• TABLE OF CONTENTS
HIDE
 Title Page
 Dedication
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Abstract
 Introduction
 Review of literature
 Field emergence of shrunken-2 corn...
 Improvement of emergence and seedling...
 Dehydration rate after solid matrix...
 Poor seed vigor of sh2 maize is...
 Reference
 Biographical sketch






Title: Seed vigor and germination of shrunken-2 maize
CITATION PAGE IMAGE ZOOMABLE PAGE TEXT
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00056217/00001
 Material Information
Title: Seed vigor and germination of shrunken-2 maize
Physical Description: xiii, 144 leaves : ill. ; 29 cm.
Language: English
Creator: Parera, Carlos Alberto, 1956-
Publication Date: 1992
 Subjects
Subject: Corn -- Seeds -- Quality   ( lcsh )
Corn -- Varieties   ( lcsh )
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
 Notes
Thesis: Thesis (Ph. D.)--University of Florida, 1992.
Bibliography: Includes bibliographical references (leaves 124-143).
Statement of Responsibility: by Carlos Alberto Parera.
General Note: Typescript.
General Note: Vita.
Funding: Electronic resources created as part of a prototype UF Institutional Repository and Faculty Papers project by the University of Florida.
 Record Information
Bibliographic ID: UF00056217
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved, Board of Trustees of the University of Florida
Resource Identifier: aleph - 001883935
oclc - 29507146
notis - AJV9069

Table of Contents
    Title Page
        Page i
    Dedication
        Page ii
    Acknowledgement
        Page iii
    Table of Contents
        Page iv
        Page v
    List of Tables
        Page vi
        Page vii
        Page viii
    List of Figures
        Page ix
        Page x
    Abstract
        Page xi
        Page xii
        Page xiii
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
    Review of literature
        Page 5
        The shrunken-2 mutant endosperm of maize
            Page 5
            Page 6
            Page 7
            Page 8
        Presowing seed treatment: Seed priming
            Page 9
            Page 10
            Page 11
            Page 12
            Page 13
            Page 14
            Page 15
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            Page 35
            Page 36
            Page 37
            Page 38
            Page 39
            Page 40
    Field emergence of shrunken-2 corn predicted by single and multiple vigor laboratory tests
        Page 41
        Page 42
        Materials and methods
            Page 43
            Page 44
            Page 45
            Page 46
            Page 47
        Results and discussion
            Page 48
            Page 49
            Page 50
            Page 51
            Page 52
            Page 53
            Page 54
            Page 55
            Page 56
        Summary
            Page 57
            Page 58
    Improvement of emergence and seedling vigor in shrunken-2 sweet corn via seed disinfection and solid matrix priming
        Page 59
        Page 60
        Materials and methods
            Page 61
            Page 62
        Results and discussion
            Page 63
            Page 64
            Page 65
            Page 66
            Page 67
            Page 68
            Page 69
            Page 70
            Page 71
        Summary
            Page 72
            Page 73
    Dehydration rate after solid matrix priming alters seed performance of shrunken-2 maize
        Page 74
        Page 75
        Page 76
        Materials and methods
            Page 77
            Page 78
            Page 79
            Page 80
        Results
            Page 81
            Page 82
            Page 83
            Page 84
            Page 85
            Page 86
            Page 87
            Page 88
            Page 89
            Page 90
        Discussion
            Page 91
            Page 92
            Page 93
        Summary
            Page 94
            Page 95
    Poor seed vigor of sh2 maize is a direct consequence of endosperm genotype
        Page 96
        Page 97
        Page 98
        Page 99
        Materials and methods
            Page 100
            Page 101
            Page 102
            Page 103
            Page 104
            Page 105
        Results
            Page 106
            Page 107
            Page 108
            Page 109
            Page 110
            Page 111
            Page 112
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            Page 114
            Page 115
        Discussion
            Page 116
            Page 117
            Page 118
            Page 119
            Page 120
            Page 121
        Page 122
        Summary
            Page 123
    Reference
        Page 124
        Page 125
        Page 126
        Page 127
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        Page 129
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        Page 142
        Page 143
    Biographical sketch
        Page 144
        Page 145
        Page 146
Full Text











SEED VIGOR AND GERMINATION OF shrunken-2 MAIZE


By

CARLOS ALBERTO PARERA

















A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1992



































To CAROL, CARLOS and VICTORIA














ACKNOWLEDGEMENTS


I would like to express my acknowledgements to Dr.

Daniel J. Cantliffe, chairman of my supervisory committee,

for his understanding, guidance, support, and friendship

during my program. Appreciation is also extended to the

other members of the supervisory committee, Dr. Peter

Hildebrand, Dr. Peter J. Stoffella, Dr. Karen E. Koch and

Dr. Donald R. McCarty for their assistance.

I would also like to thank faculty, staff, and students

in the Vegetable Crops Department who helped throughout my

graduate studies. I am also grateful to Dr. L. Curtis Hannah

and Dr. Bryan Scully for their contribution during my

research. I want also to express my gratitude to all staff

of the Synthetic Seed Laboratory for their camaraderie.

Special thanks is given to Marie Bieniek, Dr. Daniel

Leskovar, and Dr. Roy Harrell for their support and

interesting discussions.

Finally I want to thank my wife, Carol Troilo, and our

children, Carlos and Victoria, for their encouragement,

help, and love.


iii















TABLE OF CONTENTS


Page

ACKNOWLEDGEMENTS ..................................... iii

LIST OF TABLES................................. ....... vi

LIST OF FIGURES .................................... ix

ABSTRACT ................... ........................... xi

CHAPTERS

I INTRODUCTION ................................... 1


II REVIEW OF LITERATURE ............................ 5

The shrunken-2 (sh2) Endosperm Mutant of maize... 5
Presowing Seed Treatment: Seed Priming............ 9

III FIELD EMERGENCE OF shrunken-2 CORN PREDICTED
BY SINGLE AND MULTIPLE VIGOR LABORATORY TESTS.... 41

Materials and Methods............................ 43
Results and Discussion.......................... 48
Summary.... ...................................... 57


IV IMPROVEMENT OF EMERGENCE AND SEEDLING VIGOR IN
shrunken-2 SWEET CORN VIA SEED DISINFECTION
AND SOLID MATRIX PRIMING.......................... 59

Materials and Methods ........................... 61
Results and Discussion........................... 63
Summary.... ...................................... 72

V DEHYDRATION RATE AFTER SOLID MATRIX PRIMING
ALTERS SEED PERFORMANCE OF shrunken-2 MAIZE...... 74

Materials and Methods............................ 77
Results ........................................ 81
Discussion ................... .................... 91
Summary............ ........ ... ......... .......... 94

iv









VI POOR SEED VIGOR OF sh2 MAIZE IS A DIRECT
CONSEQUENCE OF ENDOSPERM GENOTYPE.................. 96

Materials and Methods.......................... 101
Results ......... ................................ 106
Discussion......................... ..... ........ 116
Summary......................................... 122


LITERATURE CITED..................................... 124

BIOGRAPHICAL SKETCH.................................... 143














LIST OF TABLES


Table Page

2-1. Selected species and osmotica reported to be
used for seed priming in osmotic solutions........ 12

2-2. Selected species and solid-matrix reported
to be used for solid matrix priming.............. 22

3-1. Means and ANOVA table for seed field emergence
of the 7 sweet corn cultivars and 7 sowing
locations and planting date...................... 49

3-1. Sowing date, soil type, bed size, fertilization,
irrigation system and average temperature for
the locations used in the study................... 50

3-3. Simple linear correlation coefficient (r)
between laboratory vigor tests and field
emergence and emergence rate index (ERI)
of 7 sh2 sweet corn.............................. 52

3-4. Factors originated after orthogonal rotation
(varimax) from laboratory vigor tests to
predict field emergence of sh2 sweet corn........ 54

3-5. Best one- or two-, and three- non collinear
factor models to predict field emergence by
laboratory vigor tests in sh2 sweet corn......... 56

4-1. Effect of seed treatments on germination of
two sh2 sweet corn cultivars in a cold
germination test................................. 64

4-2. Effect of seed treatments on emergence rate
index (ERI), emergence percentage, and dry
weight (DW) of 2 sh2 sweet corn cultivars in
a field experiment planted in October 26, 1989
at Gainesville, FL............................... 66








4-3. Effect of seed treatments on emergence rate
index (ERI), emergence percentage, and dry
weight (DW) of 2 sh2 sweet corn cultivars in
a field experiment planted in March 7, 1990
at Gainesville, FL.............................. 67

4-4. Effect of seed treatments on emergence rate
index (ERI), emergence percentage, and dry
weight (DW) of 2 sh2 sweet corn cultivars in
a field experiment planted in April 23, 1990
at Gainesville, FL................................ 68

4-5. Effect of seed treatments on emergence rate
index (ERI), emergence percentage, and dry
weight (DW) of 2 sh2 sweet corn cultivars in
a field experiment planted in November 8, 1990
at Gainesville, FL............................... 71

5-1. Dehydration rate of CNS-711 and HSII sh2
sweet corn seeds redried at different
temperatures after priming....................... 83

