CAROTENOID AND DEVELOPMENT EFFECTS ON AND VIGOR OF

TOMATO (Lycopersicon esculentum Mill.) .

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 Approved by

Dr. Mark A. Bennett, Adviser ______

Dr. Miller B. McDonald, Adviser Adviser

Dr. David M. Francis ______

Dr. Grady W. Chism Co-Adviser

Graduate Program in

Horticulture and Crop Science

ABSTRACT

The tomato crop is an important source of such as ß- and . These carotenoids play important roles in human health and nutrition. Consequently, humans benefit from the development of tomatoes with enhanced content. High pigment , 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 caused by these genes have limited their use as homozygotes in commercial varieties. The effects of these genes on plant development, including slow 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. This study evaluated the effect of fruit development on seed quality of tomato varieties with different concentrations of carotenoids under field and greenhouse conditions.

Gibberellin and norflurazon (an inhibitor of carotenoid synthesis) effects on speed of germination of varieties with different concentrations of carotenoids were also evaluated. and seeds of these varieties were assayed for total antioxidant capacity using the

Photo-induced Chemiluminescense (PCL) and the Total Equivalent

ii 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 germinated faster than the control indicating that ABA is involved in the low speed of germination of high lycopene genotypes with the dg as an expression of . 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 chemicals and equipment for seed quality evaluation.

Thanks to Soja Sekharan, Wenting Li, and Elaine 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 for providing me financial support through this dissertation process.

vi 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., Esparza- Martinez-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 ‘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

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 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...... 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-ethylbenzothiazoline- 6-sulfocin acid) diammonium salt (ABTS) was incubated with Trolox and the reduction in absorbance was determined spectro- photometrically...... 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 6- hydroxy-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 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 fruit color (i.e. yellow, orange, and red) due to different structures 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. However, increasing lycopene content by traditional and molecular biology approaches might alter other metabolic pathways and cause abnormalities in important physiological processes. For example, delayed germination and reduced plant growth have been reported in the high 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 there is evidence that high levels of carotenoids modify the synthesis of essential germination promoters such as (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. Thus, if the elevated synthesis of lycopene characteristic of 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 lycopene may be desirable for human health and nutrition due to the correlation between lycopene and reduced prostate 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 rich in antioxidants produce seeds with enhanced antioxidant levels that increase the ability to 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

b-carotene and lycopene, although other carotenoids such as

and have also received considerable attention because of

their antioxidant properties (Clinton et al., 1998; Volker et al.,

2002). In addition, and 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 b-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 . 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 by increasing levels of lycopene and other carotenoids; other metabolic pathway abnormalities are caused in important physiological processes (Croteau et al., 2000). For instance, Fray et al. (1995) reported that elevated levels of carotenoids resulted in shorter tomato plants because metabolites from the gibberellin pathway were re-directed to the carotenoid pathway. Consistent with this hypothesis, Fraser et al. (1995) reported higher content of gibberellins in plants deficient in carotenoid synthesis.

Wann (1995) observed that exogenous applications of gibberellins to high lycopene genotypes restored plant growth to levels close to those of the wild type. He claimed that high pigment lines were deficient in the synthesis of gibberellins. This observation, however, provided no conclusive evidence that gibberellins were also responsible for the low speed of germination associated with high pigment genotypes. Recently, Fraser et al.

6 (2001) noted that fruit specific expression of the synthase gene from bacteria inserted into a tomato plant resulted in non- deleterious 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 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

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 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 ABA is a ubiquitous hormone that plays a number of roles in plant development (e.g., stimulates stomatal closure, seed reserve accumulation and ) (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 biosynthesis pathway and ABA is synthesized indirectly via carotenoids, then increasing 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 (see figure 1.2) (Cutler and Krochko, 1999; Milborrow,

2001). As a result, it might be possible that the levels of

8 germination inhibitors such as ABA are over-produced in tomato

genotypes with elevated levels of carotenoids. Whether 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 vp5 mutant) (Cutler and Krochko, 1999;

Crozier et al., 2000). This observation is consistent with low

levels of ABA being necessary in the early stages of seed

maturation.

