113
Thomson, A.J., Jackson, A.C., Symonds, R.C., Mulholland, B.J.
Dadswell, A.R., Blake P.S., Burbidge, A. and Taylor, L.B. (2000a).
Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene
causes over-production of abscisic acid. The Plant Journal, 23, 363374.
Thomson, A.J., Jackson, A.C., Parker, R.A., Morphet, D.R., Burbidge,
A. and Taylor, I.A. (2000b). Abscisic acid biosynthesis in tomato:
regulation
of
zeaxanthin
epoxidase
and
9-cis-epoxicarotenoid
dioxygenase mRNAs by light/dark cycles, water stress and abscisic
acid. Plant Molecular Biology, 42, 833-845.
Wann, E.V. (1995). Reduced plant growth in tomato mutants hp and dg
partially overcome by gibberellin. HortScience, 30, 379.
Zeevaart, J. (2000). Environmental control of plant development and
its relation to plant hormones. MSU-DOE Plant Research Laboratory
Annual Report. PP. 91-97.
114
Genotype/Treatment
1
Days
after sowing
2
3
4
5
Germination (%)
‘Flora-Dade’ Control
0
68
97
100
100
‘Flora-Dade’ + GA3
0
59
96
100
100
‘Flora-Dade’ + Norflurazon
0
63
96
100
100
‘Flora-Dade’ + GA3 + Norflurazon
0
72
95
100
100
‘T4099’ Control
0
1
38
87
94
‘T4099’ + GA3
0
0
41
92
98
‘T4099’ + Norflurazon
0
1
56
92
96
‘T4099’ + GA3 + Norflurazon
0
3
68
95
97
0.01
0.05
0.05
Prob.
0.01
Table 6.1. Percentage germination (radicle protrusion) of two tomato
genotypes ‘Flora-Dade’ and ‘T4099’ treated with solutions of
gibberellin, (GA3), norflurazon, and gibberellin plus norflurazon.
115
Source
T50
GI
HL
Genotype (Gen)
10.8
***z
5136.9
***
1.52
***
Gibberellin
0.01
NS
13.5
*
5.17
***
Norflurazon
0.17
***
60.1
***
1.96
**
Gen x Gibberellin
0.07
***
11.9
NS
0.20
***
Gen x Norflurazon
0.27
***
25.5
***
0.36
***
0.001
NS
23.8
*
0.00
NS
0.001
NS
5.31
NS
0.79
***
Gibberellin x
Norflurazon
Gen x Gibberellin x
Norflurazon
Table 6.2. Means squares and significance for time to fifty percent
germination (T50), germination index (GI) and hypocotyl length (HL)
of two tomato genotypes ‘Flora-Dade’ and ‘T4099’. Seeds were treated
with gibberellin (GA3), norflurazon, or gibberellin plus norflurazon.
Germination was recorded daily when seeds showed radicle protrusion.
*, **, ***, NS= significant at the 0.05, 0.01, 0.001 probability
levels and not significant, respectively.
116
Treatment
T50
GI
HL
(Days)
(cm)
‘Flora-Dade’ + GA3+ Nor
1.96
aZ
65.0
a
3.6
b
‘Flora-Dade’ + GA3
1.95
a
61.5
a
4.7
a
‘Flora-Dade’ + Nor
1.93
a
62.4
a
3.3
c
‘Flora-Dade’ Control
1.87
a
64.0
a
3.7
b
‘T4099’ + GA3+ Nor
2.86
b
41.8
b
3.9
b
‘T4099’ + GA3
3.18
d
36.4
cd
3.9
b
‘T4099’ + Nor
2.99
c
38.4
c
2.6
d
‘T4099’ control
3.32
e
34.8
d
3.2
c
Table 6.3.
Time to fifty
index (GI) and hypocotyl
(‘Flora-Dade’ and ‘T4099’)
(GA3), norflurazon (Nor), or
Z
Means within column with
different at a=0.05.
percent germination (T50), germination
length (HL) of two tomato genotypes
treated with solutions of gibberellin
gibberellin plus norflurazon
the same letter are not significantly
117
Source
T50
GI
(Days)
Genotype (Gen)
14.26
***
5706.0
***
Experiment
0.34
***
102.92
***
Light
1.12
***
321.05
***
Gen x experiment
0.04
*
8.71
NS
Gen x light
0.13
***
3.76
NS
Light x experiment
0.066
*
23.01
*
Gen x light x experiment
0.048
*
0.49
NS
Table 6.4. Means squares and significance for time to fifty percent
germination (T50) and germination Index (GI) of two tomato genotypes
‘Flora-Dade’ and ‘T4099’. Seeds were germinated in darkness or under
8/16 h light/dark cycles (Experiment 1) and 16/8 h light/dark cycles
(Experiment 2). Germination was recorded daily when seeds showed
radicle protrusion.
*, **, ***, NS significant at the 0.05, 0.01, 0.001 probability
levels and significant, respectively.
118
Genotype
Treatment
T50
‘Flora-Dade’
8/16 L/D
1.78
‘Flora-Dade’
16/8 L/D
1.95
‘Flora-Dade’
Darkness
‘T4099’
GI
b
Z
66.1
b
c
61.5
c
1.58
a
70.3
a
8/16 L/D
3.29
e
36.0
e
‘T4099’
16/8 L/D
3.82
f
28.4
f
‘T4099’
Darkness
2.97
d
40.3
d
Table 6.5. Time to fifty percent germination (T50), germination
index (GI) of two tomato genotypes: ‘Flora-Dade’ and ‘T4099’. Seeds
were germinated under darkness or under 8/16 h light/dark cycles
(Experiment 1) and 16/8 h light/dark cycles (Experiment 2).