5-2. Imbibition rate and leakage conductivity of
CNS-711 and HSII sh2 sweet corn seeds either
redried at different temperatures after priming
and non primed .................................. 83

5-3. Glutamic acid decarboxylase activity (GADA) of
CNS-711 and HSII sh2 sweet corn seeds either
redried at different temperatures after priming
and non primed ................................... 86

5-4. Sucrose concentration in endosperm and embryo of
CNS-711 and HSII sh2 sweet corn seeds either
redried at different temperatures after priming
and non primed.................................. 86

5-5. Laboratory germination (LGE), germination
percentage after complex stress vigor test
(CST), and index of conductivity and stress
vigor test (ICS) of CNS-711 and HSII sh2 sweet
corn seeds either redried at different
temperatures after priming and nonprimed......... 88

5-6. Emergence rate index (ERI) and field emergence
percentage (14 days after sowing) of CNS-711 and
HSII sh2 sweet corn seeds either redried at
different temperatures after priming and non
primed in a field experiment sown on 3 December
1991 in Gainesville, FL.......................... 90


vii








5-7. Emergence rate index (ERI), emergence
percentage, seedling fresh (FW) and dry weight
(DW) of CNS-711 and HSII sh2 sweet corn seeds
either redried at different temperatures after
priming and non primed in a field trial sown
in 13 April 1992, in Gainesville, FL............ 90

6-1. Germination percentage, seedling fresh
weight (FW) and dry weight (DW) after complex
stress vigor test and index of conductivity and
stress vigor test (ICS) of concordant and
nonconcordant seeds.............................. 107

6-2. Pericarp thickness (PT) and endosperm:embryo
ratio of concordant and nonconcordant seeds...... 109


viii














LIST OF FIGURES


Figure Page

4-1. Average daily soil temperature (5 cm deep) for
first 7 days after sowing in fall 1989, spring
1990 and fall 1990 field experiments.............. 69


5-1. Moisture content decreased, calculated as
percentage of the original fresh weight (7-6%)
of CNS-711 (A) and HSII (B) sh2 sweet corn
seeds redried at different temperatures after
priming ............................... .......... 82

5-2. Respiration of CNS-711 and HSII sh2 sweet corn
seeds either redried at different temperatures
after priming and nonprimed (Data pooled over
the 2 cultivars). Values followed by the same
letter are not significant different at 5%
probability level by LSD test................... 85

6-1. Leakage conductivity of concordant and
nonconcordant seeds. Shrunken endosperm/colored
embryo (M),colored endosperm/ colorless
embryo (E),and colorless endosperm/colored
embryo (A). Data represent the mean of 4
measures (se) .................................. 110

6-2. Imbibition of embryo (a) and endosperm (b)
of concordant and noncorcondant seeds.
Shrunken endosperm/colored embryo (M),colored
endosperm/ colorless embryo (0),and colorless
endosperm/colored embryo (A). Data represent
the mean of 4 measures (se)..................... 111

6-3. Germination (a) and dry weight of the seedlings
(b) of embryos from concordant and noncorcondant
seeds in different medias. Shrunken
endosperm/colored embryo (B),colored
endosperm/colorless embryo (U),and colorless
endosperm/colored embryo (0).Data represent the
mean of 4 measures (se)........................ 113








6-4. Sugar concentration in embryos(a) and
endosperms (b) of concordant and nonconcordant
seeds. Shrunken endosperm/colored embryo (B),
Colorless endosperm /colored embryo (D).Data
represent the mean of 4 measures (se)............ 114

6-5. Ratio raffinose/sucrose in embryos and
endosperms of concordant and nonconcordant seeds.
Shrunken endosperm/colored embryo (B),colorless
endosperm/colored embryo (D).Data represent the
mean of 4 measures (se).......................... 115














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


SEED VIGOR AND GERMINATION OF shrunken-2 MAIZE

by

CARLOS ALBERTO PARERA

December 1992

Chairman: Daniel J. Cantliffe
Major Department: Horticultural Sciences


The direct effects of the shrunken-2 (sh2) mutation on

seed and seedling vigor of maize (Zea mays L.) are not

completely understood. The objective of this study was to

elucidate the causes) of poor seed vigor of sh2 maize via a

physiological seed treatment, solid matrix priming (SMP),

and a genetic translocation wherein the endosperm or embryo

could be isolated so that the seed part contributing to poor

seed vigor could be determined.

In order to evaluate vigor, six sh2 sweet corn

cultivars were evaluated in thirty laboratory vigor tests

and correlated with field emergence at three different

locations over two planting dates. The complex stress vigor

test and the index of conductivity and stress test (ICS) (a

combination of germination under stress conditions and seed








leakage conductivity) were the best field emergence

predictors.

Results from SMP were variable, possibly as a

consequence of the redrying technique after priming. Seeds

dried at 15 or 20C had low respiration, glutamic acid

decarboxylase activity, greater leakage conductivity, and

reduced field emergence compared to seeds dried at 30 or

40C.

A genetic manipulation created concordant and

nonconcordant seeds in embryo and endosperm for sh2. Seeds

with mutant endosperm and wild type embryo had lower seed

and seedling vigor compared to seeds with wild type

endosperm and embryo. Carbohydrate concentration was greater

in mutant endosperm resulting in rapid water uptake and

greater solute leakage. Endosperm genotype modified sugar

concentration in the embryo. The raffinose:sucrose ratio was

lower in wild type embryos excised from mutant endosperms

compared to wild type embryos excised from wild type

endosperm.

Seed membranes play a critical role in seed germination

affecting water uptake. The field emergence predictors

indirectly evaluated seed membranes after soaking stress.

The results from dehydration after SMP suggested a membrane

reorganization problem. Rapid water uptake and poor membrane

protection (low raffinose/sucrose ratio) might induce

membrane alteration in sh2 seeds. In summary, membranes play


xii








a fundamental role in seeds carrying sh2 endosperm, thus

adversely affecting the initial physiological and

biochemical steps of germination.


xiii














CHAPTER I
INTRODUCTION



Seed germination and subsequent seedling growth are

preceded by the initiation of numerous and complex

biological processes. Seed genotype, physiological maturity

at harvest, storage conditions, seed borne diseases are some

of the primary factors that singly or in combination can

affect seed germination and seedling vigor. The first step

of the germination process is imbibition. Seed water uptake

is modified by the physical and biochemical characteristics

of the seed and environmental conditions. During imbibition,

membranes control water and solute movement in and out of

seeds, playing an important role in seed germination.

The shrunken-2 (sh2) mutation of maize has been

utilized in breeding programs to increase the level of sugar

in the endosperm at maturity. Such hybrids have excellent

eating quality as well as longer shelf life after harvest

compared to other types of sweet corn. Although eating

quality is superior and demand for the product is high,

germination and seedling vigor are negatively affected by

the sh2 mutation and continue to be a problem during field

production.








2

The overall objective of this study was to predict and

improve field emergence of sh2 sweet corn seeds. More

specific objectives were: 1) to identify a laboratory test

to estimate field emergence, 2) to study the effects of the

solid matrix priming and dehydration temperature after the

treatment on sh2 seeds, and 3) to distinguish the effects of

the sh2 mutation on the embryo or the endosperm and to

determine how it might affect seed and seedling vigor.

There is a need for a rapid, reproducible, and

inexpensive laboratory test to predict field emergence of

sh2 sweet corn hybrids. The recommended tests for field corn

are not appropriate for sh2 sweet corn (Dr D. Sawyers,

Asgrow Seed Co., personal communication). The accelerated

aging test (AOSA, 1983) is not suitable for sh2 seeds

because of their high susceptibility to seed borne diseases.

The cold test, another recommended test, has repeatability

problems among laboratories and it sometimes lacks

correlation with field emergence (Fiala, 1987).

Thus, the first step of this research was to determine

a reliable laboratory test to predict seed vigor and field

emergence of sh2 sweet corn. Seeds of 7 hybrids carrying the

mutation were sown in three Florida regions at two sowing

dates to evaluate field emergence and seedling vigor. The

data were correlated with 30 different laboratory vigor

tests.








3

Solid matrix priming (SMP) combined with sodium

hypochlorite has been reported as a successful presowing

treatment to increase germination in sh2 sweet corn seeds

(Parera and Cantliffe, 1990 b). After the treatment, the

seeds must be dried to their original fresh weight to

facilitate storage and handling. Seeds of sh2 sweet corn had

fresh weight increases up to 60% during SMP treatment

(Parera and Cantliffe, 1991). Seed quality is affected by

the temperature and rate of dehydration (Herter and Burris,

1989). Subcellular systems could be severely damaged during

dehydration (Roberts, 1981). The effects of dehydration

temperature (15, 20, 30 and 40C) after SMP on seed

performance and the role of membranes during dehydration

were studied under laboratory and field conditions.