Alterations in carotenoid byiosynthesis pathway have been

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 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 endogenous levels of gibberellins lead to precocious germination.

1 0 It is interesting to note that lycopene and b-carotene accumulate late in fruit maturation, and continue accumulating even after (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 quality and fruit carotenoid content. This information is important because the tomato seed industry is currently developing genotypes differing in fruit color due 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 seed development. Those studies provided valuable

information and concepts. For example, Berry and Bewley (1991)

demonstrated that 30-day old tomato seeds had the 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 subsequent study, Berry and Bewley (1992) presented information on the effect of ABA and osmotic potential during development on the germination of tomato seeds. Precocious germination was prevented by water potential more 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 mentioned previously generated important information, but because they used dap as an 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 maturation stages that resulted in different seed quality levels. As a result, they decided to 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 (Iowa State University, 1996) and soybean (Glycine max L.

Merr.) production (Iowa State University, 1993). Thus, there is precendence for using physiological criteria in 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 the time when seeds acquire maximum seed quality. Maximum seed quality, also known as a physiological maturity (Harrington, 1972; Delouche, 1980; Powell and Matthews,

1984), coincided with the formation of the black layer ( 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 provided about the type and cause of deterioration associated with overripe fruits; however, it is possible that the elevated accumulation of lycopene in overripe fruits will result in an over production of ABA that results in delayed seed germination. It is also possible that the elevated synthesis of lycopene that results from high pigment 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 by overripe fruit maturity (Liu et al., 1996a).

Although this may be a result of altered fruit and 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 germination and vigor. Nevertheless, these authors determined seed germination under laboratory and 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. For instance, genetic differences among genotypes (Doijode, 2001), environmental and biological 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 literature review of free radical damage to human organs and resultant human diseases. He also reported 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, Kurilich et al. (1998) evaluated the levels of antioxidants such as carotenoid, tocopherol, and ascorbate in cabbage (Brassica oleracea var. capitata), 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 varieties that enhance the antioxidant potential of food. They found substantial variability within and between varieties. Kale had the highest levels of these compounds. In a similar study, Conner and Ng (1998) evaluated changes in lipid peroxidation and antioxidant status in ripening muskmelon fruits.

Their objectives were to correlate the levels of antioxidants with the developmental stage of muskmelon. They found valuable information that can be used for breeding programs aimed at increasing fruit storage potential. Thus, studies are being conducted on the potential of antioxidants as a means to increase

19 storage life or to enhance the antioxidant potential of food.

However, little research has examined antioxidants in 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 radicals and free radical scavengers on seed deterioration (Powell, 1986; Powell and Harman, 1985). The discrepancies between these reports may be a result of the methods of measuring free radical attack and antioxidant mechanisms. In addition, the evaluation of antioxidants on seed aging and deterioration has included individual antioxidants such 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 factors on seed deterioration and longevity has been studied. For instance, Sung and Jeng (1994) observed a reduction in activity levels of superoxide 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 contributions of previous researchers suggest that

enzymatic free radical protection mechanisms may increase seed

longevity and decrease seed deterioration. However, total antioxidant

capacity involves mechanisms and compounds other than enzymes (i.e.,

, 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 ideal for maximum desiccation tolerance and increased seed longevity.

23 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 of endogenous levels of ABA during the early germination process, and (4) determine whether genotypes that produce fruits with super-elevated levels of lycopene result in seeds with higher total antioxidant capacity and how 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.