Germination was recorded daily when seeds showed radicle protrusion
Z
Means with the same letter are not significantly different at a=
0.05.
119
A
Germination (%)
100
80
'Flora-Dade' 16/8
L/D
60
'Flora-Dade' 8/16
L/D
40
'Flora-Dade' dark
20
0
1
2
3
4
5
6
Days after sowing
Germination (%)
100
80
60
'T4099' 16/8 L/D
'T4099' 8/16 L/D
'T4099' dark
40
20
0
1
2
3
4
5
6
Days after sowing
Figure 6.1. Percentage germination (radicle protrusion) of two
tomato genotypes ‘Flora-Dade’ (A) and ‘T4099’ (B). Seeds were
germinated under darkness or under 8/16 h light/dark cycles and 16/8
h light/dark cycles.
120
CHAPTER 7
7. CONCLUDING REMARKS AND FUTURE STUDIES
The high pigment genes
hp-1, hp-2 and dg may be desirable for
human nutrition because they result in tomato varieties with high
carotenoid
content.
However,
they
also
affect
overall
plant
development and seed quality. Results of this research indicate that
genotypes with increased lycopene due to the combined effects of dg
ogc may result in low quality planting material as judged by lower
antioxidant capacity and delayed germination. This study found that
delayed germination was not caused by the gradual accumulation of
lycopene that accompanies fruit development because fruits harvested
at the mature green and breaker stages did not produce seeds with
greater speed of germination. Interestingly, seed from genotype ‘FG218’
dg
ogc result
in
speed
of
germination
comparable
to
that
of
normal lycopene varieties suggesting that plant genetic background can
minimize
studies
the
negative
should
accumulation
genotypes.
pattern
of
include
during
It
effects
would
a
fruit
be
carotenoid
of
the
high
comprehensive
and
seed
interesting
precursors
pigment
evaluation
development
to
such
identify
as
genes.
of
in
carotenoid
high
the
phytoene
Future
pigment
accumulation
and
other
carotenoids such as ß-carotene violaxanthin and zeaxanthin in tomato
fruits and seeds of high pigment genotypes compared to wild types.
121
The effect of norflurazon on T50 and germination index indicates
that tomato genotypes carrying the dg gene may produce higher levels
of ABA in seeds, characterized as delayed germination and dormancy.
Future
studies
should
evaluate
ABA
levels
during
fruit
and
seed
development using GC-MS to quantitatively determine the effect of high
pigment
genotypes
on
ABA
metabolism.
In
addition,
future
studies
should examine the effect of different light treatments on the postimbibition synthesis of ABA. Literature reports have shown that the
synthesis of ABA is regulated by light and mediated by the enzymes 9
cis-epoxycarotenoid dyoxygenase (NCED) and zeaxanthin epoxidase (ZEP).
Future studies should evaluate the activity of these enzymes in high
pigment tomato genotypes versus wild types.
Although information exists that seed deterioration is closely
linked
to
exists
that
longevity
a
in
decrease
in antioxidant mechanisms, little information
evaluates
a
wide
Chemiluminescence
endogenous
range
(PCL)
and
of
antioxidant
tomato
Trolox
capacity
genotypes.
Equivalent
The
and
Photo
Antioxidant
seed
Induced
Capacity
(TEAC) methodologies were shown to be good scientific approaches to
determine
the
antioxidant
capacity
of
tomato
seeds.
It
would
be
valuable to use both of these techniques to determine antioxidant
capacity during seed development and storage.
122
Future
phenolic
studies
compounds
need
or
to
evaluate
carotenoid
what
content)
factors
are
(e.g.vitamins,
responsible
for
antioxidant activity. A study of total antioxidant activity during
fruit and seed development- including evaluation of vitamins, phenolic
compounds and carotenoid- in tomato fruits and seeds will identify
whether
fruits
or
seeds
are
greater
hypothesized in this study.
123
sinks
for
antioxidants
as
BIBLIOGRAPHY
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132
APPENDIX A
P-Values for Winter 2000 Study
Variable
MG
BR
PB
RM
OR
Germination Index
0.005
0.001
0.003
0.001
0.001
Germination
0.025
0.04
0.19
0.004
0.001
SSAA
0.590
0.590
0.070
0.003
0.005
Probability values for germination index, germination, SSAA of the
genotypes ‘OH8245’ and ‘T4099’ dg ogc harvested at mature green (MG),
breaker (BR), pink breaker (PB), red mature (RM), and overripe (OR).
Winter 2000.
133
APPENDIX B
Effect of Cluster Position
Maturity
Cluster
5-day count (%)
14-day count (%)
Breaker
1
16
99
Breaker
2
1
99
Breaker
3
1
100
Red Mature
1
4
95
Red Mature
2
3
100
Red Mature
3
4
99
Effect
of
cluster
position
on
percentage
of
normal
seedlings
of
genotype T4099 dg ogc harvested at two fruit maturity stages. Seeds
were
germinated
represents
the
in
a
average
germination
of
4
chamber
replications
replication (n=7).
134
at
and
24
o
C.
three
Each
value
samples
by
APPENDIX C
Weight of One-hundred Seeds
Genotype
Maturity
Seed Weight (mg)
‘T4099’
MG
340.0 + 5.1
BR
345.7 + 7.5
PB
344.8 + 10.5
RM
354.4 + 7.2
OR
365.1 + 6.9
MG
285.1 + 6.8
BR
300.8 + 8.9
PB
315.3 + 8.0
RM
314.0 + 13.0
OR
315.7 + 5.8
‘OH8245’
One hundred seed weight of genotypes ‘T4099’ dg ogc and ‘OH8245’
harvested at five different fruit maturity stages: mature green
(MG),
breaker
(BR),
pink
breaker
overripe (OR). Winter 2000.
135
(PB),
red
mature
(RM),
and