Molecular data have shown that the sh2 mutation is

expressed only in the endosperm (Hannah and Nelson, 1976;

Giroux, 1992), although sh2 genotypes carry the mutation in

the embryo as well. The effects of the mutation on vigor at

the endosperm or embryo level were studied by using seeds

with different (nonconcordants) or equal genotype

(concordants) for these seed parts. The seeds were produced

using the capability of the supernumerary or B chromosomes

to undergo a nondisjunction at mitosis during

microsporegesis (Roman and Ullstrup, 1951). As a result, two

copies of the particular supernumerary chromosome go to the

embryo or endosperm and no copy is received by the








4

complement during fertilization. The long arm of the

chromosome 3, which contains the sh2 mutation is susceptible

to be involved in TB-A translocation. Using this genetic

manipulation, three principal seed types could be studied:

sh2 endosperm/wild type embryo, wild type endosperm and

embryo and sh2 embryo/wild type endosperm. Seed performance

was evaluated through biochemical and physiological tests in

order to determine the effect of the site of the mutation on

seed and seedling vigor.














CHAPTER II
LITERATURE REVIEW



The Shrunken-2 Mutant Endosperm of Maize



The mutant shrunken-2 (sh2) gene modifies the synthesis

of carbohydrates in maize endosperm, increasing the levels

of sucrose and decreasing starch at maturity (Creech, 1956).

According to Hutchinson (1921), the first seeds with

shrunken kernel type were observed after a self-pollination

of seeds collected from gardens of the Ponka indians in

Nebraska in 1914. Tsai and Nelson (1966) reported that the

sh2 gene, located in the long arm of chromosome 3, affects

the activity of adenosine diphosphoglucose pyrophosphorylase

(ADP-glucose pyrophosphorylase) in the endosperm. The enzyme

plays a key role in the starch biosynthesis, catalyzing the

reaction ATP + glucose-1-phosphate ADP-glucose + PP,

(Preiss, 1978). The sh2 mutation does not modify the enzyme

levels in the embryo (Hannah and Nelson, 1976).

The quality of fresh market sweet corn is associated

with the amount of sugar in the kernel. Consumers had strong

preference for sweeter corn (Showalter and Miller, 1962).

Normal sweet corn with standard sugary (sul) gene has 124 mg














CHAPTER II
LITERATURE REVIEW



The Shrunken-2 Mutant Endosperm of Maize



The mutant shrunken-2 (sh2) gene modifies the synthesis

of carbohydrates in maize endosperm, increasing the levels

of sucrose and decreasing starch at maturity (Creech, 1956).

According to Hutchinson (1921), the first seeds with

shrunken kernel type were observed after a self-pollination

of seeds collected from gardens of the Ponka indians in

Nebraska in 1914. Tsai and Nelson (1966) reported that the

sh2 gene, located in the long arm of chromosome 3, affects

the activity of adenosine diphosphoglucose pyrophosphorylase

(ADP-glucose pyrophosphorylase) in the endosperm. The enzyme

plays a key role in the starch biosynthesis, catalyzing the

reaction ATP + glucose-1-phosphate ADP-glucose + PP,

(Preiss, 1978). The sh2 mutation does not modify the enzyme

levels in the embryo (Hannah and Nelson, 1976).

The quality of fresh market sweet corn is associated

with the amount of sugar in the kernel. Consumers had strong

preference for sweeter corn (Showalter and Miller, 1962).

Normal sweet corn with standard sugary (sul) gene has 124 mg








6

of sugar/g of dry weight (DW) compared to approximately 330

mg of sugar/g DW in sh2 kernels (Burgmans and Lill, 1987). A

sensory evaluation panel showed that hybrids with sh2

endosperm produced high quality canned, frozen and fresh

market products compared to su hybrids (Garwood et al.,

1976).

A concern to a broader use of hybrids carrying the sh2

mutation is the poor seed and seedling vigor, specially

under conditions of stress associated with this genotypes.

Seeds of sh2 maize are less uniform, lighter, and easier to

damage compared to wild type (Styer and Cantliffe, 1983).

The ratio of endosperm to embryo dry weigh was two times

greater in su than sh2 seeds (Styer and Cantliffe, 1984a).

Lower seed vigor of sh2 was related to small endosperm and

low availability of nutrient reserves for the embryo during

germination (Wann, 1980) and susceptibility to pericarp

damage during harvest (Wann, 1986). Styer and Cantliffe

(1984 a) reported a dysfunction of the scutellum or axis in

relation to carbohydrate metabolism and utilization.

Recently, Harris and DeMason (1989) reported that the low

vigor of sh2 seeds is associated with inadequate aleurone-

controlled reserve mobilization. Despite more than 25 years

of research, the reason of poor seed and seedling vigor is

not completely understood.

Seed and soil borne diseases were reported initially as

one of the main reasons for poor stand and seedling vigor








7

(Berger and Wolf, 1974; Pieczarka and Wolf, 1978). However,

a more direct effect lies in the physical characteristics of

the seeds and the high levels of leakage during imbibition.

Small cracks on the pericarp are potential areas for

pathogen penetration (Styer and Cantliffe, 1984b). The

pathogens, mainly Fusarium moniliforme, Aspergillus spp. and

Penicillium spp. can infect both the endosperm and embryo,

reducing germination under stress conditions. The high

levels of metabolite leakage can further contribute to

pathogen growth and development (Schrot and Cook, 1963;

Pollock and Toole, 1966; Nordin, 1984). Seed of sh2 maize

had higher leakage than su endosperm (Wann, 1986). Parera

and Cantliffe (1991) reported a high negative correlation

between imbibition, seed leakage conductivity, and

germination in sh2 hybrids.

Many presowing treatments have been proposed to

increase field emergence and seedling vigor of sh2 sweet

corn. Berger and Wolf (1974), Cantliffe et al. (1975),

Pieczarka and Wolf (1978), and Cantliffe and Bieniek (1988)

recommended fungicide seed treatments to improve field

emergence and seedling vigor of sh2 hybrids. In a more

recent report (Parera and Cantliffe, 1990 a), it was

necessary to combine four different fungicides to reach an

appropriate stand in the field.

Environmental concerns for contamination and

indiscriminate use of pesticides is forcing the utilization








8

of alternative measures for pest control. Biocontrol of

pathogens affecting seeds and seedlings of sh2 sweet corn

have been studied. Trichoderma spp have been used as a

biocontrol agent for soil borne diseases in many species

(Hadar et al., 1984). Harman and Taylor (1988) reported

better final stand and seedling vigor in two sweet corn

cultivars after seed treatment with a combination of solid

matrix priming and two strains of Trichoderma. In the other

hand, Parera and Cantliffe (1990 a) reported no effect on

stand establishment and seedling vigor after the same

treatment under Florida conditions. Callan et al. (1990,

1991) reported less symptoms of preemergence damping-off

after on seeds treated with Pseudomonas fluorescens and

imbibed in warm water.

Reports on presowing osmotic treatments in sweet corn

had contradictory results. Seed moisturizing and soaking

improved early emergence and seedling vigor of sh2 sweet

corn while seed priming had negative effects (Bennet and

Waters, 1987). Murray (1990) reported more and early

germination in two sh2 hybrids after priming in PEG osmotic

solution. Parera and Cantliffe (1990 b) improved field

emergence and seedling vigor of sh2 sweet corn hybrids by

combining solid matrix priming and seed disinfection with

sodium hypochlorite. A direct consequence of this treatment

was reduced seed water uptake and leakage conductivity after

priming (Parera and Cantliffe, 1991). Chern and Sung (1991)








9

also reported improvement of germination in sh2 sweet corn

hybrids by controlling water uptake. The last two reports

open a new perspective to the germination problem in maize

with sh2 endosperm.



Presowing Seed Treatment: Seed Priming

Rapid and uniform field emergence are two essential

prerequisites to increase yield and quality in annual crops.

Uniformity and percentage of emergence of direct seeded

crops have a major impact on final yield and quality (Wurr

and Fellow, 1983). Both factors are inherent to seed quality

and environmental conditions during seed emergence. Slow

rate of emergence results in smaller plants (Ellis, 1989)

and seedlings which are more vulnerable to soil borne

diseases (Gubels, 1975, Osburn and Schroth, 1989). Extended

emergence periods expose the soil to bed deterioration and

compaction (Heydecker, 1978), particularly under adverse

conditions.

Various presowing seed treatments have been proposed

and used to reduce the time between sowing and emergence and

improve synchronization of field emergence in annual crops.

Evenary (1980) reported that ancient Greek farmers presoaked

cucumber seeds in water and honey to increase germination

rate and emergence. In the 17th century, presowing

treatments with salt solutions were a common practice among

Russian farmers (Yapparov and Iskhakov, 1974). Soaking seeds








10

in water before planting has been an old-time farmer

practice to reduce the time between planting and emergence.

One early report in 1918 recommended to place seeds of

radish, bean, corn, cucumber, and squash in lukewarm water

overnight to increase germination velocity, (Wilkinson,

1918).

In the last two decades, seed priming has become a

frequent seed treatment to increase emergence rate and

improve uniformity in many vegetable and flower species.

Heydecker (1973) acknowledged the use of the term 'priming

of seeds' by Malnassy (1971) to describe a presowing seed

treatment to enhance germination and increase uniformity

under adverse conditions. In the same report, terms such as

halopriming (soaking in salt solutions) or osmopriming

(soaking in other osmotic solutions) were proposed as

alternative to priming. Osmoconditioning or osmotic

conditioning are also used to describe the same treatment

(Khan et al., 1978). Kubik et al. (1988) and Taylor et al.