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31 geranylgeranyl IPP Tocopherols diphosphate synthase Chlorophyll

(Ggps) Gibberellins GGPP Phytoene synthase (Psy)

Phytoene

Phytoene desaturase (Pds)

?-Carotene Carotene desaturase (Zds) Lycopene Lycopene Lycopene epsilon beta cyclase cyclase (crtL-e) (crtL-b)

d-Carotene ? -Carotene Lycopene beta Lycopene cyclase beta (crtL-b) cyclase a-Carotene (crtL-b) ß -Carotene Ring

hydroxylases Ring beta and epsilon hydroxylase (crt-R-b) beta (crt -R-e) (crtR-B-2) Lutein

Zeaxanthin

Zeaxanthin

epoxidase (ZEP)

AntheraxanthinViolaxanthin

9-cis-epoxycarotenoid

dioxygenase (NCED)

Neoxanthin

Xanthin ABA

Figure 1.1. The carotenoid pathway

32

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 hp-

2) 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. This study evaluated whether harvesting early during fruit and seed maturation would result in 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 of the gradual accumulation of lycopene in fruit tissues and that speed of germination of dg genotypes 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, increasing carotenoid and lycopene content using traditional breeding techniques and molecular manipulation might also alter other metabolic pathways and cause undesirable abnormalities in plant development (Croteau et al., 2000). For example, delayed germination and reduced plant growth have 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 (2-

3X) than normal tomato genotypes (Jarret et al., 1984; Wann et al.,

1985; Berry and Uddin, 1991). In addition, high levels of

35 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. As a result, additional research is needed to determine whether a direct relationship exists between 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, fruit development can be influenced by environmental conditions making it 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 tomato genotypes differing in seed vigor and 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 8-

10 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 seeds were fermented in a beaker at room temperature (~24 0C) 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 O C until evaluation.

In summer 2001, the recurrent parent ‘Flora-Dade’ and the experimental line ‘T4099’ dg ogc were planted in 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 the winter 2000 study, seed quality was evaluated by standard germination, germination index and the saturated salt accelerated aging (SSAA) tests (Jianhua and McDonald, 1996). The germination index (GI) was calculated for each treatment by 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 cases, seeds were recorded as

39 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 germination test, which was conducted on dry and fresh seeds (seeds extracted after the fermentation process 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 differences between genotypes at each stage of fruit maturity (Hinkelmann and Kempthorne, 1994). Data expressed in percentage were transformed using arcsine square (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 significant effects of genotype and interaction of genotype by maturity for GI were observed (Table 2.1). The main

40 effect of genotype indicated a clear difference in seed quality between ‘OH8245’ and ‘T4099’. The genotype x 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 that ‘T4099’ seed was more susceptible to artificial deterioration, especially seeds from overripe 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 overripe ‘T4099’ fruits had 85% and 63% germination before and after the treatment, respectively, while 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 and accumulation, the question of whether harvesting ‘T4099’ in the early stages of fruit 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). This finding suggests that ‘T4099’ might acquire maximum germination capacity in early fruit maturity stages and that red mature and 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 2.2). Fruits were classified based on color and firmness and since fruits of ‘T4099’ possessed greater firmness

(Wann, 1996), seeds within the late red mature stage might have initiated deterioration with no visual symptoms in the fruit as reported in previous studies for overripe fruits (Valdes and Gray,

1998; Demir and Samit, 2001).

In the summer study, the recurrent parent ‘Flora-Dade’ replaced ‘OH8245’ to minimize the effect of different genetic background between ‘OH8245’ and ‘T4099’. We also evaluated whether planting seeds without desiccation would affect the speed of germination of ‘T4099’. In addition, normal seedling and 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 higher germination than

‘T4099’ in all fruit 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. This difference at 5 d between ‘Flora-Dade’ and ‘T4099’, which is more obvious for normal seedlings, was consistent with the low GI found in the winter study and demonstrates the low seed vigor of ‘T4099’. Similarly, for fresh seeds, the difference in percentage germination was greater for normal seedlings compared to radicle protrusion. Based on the germination for fresh and dry seeds, drying affected seeds from

45 ‘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 normal seedlings. This indicates that the greatest difference between these two genotypes is limited to seedling development rather than germination (radicle 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 and because ABA is a

47 derivative from carotenoid precursors (Zeevaart et al., 2000); it is also possible that dg genotypes produce greater levels of ABA causing delayed germination. Thus, both decreased gibberellins and/or increased ABA may be involved in the slow 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.