(1988) introduced the term solid matrix priming (SMP). The

expression is used for a presowing treatment where a solid-

matrix instead of an osmotic solution is used.

Matriconditioning was proposed by Khan et al. (1990) as an

alternative term to SMP. Recently, Callam et al. (1990)

coined the word 'bioproming', a treatment where sweet corn

seeds are coated with a bacteria and soaked in warm water

until the seed moisture content increased to 35-40%. In








11

the discussion which follows the original priming and solid

matrix priming terms will be used. Through the discussion it

will be shown that the use of salts as an osmoticum actually

increases seed water potential. The cadre of other terms has

actually led to confusion of what seed priming is and what

is needed to obtain consistent positive effect from priming.

Heydecker (1973) defined seed priming as a presowing

treatment where the seeds are soaked in an osmotic solution

that allows them to imbibe water and go through the first

steps of germination but does not permit radicle protrusion

through the seed coat. The seeds are then dried to their

original moisture content and stored or planted via

conventional techniques. This definition can be partially

extended to SMP. The main difference between the two

treatments is that in SMP a solid-matrix regulates seed

water uptake instead of the osmotic potential of a solution.

Seed Priming and Osmotic Solutions

Several materials have been used as an osmoticum for

priming treatment (Table 2-1). Inorganic salts, such as KNO3

(Bradford et al., 1988), K3PO4 (Cantliffe, 1981), and a

chemically inert compound, polyethylene glycol 6000 (PEG

6000) (Hegarty, 1977; Bodsworth and Bewley, 1981) or 8000

(PEG 8000) (Ali et al., 1990; Adegbuyi et al., 1981) are

materials most commonly used to adjust the osmotic potential

of the solution.












Table 2-1. Selected species and osmotica reported to be used
for seed priming in osmotic solutions.


Crop Osmoticum Reference


Asparagus
Asparagus
officinalis L.

Aubergine
Solanum melongena


Beet
Beta vulgaris L.

Beet (sugar)

Cabbage
Brassica oleracea L.
var. capitata

Carrot
Daucus carota L.












Celery
Apium graveolens L.


Field Corn
Zea Mays L.


PEG, NaNO3,
Saline Seawater


Mannitol



PEG, MgSO4


PEG, NaCl


PEG




Sodium
Polypropionate
KH2PO4, Glycerol

KNO3 + KH2PO4
KNO3, K2HPO4, K3P04
KNO3+K3PO4,
KNO3+K2HPO4

PEG



Sodium Polypropionate
KH2PO4, Glycerol

KNO3 + KH2PO4 + GA
KNO3 + K3P04, K3P04


PEG, KH2PO4, K2HP04
KNO3+KH2PO4


Pill et al.,1991



Passam et al.,1989


Taylor et al.,1985


Osburn and Schroth,1989


Hill et al.,1989
Perkins-Veazie
et al.,1989


Brocklerhurst and
Dearman,1984
Gray et al.,1990
Hill et al.,1989
Murray,1989

Zuo et al.,1988 a,b
Brocklerhurst and
Dearman,1984
Globerson and Feder,1987
Haigh and Barlow,1987

Haigh et al.,1986

Brocklerhurst and
Dearman,1984
Singh et al.,1985
Tanne and Cantliffe,1989
Zuo et al.,1988 a,b
Blocklerhurst and
Dearman,1984
Globerson and Feder,1987
Tanne and Cantliffe,1989

Basra et al.,1988 a,b


Corn cockle
Agrostemma ghitago L.

Cucumber
Cucumis sativus L.

Dusty miller
Senecio cineraria DC.


PEG


De Klerk,1986


Mannitol
NaCl, Milk


Passam et al.,1989
Thanos and Georghiou, 1988


Carpenter,1990


PEG













Continuation table 2-1.


Crop Osmoticum Reference


Kale
Brassica oleracea L.
var. acephala


Leek
Allium porrum L.




Lettuce
Lactuca sativa L.


Muskmelon
Cucumis melo L.



Onion
Allium cepa L.


Pansy
Viola x wittrockiana
Gais.

Parsley
Petroselinum hortense
Hoff.


Parsnip
Pastinaca sativa L.


Rao et al.,1987


PEG


Mannitol


PEG



K3PO4


Mannitol
KNO3
KH2PO4 + KNO3


PEG



PEG + Enriched air
Mannitol, NaC1
Glycerol


KN03+K3P04,
KNO3+K2HPO4
K2HPO4
K3PO4, KNO3


PEG


PEG


Bray et al.,1989
Gray et al.,1990
Parera and Cantliffe,1992
Parera and Cantliffe, 1992


Cantliffe,1981
Hill et al.,1989
Tarquis and Bradford, 1992
Valdez and Bradford,1987
Cantliffe,1981
Cantliffe et al.,1984
Guedes and Cantliffe,1980
Perkins-Veazie and
Cantliffe,1984
Wurr and Fellows,1984

Passam et al.,1989
Bradford et al.,1988
Nerson and Govers,1986


Ali et al.,1990
Dearman et al.,1986
Gray et al.,1990
Haigh and Barlow,1987
Bujalsky et al.,1989
Furutani et al.,1986
Broclehurst and
Dearman,1983 a,b


Haigh et al.,1986
Haigh and Barlow,1987
Haigh et al.,1986


Carpenter and
Boucher, 1991 a,b


Akers et al.,1987
Pill,1986
Rabin and Berkowitz,1988


Gray et al. 1984


PEG













Continuation table 2-1.


Crop Osmoticum Reference


Pea
Pisum sativum L.
var.sativum


Peanut
Arachis hypogea L.

Pepper
Capsicum anuum L.


PEG

Sodium Polypropionate


PEG


PEG


Mannitol

Sodium polypropionate
KNO3





KH2PO4
KNO3+K2HPO4
NaCl, MgSO4

KC1, K2SO4, Na2SO4,
CaCI, Na2HPO4, K2HPO4,
NaC1+CaCl2


Purple Coneflower
Echinacea purpurea (L.)
Moench.

Ryegrass
Lolium perenne L.


KN03



PEG


Dell'Aquila and
Bewley,1989
Zuo et al.,1988 a,b


Fu et al.,1988


Cantliffe and
Watkins, 1983
Aljaro and Wyneken,1985
O'Sullivan and Bow,1984
Rivas et al.,1984
Stofella et al.,1992
Georghiou et al.,1987
Passam et al.,1989
Zuo et al.,1988 a, b
Bradford et al.,1990
Globerson and Feder, 1987
Rivas et al.,1984
Smith and Cobb.,1991
Sundstrom and
Edwards,1988-89
Globerson and Feder,1987
Jones and Sanders,1987
Aljaro and Wyneken,1985
Smith and Cobb,1991


Smith and Cobb,1991

Samfield et al.,1990-91



Dannenberg et al., 1992


Sorghum
Sorghum
Moench.

Soybean
Glycine


bicolor (L.)



max (L.) Merr.


Spinach
Spinacea olereacea L.


PEG, KNO3, K2HPO4,
K3PO4, KNO3+K3P04,
KNO3+K2HPO4

PEG, Mannitol
PEG + Ga3 + Kinetin
Sodium polypropionate

Sodium polypropionate


Haigh and Barlow,1987

Helsel et al.,1986
Lorenz et al.,1988
Zuo et al.,1988 a,b

Zuo et al.,1988 a,b


Sweet Corn
Zea mays L.


Tabasco pepper
Capsicum frustecens L.


PEG


Murray,1990


PEG, KNO3
KNO3+Ga


Rivas et al.,1984
Sundstrom et al.,1987












Continuation table 2-1.


Crop Osmoticum Reference


Tickseed
Coreopsis lanceolata L.

Tomato
Lycopersicon esculentum
Mill




























Turnip
Brassica rapa L.
var. rapa

Watermelon
Citrullus lanatus
(Thumb.) Matsumi&Nakai


KNO3


PEG






Mannitol, NaCl, Milk,
Sucrose

Sodium polypropionate
KNO3






K2HPO4, K3PO4
KNO3+K3PO4, KNO,+K2HPO4
KNO3+K2HPO4




KNO3+K3PO4


KNO3+K3PO4+
Uniconazole

PEG


KNO3
KNO3+KIPO0


Samfield et al.,1990-91

Ali et al.,1990
Avarado et al., 1987
Alvarado and
Bradford,1988 a,b
Bino et al.,1992
Haigh and Barlow,1987
Hill et al.,1989

Thanos and
Georghiou,1988
Zuo et al.,1988 a,b
Ali et al.,1990
Alvarado et al.,1987
Alvarado and
Bradford,1988 a,b
Argerich and
Bradford,1989
Haigh and Barlow, 1987
Haigh and Barlow, 1987
Haigh and Barlow, 1987
Argerich et al.,1989
Argerich and
Bradford,1989
Haigh et al.,1986
Haigh and Barlow,1987
Haigh et al.,1986
Haigh and Barlow,1987
Odell and Cantliffe,1986

Davis et al.,1990

Rao et al.,1987


Elmstrom,1985
Elmstrom,1985
Sachs,1977








16

Various inorganic salts were first used as osmotica for

seed treatments. In early work, Kotowski (1926) reported

greater germination of pepper seeds, after they were soaked

in solutions of NaNO3, MnSO4 and MgCl2. Ells (1963), using a

K3PO4 and KNO3 solution improved the germination rate of

tomato at low temperatures. However, this and other early

reports (Dastur and Mone, 1958) attributed the advantages of

the treatments to a nutritional effect of the salts. In

fact, Ells in his 1963 report coined the term vigorization

wherein he suggested that the seeds had improved vigor. One

U.S. seed company sold 'vigorized' tomato and pepper seeds

for over 15 years which were actually primed via the

process. The utilization of inorganic salts as an osmotica

has generated conflicting recommendations. Seeds of leek and

carrot had a reduced germination percentage when they were

primed with KH2PO4 compared with PEG 6000 (Brocklehurst and

Dearman, 1984). Conversely, Haig and Barlow (1987) reported

that primed carrot seeds performed better when inorganic

salts (KNO3, K3PO4) were used as an osmotica instead of PEG.

Tomato seeds primed in KNO3 solution germinated more rapidly

at 15C compared with seed primed in PEG 8000 solutions

(Alvarado et al., 1987). There were no differences in the

rate of germination of pepper seeds, when PEG, MgSO4 or NaC1

were used as osmotica (Aljaro and Wyneken, 1985). Pepper

seeds emerged more rapidly after priming in a solution of

KNO3 + K3PO4 compared to seeds primed in PEG 6000 (O'Sullivan








17

and Bouw, 1984). Seedlings from 'Jalapeno' pepper seeds

primed in PEG 6000 solution had lower dry weight compared

with seedlings from seeds primed in salt solutions (Rivas et

al., 1984).

Some of the reported advantages of the inorganic salts

over PEG are that they are easy to aerate and remove from

the seed after treatments and are less costly. Other authors

included as a benefit to the use of inorganic salt, a

nutritional effect from priming. Dastur and Mone (1958)

found that nitrogen, potassium or phosphate concentration in

embryos did not change after soaking cotton seeds in salt

solutions; however, an increase in the concentration of

micronutrients such as manganese and cooper was observed.

The size, structure, biochemical constitution, position

of seed protecting layers, and the type of salt and soaking

time are the principal factors influencing embryo ion

penetration. High ion concentration in the embryo may

adversely affect germination when inorganic salts are used

as osmotica in the seed priming treatment. Brocklehurst and

Dearman (1984) reported lower germination percentage of leek

seeds primed in KH2PO4 solution compared to seeds primed in

PEG solutions. In the same experiment, seeds of carrot,

leek, celery and onion absorbed less water when they were

primed in PEG or glycerol solutions compared to KH2PO4

solutions. Lettuce seeds primed in K3PO4 solution absorbed

more water compared to seeds primed in PEG or PEG + K3P04








18

solution or soaked in water (Guedes et al., 1979). These

results confirmed that ions are be able to penetrate and

accumulate inside of the seeds during priming, when

inorganic salts solutions are used. The accumulated salt

might then reduce the osmotic potential of the seed and

induce more water absorption as the treatment progresses.

Polyethylene glycol (PEG) is also commonly used as an

osmotica. PEG has advantages over inorganic salts because it

is chemically inert and does not have adverse effects on the

embryo (Cantliffe, 1983). The size of the PEG molecule

prevents penetration to seed tissues (Brocklehurst and

Dearnan, 1984) thus reducing toxic side effects on seeds

(Heydecker and Coolbear, 1977).

In addition to inorganic salts or PEG, oligosacharides

such as mannitol have been successfully utilized to regulate

the osmotic potential of the priming solution. Georghiou et

al. (1987) improved germination rate and final germination

in pepper seeds after priming in a mannitol solution.

Similarly, Passan et al. (1989) reported rapid germination

and larger seedlings when seeds of aubergine, pepper, and

melon were primed using a mannitol solution compared to

nonprimed seeds. When sown at high temperature, the

germination rate and final germination of leek seeds was

improved by using mannitol as an osmotica (Parera and

Cantliffe, 1992). Seeds of tomato and cucumber which were

germinated under unfavorable light conditions (continuous








19

far-red) had faster and better final germination compared to

untreated seeds after priming in a mannitol solution (Thanos

and Georghiou, 1988).

Other products such as sodium polypropionate (Zuo et

al., 1988 a,b), glycerol (Brocklehurst and Dearman, 1983 a,

b), and synthetic seawater (Pill et al., 1991) have also

been used as alternative osmotica to salts and PEG in seed

priming.

Several authors combined osmotic and other nonosmotic

products to improve the effects of seed priming. A mixture

of various salts in the solution is a common recommendation

in many species (Elmstrom, 1985; Gray and Steckel, 1977;

Globerson and Feder, 1987; Haigh et al., 1986). The addition

of fungicide to the priming solution has been successfully

used to prevent pathogen grown during priming (Szafirowska

et al., 1981; Leskovar and Sims, 1987).

PEG or salts combined with growth regulators has been

reported as an effective combination to improve germination

under stress conditions. Under suboptimal temperatures and

soil crusting, Lorenz et al. (1988) observed greater

emergence of soybean seeds, when they were primed in a

solution of PEG and gibberellin (GA). When celery seeds were

primed in PEG with ethephon and GA, high temperature

dormancy was overcome and germination was synchronized

(Brocklehurst et al., 1982). Thermodormancy of lettuce was

reduced by priming the seeds in a solution of K3PO4 and








20

benzyladenine (BA) (Cantliffe, 1992). The incorporation of

BA (100 ppm) to a PEG (-12.5 bars) priming solution

increased the germination of 'Earlybelle' celery at 25C to

43% compared to 27% when PEG alone was used (Tanne and

Cantliffe, 1989). Priming pepper seeds in PEG + GA solution

slightly increased germination rate of pepper at low

temperature compared to seeds primed in PEG alone (Cantliffe

and Watkins, 1983). Antirrhinum majus seeds (Kepczynski,

1979) had improved germination at low temperature (15C)

after priming in PEG + GA solution compared to nonprimed or

primed seed in PEG.

Solid Matrix Priming

The considerable volume of solution required per seed

and problems with aeration of the solution can become

obstacles to extend seed priming via osmotic solutions to a

large seeded species or to large seed quantities. Further,

the cost of the osmotica such as PEG is high, and osmotica

must be removed from the seed before dry back. Both salts

and PEG present environmental concerns when disposal of the

osmotica is considered.

Solid Matrix Priming (SMP) treatment consists of mixing

seeds with an organic or inorganic carrier and water for a

prescribed period of time. The moisture content of the

mixture is brought to a level just below that required for

radicle protrusion (Harman and Taylor, 1988). Seed water








21

uptake is regulated by the solid-matrix potential (Kubik et

al., 1988).

Different types of materials have been used as a solid-

matrix for this treatment (Table 2-2). The ideal

characteristics of the materials to be used in SMP were

summarized by Taylor et al. (1988) and included: 1) no

toxic, 2) high water holding capacity, 3) remain friable at

different moisture content, and 4) easy to remove from seeds

after treatment. Khan et al. (1992 b) reported that Micro-

cell A was more effective to induce early emergence of beet

compared to Agro-lig or expanded vermiculite # 5. Sphagnum

moss was difficult to remove from tomato seeds after priming

compared to leonardite shale (Taylor et al., 1988). The SMP

has a potential application to both large and small seeded

species and large amounts of seeds can be primed at one

time. Promising results from SMP have been reported in many

species. The SMP treatment reduced the time between seeding

and emergence of tomato, onion and carrot seeds compared to

nonprimed seeds (Taylor et al., 1988). After SMP treatment,

using calcined clay as a matrix-solid, pepper seeds had more

rapid (low T50) and final germination compared to nonprimed

seeds (Kubik et al., 1988). Primed table beet seeds via SMP

had a rapid emergence and better final stand compared to

nontreated seeds (Khan et al., 1990 a).

The characteristics of the SMP treatment minimizes

aeration problems and facilitates the incorporation of other












Table 2-2. Selected species and solid-matrix reported to be
used for solid matrix priming.



Specie Solid Reference


Beet
Beta vulgaris L.

Carrot
Daucus carota L.

Celery
Apium graveolens L.

Cucumber
Cucumis sativus L.



Peas
Pisum sativum L.
var. sativum

Onion
Allium cepa L.

Pepper
Capsicum anuum L.

Snap bean
Phaseolus vulgaris L


Sweet Corn
Zea mays L.