Literature Cited

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Berry, T. and Bewley, D.J. (1991). Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation. Planta, 186, 27-34.

Berry, T. and Bewley, D.J. (1992) A role for the surrounding fruit tissue in preventing the germination of tomato (Lycopersicon esculentum) seeds. Plant Physiology, 100, 951-957.

48 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.

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.

Croteau, R., Kutchan, T.M., Lewis, G.N., Crozier, A., Kamiya Y., Bishop, G. and Yokota, T. (2000). Natural products (Secondary metabolites). In: Biochemistry and Molecular Biology of Plants. (Eds.) Buchanan, B.B., Gruissem, W., Jones, R.L. pp. 1250-1316. American Society of Plant Physiologists, Rockville, MA.

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.

Fray, R.G., Wallace, A., Fraser, P.D., Valero, D., Hedden, P., Bramley, P.M. and Greierson, D. (1995). Constitutive expression of a fruit phytoene synthase gene in transgenic tomatoes causes dwarfism by redirecting metabolites from the gibberellin pathway. Plant Journal, 8, 693-701.

Giovanelli, G., Lavelli, V., Peri, C. and Nobili, S. (1999). Variation in antioxidant components of tomato during vine and post harvest ripening. Journal of Science of Food and Agriculture, 79, 1583-1588.

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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 for seed testing. Seed Science and Technology, 27, supplement.

49 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.

SAS Institute, Inc., (2001). Cary, NC

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Van Den Berg, H., Faulks, R., Granado, H.F., Hirschberg, J., Olmedia, B., Sandmann, G., Southon, S. and Stahl, W. (2000). The potential for the improvement of carotenoid levels in foods and the likely systemic effects. Journal of the Science of Food and Agriculture, 80, 880-912.

Wann, E.V., Jourdain, E.L. Pressey, R. and Lyon, B.G. (1985). Effect of mutant genotypes hp ogc and dg ogc on tomato fruit quality. Journal of the American Society for Horticultural Science, 110, 212- 215.

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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 BR PB

GI FG SSAA GI FG 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 0.04 0.59 0.01 0.19 0.07

RM OR

GI FG SSAA GI FG SSAA

'OH8245' 19.5 99.2 97.5 18.9 96.2 95.0

'T4099' 12.2 90.5 75.0 11.0 85.2 63.0

Prob. 0.01 0.01 0.01 0.01 0.01 0.01

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’ 73.2 a 93.0 a 76.0 a 97.0 a 59.0 b

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’ 15 a 35.5 az 75.5 a 92.0 a 92.7 a

‘T4099’ 0 b 3.75 b 21.2 b 10.0 b 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’ 55.2 b 61.2 b 83.0 b 83.0 b 85.3 a

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’ 0 1.0 b 13.7 b 4.9 b 24.0 b

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 study evaluated the effect of fruit maturity and production environment on germination and vigor of four tomato genotypes varying in fruit color and lycopene content. 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 OC for 48 h. Seed quality was evaluated as percentage germination 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 stage was reduced in genotypes ‘Flora-Dade’,

‘OH9242’ (ogc) and ‘FG-218’ (dg ogc). Variance component analysis indicated that fruit maturity 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 maturity stages. These results suggest that harvesting tomato fruits from the breaker to red mature 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 Ellis, 1992; Liu et al., 1996). However, most of the studies on fruit development have evaluated one genotype and a single environment. Valdes and Gray (1998) evaluated the effect of fruit maturity on seed quality of four tomato genotypes produced under different environments. They observed differences among genotypes for the effect of fruit maturity, especially at the overripe fruit maturity stage. However, they failed to provide 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 seed industry is currently developing tomato genotypes with high carotenoid content, including lycopene, that produce fruits with higher red color intensity compared to 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 conditions. Although seed vigor is genetically determined, it is also highly modified by the 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 these factors in seedling establishment has not been evaluated. The objective of this study was to determine the influence of fruit maturity, genetic and production 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 lycopene, but are paired by

57 pedigree. Variety ‘T4099’ was described by Wann (1996), and was developed by crossing the dg ogc genes into a ‘Flora-Dade’ background. The two genotypes share 97% of genes by descendent.