Tomato
Lycopersicon
sculentum Mill


Microcell E.+Fungicide
Agro-lig, Vermiculite #5

Microcel and Vermiculite#5
Leonardite Shale

Calcined Clay + NaOCl


Leonardite shale+Trichoderma

Bituminous coal, Sphagnum moss
+Enterobacter cloacae+Trichoderma

Leonardite shale+Trichoderma



Leonardite shale


Microcell E, Vermiculite #5
Calcined clay+Fungicide

Leonardite shale+Trichoderma



Calcined clay + NaOCl

Leonardite shale+Trichoderma



SMP+Bacteria SMP+Fungicide


Leonardite shale
Sphagnum moss, soft coal
Calcined clay+Fungicide
Microcell, Vermiculite #5
Leonardite shale+Trichoderma

Bituminous charcoal+Trichoderma

Sphagnum moss+Trichoderma


Khan et al.,1992a


Khan et al.,1992b
Taylor et al.,1988

Parera et al.,1992


Harman and
Taylor,1988
Harman and
Taylor,1989

Harman et al.,1989



Taylor et al.,1988


Khan et al.,1992b
Kubicket al.,1988

Harman et al.,1989



Parera and
Cantliffe,1990b
Harman and
Taylor,1988
Parera and
Cantliffe,1990a
Cantliffe and
Bieniek,1988

Taylor et al.,1988

Kubicket al.,1988
Khan et al.,1992b
Harman and
Taylor,1988
Harman and
Taylor,1988
Harman and
Taylor,1988








23

types of products in to the mix. Harman and Taylor (1988)

utilized this advantage and combined leonardite shale or

bituminous coal with Trichoderma strains or Enterobacter

cloacae to control seed and soil borne diseases in cucumber

and tomato. Germination rate was increased and

postemergence damping-off was reduced in both species after

the treatment. Parera and Cantliffe (1990 a) reported no

effect of SMP with Trichoderma strains on the rate and final

emergence, and seedling vigor of sh2 sweet corns. However,

the same authors (Parera and Cantliffe, 1990 b) successfully

improved seed emergence and seedling growth of the same sh2

sweet corn, when the seeds were primed using calcined clay

as osmoticum and sodium hypochlorite as a seed disinfectant.

SMP and sodium hypochlorite also increased rate and final

germination of celery at high temperatures (Parera et al.,

1992). Khan et al. (1992a) reported an improvement on rate

of germination and marketable yield of table beet after SMP

using a combination of synthetic calcium silicate as solid-

matrix and fungicides.



Factors Affecting Seed Priming

Aeration

Several factors must be controlled during the priming

treatment in order to improve seed performance. Heydecker et

al. (1975) demonstrated that proper aeration of the osmotic

solution is essential to living seeds and helps synchronize








24

seed germination. However, later reports showed that the

response to aeration during treatment varies according to

the species. Onion seeds primed in aerated solution of PEG,

by using enriched oxygen, had greater germination compared

to seeds primed in nonaerated solutions (Bujalski et al.,

1989). Similarly, Heydecker and Coolbear (1977) reported

lower germination of onion seeds after priming in nonaerated

solution of PEG. No differences on final germination of

lettuce was observed after seed priming in aerated or

nonaerated solution of K3P04 (Cantliffe, 1981). The aeration

of the solution also modified the duration of the priming.

Regardless of the type of solutions or temperature used in

lettuce seed during priming, the seeds primed in aerated

solution required less soaking time to achieve higher

germination (Guedes and Cantliffe, 1980).

Light

The beneficial effects of seed priming could be

modified by the quality of light during the treatment. When

endive seeds were primed in a KNO3 solution under continuous

red light, they had higher germination percentage and lower

spread of germination compared to seeds primed in the dark

(Bekendam et al., 1987). Guedes and Cantliffe (1980)

reported that light during priming did not affect the seed

germination of lettuce 'Mesa 659' compared to seeds primed

in the dark. However, in other two cultivars 'Minetto' and

'Ithaca', the seeds had higher germination if they were








25

primed in light. Seed priming in a K3PO4 solution improved

germination of 'Valmaire' lettuce at high temperature

regardless of the light conditions. However, the seeds

germinated more rapidly if they were primed in the dark

(Cantliffe et al., 1981). Celery seeds primed in a solution

of PEG + benzyladenine had better germination at high

temperature when the priming treatment was done under light

(Nakamura et al., 1982).

Duration of the Treatment

The ideal duration of the treatment varies according to

the type of osmotica, osmotic potential of the solution,

temperature during the treatment, and specie. If radicle

protrusion occurs during seed priming, irreversible embryo

damage could be expected during dehydration after treatment.

The effect of duration of priming on pepper seed performance

was clearly demonstrated by Cantliffe et al. (1987). The

seeds where primed in a PEG solution for 4, 5 and 6 days.

The seeds primed for 6 days had the lowest germination rate,

but they produced almost 60% of abnormal seedlings compared

to 14% or 0% in seed primed for 5 or 4 days, respectively.

In some species such as lettuce the duration of priming

might be hours. Lettuce seeds need only 6 hours of soaking

in a Na2HPO4 solution to overcame thermodormancy (Guedes and

Cantliffe, 1977). Other species require weeks of soaking to

produce significant effects. Ely and Heydecker (1981)

recommended 3 weeks of soaking in PEG 6000 solution to








26

improve the germination rate of parsley. In tomato, one week

of priming in a PEG 6000 solution was the ideal seed priming

period to increase germination rate (Wolfe and Sims, 1982).

Argerich and Bradford (1990) recommended 5 days of priming

at 20C in a K2HPO4 + KNO3 solution to improve seed vigor.

Spinach seeds required 14 days of priming to improve

emergence compared to nonprimed seeds (Atherton and Faroque,

1983).

It is clear, that duration of seed priming depends on

the specie or seed type. However, temperature during

treatment and osmotic potential of the solution can affect

the length of the soaking period.

Temperature

Temperature during seed priming also influences the

effectiveness of the seed priming. If the temperature is

maintained below the optimum range for germination, it may

prevent seed germination during priming. At 25C, a high

percentage (63%) of carrot seeds germinated in the priming

solution (-0.5 MPa PEG), and no germination was observed at

15C (Elballa, 1987).

The temperature during treatment also appear to be

related to the osmotic potential of the solution, type of

osmotica, and length of the treatment. Heydecker et al.

(1975) reported different optimum temperatures during

priming according to the specie: 10C for onion, 15C for

beet, and 20C for carrot. Regardless of the osmotic








27

potential of a mannitol solution, onion seeds primed at 10C

had faster and greater final germination compared to seeds

primed at 24C (Furutani et al., 1986). The germination

percentage of lettuce seeds was greater after seed priming

at 15C compared to seeds primed at 5C or 25C (Cantliffe,

1981). Temperature during priming may alter the soaking

period in many species. Seed priming at 15C for 14 days was

more effective to improve seed germination of tomato,

carrot, and onion than 14 day at 25C (Haigh et al., 1986).

In the same experiment, 7 days of priming at 25C had the

same effect on seed germination of carrot and tomato than 14

days at 15C.

Osmotic Potential

Osmotic potential of the solution is another factor

affecting the length and effectiveness of the priming

treatments. Seed priming in low osmotic potential solutions

may induce seed germination during the treatment because a

deficient water uptake control. Increasing the osmotic

potential from -0.5 to -1.0 MPa prevented the germination of

carrot seeds in the priming solution (Elballa, 1987). A wide

range of osmotic potentials have been used with different

responses. Tomato seeds germinated more rapidly when the

osmotic potential of the solution was -0.58 or -0.86 Mpa

compared to -1.19 or -1.49 MPa, however no differences were

observed in onion seeds at the same range (Ali et al.,

1990). A small change in the osmotic potential of the








28

solution may affect the effectiveness of the treatment. When

pansy seeds were primed in a -0.8 MPa PEG solution, a

percentage of the seeds germinated during the treatment

(Carpenter and Boucher, 1991a). If the same seeds were

primed in a lower osmotic potential solution (-1.0 MPa) no

germination was observed during priming and the seeds had

improved emergence at high temperature stress compared to

nonprimed seeds. Higher germination rate of celery seed was

reported after priming in lower concentration (300 g/l) of

PEG 6000 compared to seed primed at more higher

concentration (400 g/l) (Singh, et al., 1985).

Seed Quality

Different response to seeds priming among seed lots has

been reported by numerous authors (Haigh and Barlow, 1987;

Heydecker and Coolbear, 1977; Bradford et al., 1990).

Perkins-Veazie and Cantliffe (1984) clearly demonstrated

that seed vigor is another factors influencing seed priming.

They reported that artificially aged seeds of 'Green Lakes'

and 'Montello' lettuce did not respond to priming treatment

in term of circumventing thermodormancy. Dearman et al.

(1986) after priming aged onion seeds concluded that the

loss of viability during ageing can not be restored by the

seed priming treatment. However, the seeds which are still

viable after ageing could improve germination rate after the

priming. Elballa (1987) recommended to use high quality

carrot seeds to obtain the best results from priming. High








29

vigor and free pathogen seeds are both essential requisites

for good priming results (Cantliffe et al., 1987). Suena

(1990) concluded, after study the relationship between

priming and seed vigor in 20 tomato genotypes and seed lots,

that priming accelerated the rate of germination of both

high and low seed lots. The difference response was a

function of the genotype rather than vigor of the seeds.