‘OH9242’ is a crimson (ogc) breeding line with moderately elevated levels of Lycopene (Francis, in progress) and excellent color.

Experimental line ‘FG-218’ was developed by ‘OH9242’ and ‘T4099’ followed by two backcrosses with selection for dg. ‘OH9242’ and ‘FG-

218’ 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 OC) to approximately 8% seed moisture content (fresh weight basis).

58 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 analysis was conducted as a combined experiment including two replications by location (Fremont and Wooster) and four genotypes. Genotypes were considered as fixed effects while replications and locations were random effects. The analysis was conducted using the SAS procedure GLM (SAS Institute, 2001) as repeated measures to determine statistical differences among genotypes at a given fruit maturity stage (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 than ‘Flora-Dade’,

‘OH9242’ogc, and ‘FG-218’ dg ogc (Table 3.2). 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, in this experiment, germination of red mature and overripe fruits was not affected. It is possible that 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 carry the dg gene, which can result in slow germination (Jarret et al., 1984; Berry and Uddin, 1991). 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. This genotype difference effect on GI versus

germination percentage is possibly a result of the low 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 x genotype x maturity and maturity explained

approximately 86% of the variance for GI (Table 3.1). Thus,

61 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). Seeds of tomato (Lycopersicon esculentum Mill.) which develop in a fully hydrated environment in the fruit switch from a developmental to a germinative mode without a requirement for desiccation. Planta, 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 for seed testing. Seed Science and Technology, 27, supplement.

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 and procedures of statistics. P. 666, McGraw-Hill, New York.

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 Var Sources P-Value (%) 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

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 BR RM OR Genotypes GI GP (%) GI GP (%) GI 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) with fruit maturation remain poorly understood. This study examined the effect of fruit maturity on tomato seed quality after the red mature stage. The variety ‘Flora-

Dade’ 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 and fruit firmness. Seeds were extracted by fermentation at 24 OC for 48 h. Seed quality was assayed using the standard germination 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 effects on tomato seed quality have been extensively studied. Seeds extracted from red tomato fruits have maximum seed 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 quality of overripe fruits. In addition it is still unclear when fruits initiate substantial changes 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 ‘Flora-

Dade’ 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 and harvested at 55, 60, 70, 80 and 90 days after pollination (dap). Fruit age was determined based on dap, but visual changes in fruit firmness, color, lycopene content, and degree of fruit deterioration were also evaluated.

68

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 (~24 0C) for 48 h. Seeds were then rinsed and surface dried at room temperature to approximately 8% moisture content (dry weight basis). 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 chloride as the saturated salt solution to achieve the desired relative humidity. After the SSAA treatment, seeds were evaluated for standard germination as described above.

69 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 day old tomato fruits were red mature and firm while 60-day-old fruits, although still firm, were 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, red color remained unchanged after the red mature stage, which was verified by lycopene content (Table 4.1). Thus, fruit firmness, especially after fruits reach color, appears a 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 mother plant. Tomato seeds do not germinate in the mother 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 to test the dormancy model in

72 overripe tomato fruits. This study demonstrates, however, that overripe tomato fruits result in seeds with low germination, although this effect may be an indication of 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 fruits are more susceptible to fermentation longer than 24 h and might have lower germination percentage (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 for seed testing. Seed Science and Technology, 27, supplement.

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 µ-carotene, B-carotene, lycopene and Zeaxanthin. Journal of Food and Agricultural Food Chemistry 50, 221- 226.

7 4

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’, which produces elevated levels of lycopene and ascorbic acid, and the recurrent parent ‘Flora-Dade’ were 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 number (Jarret et al.,

1984; Sacks and Francis, 2001). The conversion of 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 super- elevated 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 have reported that levels of free radicals are at least partly responsible for differences in seed longevity

(Thapliyal and Connor 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 radical scavengers therefore may increase seed deterioration. Thus, seeds with enhanced levels of 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 of the high lycopene line ‘T4099’ dg ogc and its

recurrent parent, the open pollinated variety ‘Flora-Dade’, 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 0C 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 oC and 70% RH

until analysis for antioxidant capacity.