Because a few or no enough information about seed quality

and priming, all seed companies will not prime any seed lot

without a previous seed vigor analysis.

Dehydration After Priming

In order to achieve the maximum potential of priming

requires strict control of all the factors previously

discussed. However, following priming, dehydration and

subsequent seed storage become crucial components of the

technology for commercial application. To facilitate

handling and storage, the seeds should be redried to their

original fresh weight after the priming treatment.

Seed water uptake during priming is influenced, as

discussed above, by the osmotic potential of the solution,

type of osmoticum, duration of the treatment and /or the

physical characteristic of the seeds. As an example, seeds

of 'Mesa 659' lettuce imbibed more water, regardless of the

type of osmoticum used during priming, compared to 'Minetto'

or 'Ithaca' (Guedes et al., 1979). Knypl and Khan (1981)

reported that soybeans seeds primed in PEG solution (25








30

g/100 ml water) increased their original fresh weight 151%

after 4 days of treatment. Lettuce seeds increased up to 77%

their fresh weight after 6 h of priming (Guedes and

Cantliffe, 1980). Seeds of sh2 sweet corn primed via solid

matrix priming increased their original fresh weight up to

50-55% (Parera and Cantliffe, 1991).

Some controversial results of the effect of dehydration

after priming have been reported. According to Nerson and

Govers (1986), the beneficial effects of priming were

maintained in muskmelon 'Persia 202'. However in the

cultivar 'Noy Yizre'el' drying the seeds to the original

fresh weight significantly reduced germination. Seeds of

maize, soybean, wheat and barley primed in PEG solution had

earlier germination compared to nonprimed seeds; however,

after drying all the advantages of the priming treatment

were lost (Bodsworth and Bewley, 1981). The advantages of

seed priming were maintained after drying back Lolium

perenne seeds, which was contrary in five other primed

herbage grass seeds (Poa trivialis, Festuca rubra, Lolium

multiflorum, Phleum pratense, and Festuca ovina) wherein

final germination was reduced after priming and dehydration

(Adegbuyi et al., 1981).

Although temperature and rate of dehydration are both

critical factors affecting seed quality, little information

is available about their effects on primed seeds. Generally,

seeds are dried at room temperature and there are no








31

specifications regarding temperature and/or relative

humidity. When dehydration conditions are described, a wide

range of temperature and/or relative humidity were used. For

example, tomato seeds were dried at 30C after priming but no

specific relative humidity or length of drying back time

(Alvarado et al., 1987). After priming, cabbage seeds were

dried to their original moisture content at 7C and 45%

relative humidity (Perkins-Veazie et al., 1989). Onion

(Bujalski et al., 1989) and watermelon seeds (Sachs, 1977)

were dried to their original fresh weight at 15C after

priming. Primed lettuce seeds were dried at 21C and 40%

relative humidity (Guedes and Cantliffe, 1977). In all of

the above citations, conditions for dry back were not

studied, this seeds were just 'dried back'. Since critical

modifications at the seed membrane level occur during seed

dehydration (Burris and Navratil, 1989), more precise

information about the effect of temperature and/or relative

humidity after priming is necessary.

After seed priming and dehydration, seeds are usually

stored before planting. The conditions and duration of

storage may alter the advantages achieved during priming.

Primed spinach seed maintained a high germination at

stressful high temperatures after 1 month of storage

(Atherton and Faroque, 1983). Odell and Cantliffe (1986)

primed and stored (10C and 45% RH) tomato seeds for 3, 7, 10

and 19 months and evaluate the germination at 25 and 35C. At








32

25C, 10 months of storage did not affect final germination,

however under stress germination conditions (35C) after 7

months of storage the seeds had reduced germination. Akers

et al. (1987) reported that the priming effect in parsley

seeds, primed in an aerated solution of PEG 8000, was not

modified after 8 month of storage. Leek and carrot seeds

primed in PEG solution and stored at 10C for 12 months

maintained all the advantages achieved during the treatment

(Dearman et al., 1987). Some authors reported an interaction

between the osmotica used during the treatment and potential

storage capacity. Seeds of tomato primed in a KNO3 solution

had a low capacity to tolerate high temperature storage

(30C) compared to seeds primed in a PEG solution (Alvarado

and Bradford, 1988 a). Germination velocity was not affected

in nonprimed tomato seeds maintained for 6 months at 30C, in

the same period and condition primed seed in PEG or KNO3

lost almost 50% of the (Alvarado and Bradford, 1988 b).

Primed pepper seeds maintained at 35C for six months

maintained the higher rate of germination and final

germination compared to nonprimed seeds (Georghiou et al.,

1987). Viability and germination of primed tomato seeds were

severely deteriorated after six months of storage at 30C; no

deleterious effects was observed in seeds maintained at 4C

for the same period of time (Argerich et al., 1989). Eight

months of storage at 5C reduced germination of primed salvia

seeds (Carpenter, 1989). In the same experiment low (11%) or








33

high (95%) relative humidity storage conditions accelerated

the deterioration compared to 50% and 70% relative humidity.

The same author (Carpenter, 1990) reported no changes in

primed dusty miller (Senecio cineraria) seed performance

after 16 weeks of storage at 5C and 52% relative humidity.

Watermelon seeds primed in salt solutions maintained the

ability of rapid germination up to 20 weeks under dry

storage conditions (Sachs, 1977). Two months of vacuum

storage of primed Echinacea purpurea seeds declined the

advantages achieved after priming (Samfield et al., 1990).

Seed viability and the improvement of the germination rate

of primed onion seeds were not modified after 18 months of

storage at 10C (Dearman et al., 1986). Evidently, after the

analysis of the above information, the effect of storage

conditions after priming have been extensively studied. More

information about seed dehydration condition after priming

and potential effect on seed behavior are required.



Effect of Seed Priming on Seedling Growth and Development

Seed priming treatments modify axes growth and

subsequent seedling development. The response varies

according to the specie and priming conditions. Embryo

volume and cell number per embryo of leek and onion were not

modified by a priming treatment (Gray et al., 1990). In the

same experiment and conditions, the embryos of carrot

increased their volume almost 50% and the number of cells








34

two fold. The endosperm of 'Minetto' lettuce had a

progressive rupture of membrane after 9 h of priming and was

one of the potential factors to increase germination at high

temperature in this specie (Guedes et al., 1981).

The root growth of primed pepper seeds was analyzed by

Stoffella et al. (1992). They concluded that the number of

basal and lateral roots and taproot length, 14 days after

seeding, were not modified by seed priming. The root length

of primed lettuce seeds germinated at 35C was greater

compared to nonprimed seeds (Wurr and Fellows, 1984). The

root dry weigh of primed and nonprimed bell peppers, grown

with sprinkler irrigation or drip irrigation, was similar

after 50, 70 and 90 days after planting (Leskovar and

Cantliffe, 1992). Seed priming did not affect the rate of

radicle growth and degree of branching in tomato seeds after

germination (Odell and Cantliffe, 1986). Primed parsley

seeds yielded 52% more shoot fresh weight compared to

nonprimed seeds 24 days after sowing (Pill, 1986). The

differences in root or shoot growth between primed seeds and

nonprimed seeds is more evident under stressful conditions

and it was clearly demonstrated by the two following

reports. The root growth of perennial ryegras primed seeds

germinated at low temperature (5, 10, or 15C) was greater in

primed seeds compared to nonprimed seeds and no differences

was observed at 25C (Danneberg et al., 1992). Shoot dry

weight was higher in primed tomato germinated at stressful








35

conditions compared to nonprimed seeds (Odell et al.,

1992b).



Effect of Seed Priming on Yield and Harvest Quality

Synchronization and fast seed emergence are both

commonly reported benefits of seed priming. Particular

advantages of seed priming are augmented under adverse

conditions (Wiebe and Muhyaddin, 1987; Knypl and Khan,

1981). However, when yield and plant quality characters were

analyzed the effect of priming becomes more elusive.

Seed priming promoted early growth of aubergine,

pepper, cucumber and melon plants, but no differences were

detected in early and final yield, and fruit size between

primed and nonprimed seeds (Passam et al., 1989). Flowering

of processing tomato was earlier in primed plots compared to

nonprimed plots, but this was not reflected in early

maturation, yield and fruit soluble solid (Alvarado et al.,

1987). Similar result in the same specie was reported by

Argerich and Bradford (1990). Early growth of tomato was

improved by seed priming, but only under more stressful

conditions the priming treatment increase marketable yield

(Odell et al., 1992a). Seed priming did not increase final

yield of celery (Rennick and Tiernan, 1978; Tiernan and

MacNaidhe, 1981). Early yield in lettuce was higher in

primed seeds compared to nonprimed seeds (Cantliffe et al.,

1981). However, the number of marketable heads of lettuce








36

was not improved by priming treatment (Seale and Cantliffe,

1986). Soybeans primed seeds did not yield greater growth or

altered maturation dates (Helsel et al., 1986). Similar to

seedling growth and development, beneficial effects of

priming on yield and quality was only observed under stress

conditions.