80

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 0C 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 lipid- soluble 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 oC until used. For water-soluble antioxidant extraction, 20 ml of pure methanol were added to the aqueous phase. Samples were then shaken at 30 oC for 30 min and filtered using a vacuum with a square funnel and Whatman paper no.

1. The filtrate was then stored at –20 oC 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 ml 6-hydroxy-2,5,7,8-

tetramethylchroman 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 ml) 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 ml of the methanol (66%) solution containing water- soluble 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 presence of antioxidants, which permits their quantification. The quantification is performed using a standard compound such as trolox (lipid soluble antioxidants) or ascorbic acid (water soluble antioxidants). In this study, the antioxidant capacity was evaluated using the Photochem® instrument and ACL

(antioxidant capacity of lipid soluble) and ACW (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 ml) was mixed with

2.3 ml of ACL-1, 200 ml of ACL-2 and 25 ml 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 later adjusted for seed concentration.

84 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 and standard were injected into the Photochem® instrument to obtain the antioxidant capacity, 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 sub-samples of 10 fruits per replication. Total antioxidant capacity for fruit tissue was evaluated with 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 water- soluble 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’ for lipid and water-soluble antioxidants, respectively (Table 5.8). The PCL method distinguishes the antioxidant capacity of seeds of these two genotypes in both water- and 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 is important to emphasize that fruit water-soluble antioxidants were not assayed in this study. The 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 correlation exists between ascorbic acid content and water soluble antioxidant capacity (Popov and Lewin, 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 TEAC results demonstrate that ‘T4099’ fruit tissue had higher levels of antioxidants while seeds from 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 et al., 2000). In addition, a decrease in enzymatic antioxidant protection has been associated with lipid peroxidation in cocoa (Theobroma cacao L.) seeds (Li and Sun, 1999) and lipid peroxidation is considered as a cause of seed deterioration (Chiu et al., 1995; McDonald, 1999). In 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 is hypothesized that the selection of tomatoes with elevated lycopene content, although desirable for human nutrition and health, may result in seeds with lower antioxidant levels. This may result in lower quality planting seed compared to wild type genotypes as previously reported (Ramirez-Rosales, 2002 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.

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90 Miller, N.J., Sampson, J., Candelas, L.P., Bramley, P.M. and Rice- Evans, C.A. (1996). Antioxidant activities of carotenes and . FEBS Letter, 384, 240-242.

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Pellegrini, N., Re, R., Yang, M. and Rice-Evans, C. (1999). Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying 2,2’-azinobis(3- ethylenebenzothiazoline-6sulfonic acid radical cation decolorization assay. In: Methods of Enzymology, Oxidants and Antioxidants. (Ed.) Packer, L. Vol 300, pp. 379-389. Academic Press, San Diego.

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Sacks, E. and Francis, D. (2001). Genetic and environmental variation for tomato flesh color in a population of modern lines. Journal of the American Society for Horticultural Science, 126, 221- 226.

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91 Volker, B., Puspitasari-Nienaber, N.L., Ferruzzi, M. and Schwartz, S.J. (2002). Trolox equivalent antioxidant capacity of different geometrical isomers of µ-carotene, b-carotene, lycopene and zeaxanthin. Journal of Food and Agricultural Food Chemistry, 50, 221-226.

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92 Source DF MS F CALC P

Genotype 1 146.66 99.80 < 0.0001

Replications 2 2.9213 2.64 0.112

Sample 2 2.1944 1.98 0.1803

Error 16 23.51

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

LSD (0.05) 1.20 44.8

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 P

Genotype 1 599545.7 219.42 < 0.0001

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

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 PCL

Genotype ACW 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 ‘Flora- Dade’ and ‘T4099’ tomato genotypes.

96 250

200 y = 0.0381x - 39.148 R 2 = 0.8532 150

TEAC 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 A y = 2.4779x + 0.0108 2 0.30 R = 0.9921 0.20 0.10

Absorbance 0.00

(ABTS- Trolox) 0 0.025 0.05 0.075 0.1 Trolox concentration( mm/L)

B y = 2.6643x + 0.0666 R2 = 0.9997 0.30 0.20 0.10

Absorbance 0.00 (ABTS-Trolox) 0 0.025 0.05 0.075 0.1 Trolox concentration (mmol/L)

C y = 2.2024x + 0.1913 R2 = 0.9918 0.4 0.3 0.2 0.1 - Trolox) 0

Absorbance (ABTS 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 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-ethylbenzothiazoline-6-sulfocin acid) diammonium salt (ABTS) was incubated with Trolox and the reduction in absorbance was determined spectro-photometrically.

98 A y = 0.2098x + 0.3772 R2 = 0.9828 1.00 0.80 0.60 0.40 0.20 0.00 Inhibition (%) 0 0.5 1 1.5 2 2.5 Trolox (nmol/L)

y = 50.131x + 5.0745 B 2 R = 0.9758 200

150

100

50

0 Lag phase (seconds) 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 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 6-hydroxy-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 CHAPTER 6

Gibberellin plus Norflurazon Enhance the Germination of Dark Green Tomato (Lycopersicon esculentum Mill.) Genotypes

Summary

Tomato (Lycopersicon esculentum Mill.) varieties rich in lycopene are desirable for human nutrition because of 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 experiment, tomato seeds were germinated under different light conditions. Germination was recorded daily as radicle protrusion. Speed of germination was evaluated as time 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 available in improved tomato germplasm. However, these genes cause undesirable pleiotropic effects such as reduced plant growth, slow germination and brittle stems (Jarret 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 reduced plant growth of dg and hp genotypes has been associated with low endogenous gibberellin content (Wann, 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 et al., 2000a) that resulted in delayed seed germination. Even though these are examples of the relationship between 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 (Debeaujon and Koornneeef,

2000), Nicotiana plumbaginifolia (Grappin et al., 2000), wheat

(Triticum aestivum L.) (Garello and Le Page-Degivry, 1999) yellow cedar (Chamaecyparis nootkatensis) (Schmitz et al., 2000), and tomato

(Thomson et al., 2000b). Since dg and hp genotypes produce elevated tomato fruit carotenoid content, it is possible that not only decreased gibberellins but also increased ABA levels 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 dormancy with carotenoid inhibitors demonstrates the hypothesis that ABA is also synthesized de novo in seeds during imbibition. Norflurazon has been successfully used in transgenic tomato lines resulting in lower ABA content and decreased seed dormancy (Thompson et al., 2000b). But, it has not been used to date in tomato genotypes with increased lycopene.

The delayed speed of germination of an hp genotype is also caused by higher sensitivity to light mediated by 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 ‘Flora-

Dade’.

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

-4 (control), 10 M gibberellin (GA3) (Aldrich, Milwaukee, WI), 20 mg/L

of norflurazon (Supelco, Bellefonte, PA) or gibberellin plus

norflurazon. 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, gibberellin and norflurazon. All main factors had two levels: ‘Flora-Dade’ and ‘T4099’ for genotype, and presence 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 ‘Flora-

Dade’ compared to ‘T4099’.

107

‘Flora-Dade’ showed lower T50 and higher germination index values than ‘T4099’ regardless of gibberellin and 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 plus norflurazon compared to the control. The greatest reduction (approximately 0.5 days) in T50 between any treatment and control was observed for gibberellin plus norflurazon.

For germination index, gibberellin plus norflurazon showed the highest value (41.8) (P<0.05) followed by norflurazon (38.4).

Germination index of ‘T4099’ was not significantly different for gibberellin compared to the control (P<0.05). Thus, gibberellin alone influenced speed of germination as measured by T50, but not germination index (Table 6.3)

Hypocotyl length was greater in the presence of exogenous gibberellin in both genotypes, and the difference between the control and gibberellin treatment was greater for ‘Flora-Dade’ compared to ‘T4099’. Norflurazon resulted in 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 (Breitenbach et al., 2001) and

108 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 reason that norflurazon or gibberellin plus norflurazon resulted in lower T50 and greater germination index values compared to gibberellin alone. Once post-imbibition ABA action is prevented by norflurazon, gibberellin 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 that exogenous application of

gibberellin resulted in plant growth of high pigment genotypes

similar to the wild type. However, a similar response was not

observed for germination, suggesting that germination not only

involves gibberellins, but also ABA (Bewley and Black, 1994; Bewley,

1997). Since norflurazon is an inhibitor of carotenoid synthesis

(Breitenbach et al., 2001), and consequently ABA, the 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 germination. Tomato fruits with

109 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 were highly significant differences for main effects

(genotype, experiment and light) for T50 and germination 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 germination were different for ‘Flora-Dade’ compared to

‘T4099’. For example, the difference between ‘T4099’ between

110 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 improved by darkness and that this effect is higher in ‘T4099’. These results also suggest that ‘T4099’ shows a higher light sensitivity than

‘Flora-Dade’, and this hypersensitivity is probably mediated by phytochrome A as previously observed for an hp tomato 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 those for darkness (2.97 and 40.3, respectively) (Tables 6.3 and 6.5). This finding suggests that darkness and gibberellin plus norflurazon improve the 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 evaluated the mRNAs levels of NCED and ZEP at different light/dark cycles and concluded that 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 desaturase inhibits the desaturation of phytoene resulting in a higher accumulation of 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.

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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 endo- ß-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.

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Grappin, P., Bouinot. D., Sotta, B., Miginiac, E. and Julien, M. (2000). Control of seed dormancy in Nicotiana plumbaginifolia: post- imbibition abscisic acid synthesis imposes dormancy maintenance. Planta, 210, 279-285.

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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.

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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, 363- 374.

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114

Genotype/Treatment Days after sowing

1 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

Prob. 0.01 0.01 0.05 0.05

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 ***

Gibberellin x 0.001 NS 23.8 * 0.00 NS

Norflurazon

Gen x Gibberellin x 0.001 NS 5.31 NS 0.79 ***

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)

Z ‘Flora-Dade’ + GA3+ Nor 1.96 a 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 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 Z Means within column with the same letter are not significantly different at a=0.05.

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 GI

‘Flora-Dade’ 8/16 L/D 1.78 b Z 66.1 b

‘Flora-Dade’ 16/8 L/D 1.95 c 61.5 c

‘Flora-Dade’ Darkness 1.58 a 70.3 a

‘T4099’ 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.

119 100 A

80 'Flora-Dade' 16/8 L/D 60 'Flora-Dade' 8/16 L/D 40 'Flora-Dade' dark

Germination (%) 20

0 1 2 3 4 5 6 Days after sowing

100

80

60 'T4099' 16/8 L/D 'T4099' 8/16 L/D 40 'T4099' dark

Germination (%) 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 ‘FG-

218’ dg ogc result in speed of germination comparable to that of normal lycopene varieties suggesting that plant genetic background can minimize the negative effects of the high pigment genes. Future studies should include a comprehensive evaluation of carotenoid accumulation during fruit and seed development in high pigment genotypes. It would be interesting to identify the accumulation pattern of carotenoid precursors such as phytoene 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 post- imbibition 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 a decrease in antioxidant mechanisms, little information exists that evaluates endogenous antioxidant capacity and seed longevity in a wide range of tomato genotypes. The Photo Induced

Chemiluminescence (PCL) and Trolox Equivalent Antioxidant 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 studies need to evaluate what factors (e.g.vitamins, phenolic compounds or carotenoid content) are 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 sinks for antioxidants as hypothesized in this study.

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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 in a germination chamber at 24 oC. Each value represents the average of 4 replications and three samples by replication (n=7).

134

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

‘OH8245’ 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

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 (PB), red mature (RM), and overripe (OR). Winter 2000.

135