Molecular and Physiological Aspect of Seed Priming

The physiological and biochemical effects of seed

priming during or after the treatment are not completely

understood. Many reports related seed priming treatment and

changes in seed nucleic acids. In a pioneer report, Sen and

Osborne (1974) demonstrated that during hydration of rye

embryos, synthesis of RNA, proteins and low levels of DNA

occur as a normal germination process. However, they

observed that these metabolites are stable after dehydration

and account for the ability of the embryo to rapidly

synthesize proteins and RNA after rehydration. Lettuce

seeds, primed in PEG 6000, had increased enzymatic activity

(acid phosphatase and sterase) and reduced time for RNA and

protein synthesis compared to nonprimed seeds (Khan et al.,

1978). There was no change on protein quality of pea axis

after soaking in osmotic solutions (Dell'Aquila and Bewley,

1989). The activity of amylase during germination of primed

pea and tomato, and peroxidase in primed tomato and spinach

was higher compared to nonprimed seeds (Zuo et al., 1988 a).








37

Coolbear and Grierson (1979) concluded that the high levels

of nucleic acids of primed tomato seeds may account for the

rapid germination of primed seeds. They observed that the

increase in nucleic acid amount was due to rRNA during and

after priming treatment. Rapid and high incorporation of 32P

by the RNA and DNA from embryonic axes of peanut during

priming was reported by Fu et al., (1988).

Tomato seeds started DNA replication after 2 days of

priming and reached a steady state at 14 days (Bino et al.,

1992). In the same study, the authors reported an increase

of the 4C DNA level in root tip cells. In leek embryos,

during 14 days of priming, reduced DNA synthesis activity

levels was observed and no cell division was detected (Bray

et al., 1989). However, when the seeds were germinated,

after 6 to 9 h of imbibition, DNA synthesis was greatly

intensify. Similar results were reported by Khan et al.

(1980) for lettuce seed. In this specie the lag phase to

initiate DNA synthesis was 6 h. In wheat embryos,

Dell'Aquila and Taranto (1986) reported a fast increased of

DNA synthesis after 12 h of imbibition.

Physiological studies showed an increase and

improvement of metabolic processes involved in germination

when primed seeds are rehydrated. Respiration of pepper

seeds was stimulated after priming (Malnassy, 1971). After

rehydration, respiration and ATP production were higher in

primed seeds of spinach, kohlrabi, pepper and eggplant








38

compared to nonprimed seeds (Mazor et al., 1984). In the

same experiment, dehydration of the primed seeds reduced

seed ATP levels but not below nonprimed seeds. Sundstrom and

Edwards (1989) reported more respiration in primed

'jalapeno' pepper seeds compared to control, however the

treatment reduced the rate of respiration in Tabascoo'

pepper seed. Primed seeds of pea and tomato (Zuo et al.,

1988); and peanut (Fu et al., 1988) released more ethylene

after rehydration compared to nonprimed seeds. Less

cytokinin activity was detected in celery seeds after

priming treatment (Thomas, 1984).

Maize embryos excised from primed seeds have more

phospholipids and steroles (76.2 and 8 gg respectively)

compared to embryos from nonprimed seeds (36.2 and 5 Ag)

(Basra et al., 1988 a,b). The same authors also reported

changes in phospholipids quality. Diphosphatidylglycerol was

the predominant in primed embryos and phosphatidylcholine

was in nonprimed embryos. The quality of membrane lipids of

peanut seeds was modified also by seed priming, where the

index of unsaturated fatty acid (IUFA) was significantly

higher in primed seeds compared to nonprimed seeds (Fu et

al., 1988).

Conclusions

There is no doubt about the beneficial effects of the

priming treatment on rate and synchronization of the

germination. The success or failure of the priming treatment








39

are influenced by a complex interaction of factors such as

species, osmoticum, osmotic potential of the solution,

duration of the treatment, temperature, seed vigor,

dehydration and storage conditions.

Despite of the quality and quantity of studies

reporting the molecular and physiological effects of seed

priming, the biochemical mechanism on priming is still

unclear. Bradford (1986) postulated that during the

treatment there is solute accumulation in the seed and no

germination occurs because the osmotic potential of the

solution is below the threshold required for embryo growth.

During dehydration the level of solutes in the embryo are

maintained, thus during reimbibition the low osmotic

potential and higher turgor potential of the embryo may

induce fast hydration and growth. Gray et al.(1990)

concluded that the production of osmoticc solutes actives'

was not the main reason for improving germination rate in

primed seeds. They suggested that during priming the seeds

are artificially maintained in phase II of imbibition, and

the substances generated in this latent period may increase

cell wall extensibility or removing restrictions for radicle

growth. Karssen et al. (1989) arrived at the same conclusion

after study the effect of priming in tomato, celery and

lettuce.

Although the active participation of membranes in seed

hydration and dehydration mechanisms, their role during and








40

after priming have been not extensively studied. Recently,

Basra et al. (1988 a,b) reported changes in quantity and

quality of membrane phospholipids during and after priming.

Parera and Cantliffe (1991) demonstrated that sweet corn

seeds primed via SMP had less seed leakage and reduced water

uptake rates during early imbibition compared to nonprimed

seeds. Similar results were reported by Zuo et al. (1988 b)

in pea seeds after priming. The role of membrane role during

and after priming is still an open question.

Finally, more practical and essential subjects such as

seed vigor and priming effectiveness, effects of dehydration

after priming, storage conditions, ideal combination of

matrix-solid, water, and seeds in SMP treatments are in

need of further studies.














CHAPTER III
FIELD EMERGENCE OF shrunken-2 CORN PREDICTED BY
SINGLE AND MULTIPLE VIGOR LABORATORY TESTS



The poor seed emergence and vigor in sweet corn with the

shrunken-2 (sh2) gene has been attributed to a disfunction

of the scutellum in relation to carbohydrate utilization

(Styer and Cantliffe, 1984a), high susceptibility to seed

and soil-borne diseases (Berger and Wolf, 1974), and

potential imbibitional injury potentially affecting membrane

integrity (Chern and Sung, 1991; Parera and Cantliffe,

1991).

High quality seeds are essential to achieve a proper

plant population and crop uniformity, and both are primary

factors affecting crop yield and quality in sweet corn

wherein the crop is harvested once over. The standard

(towel) germination test is poorly correlated with field

emergence in some species (Perry, 1978). Perry (1981)

defined vigor tests as representing field performance of the

seed more completely than germination tests. Several

attempts have been made to develop a standardized vigor test

to predict field emergence for various species. To date,

after 40 years of research, only the conductivity test for

one specie, garden peas (Pisum sativum L.), is








42

internationally accepted as indicator of seed vigor (Hampton

and Coolbear, 1990). An ideal vigor test must be repeatable,

rapid and inexpensive, and able to reflect the potential of

the seed lot under unfavorable as well as favorable

conditions (Matthews, 1981).

The soil cold test is one vigor test recommended by the

Association of Official Seeds Analysts (AOSA) (1983) to

predict seed vigor in corn. Stress conditions such as low

temperature and high soil moisture content are simulated

under laboratory conditions. High correlation between the

test and field emergence of corn has been reported (Martin

et al., 1988), however, contradictory results were reported

by Fiala (1987). The cold test has produced variable results

in corn among different laboratories (Burris and Navratil,

1979). Biochemical activity of the seed may predict seedling

performance (Perl, 1987).

The conductivity test is a biochemical test recommended

by AOSA (1983) as another potential indicator of vigor

status of corn seeds. This test is quick in that the seeds

are soaked and the electric conductivity of the leachate is

measured after a short period of time. Negative correlations

between the conductivity test and field emergence of sugary

(su) and sh2 sweet corn have been reported in soils with

temperatures below 20C (Waters and Blanchette, 1983; Tracy

and Juvik, 1988). Activity of seed enzymes such as glutamic

acid decarboxylase has also been correlated with seed vigor








43

(Grabe, 1964) or seed deterioration in corn (Bautista and

Linko, 1962).

The complex stressing vigor test is a successful

laboratory test for predicting field emergence in wheat

(Triticum aestivum L.) and field corn (Barla-Szabo and

Dolinka, 1988). The test is divided into a stress period,

24h soaking at 25C and 24h at 5C, followed by germination in

a paper towel at 25C. The accelerated ageing test is

another approach proposed to predict field emergence. The

test has a wide acceptance for evaluating seed quality of

soybean (TeKrony, 1973). Repeatability problems of the

accelerated aging test have been reported in sh2 sweet corn

(Wilson and Trawatha, 1991) and other species (Perry, 1984).

In a preliminary experiment, sh2 sweet corn seeds were

severely affected by pathogens during high humidity storage,

thus giving misleading results concerning the potential

behavior of the seeds in the field.

Our objective was to develop a reliable, rapid, and

economic laboratory test or series of tests to predict field

emergence and vigor of sh2 sweet corn grown in diverse

environments and soil types.



Material and Methods

Six sh2 sweet corn cultivars: Sweet Belle, Even Sweeter,

Dazzle, and Challenger from Asgrow Seed Co., and How Sweet

It Is and Crisp N'Sweet 711 from Crookhan Seed Co., and









44

breeding line XPH-3009 from Asgrow Seed Co. were used in

this study. The seeds were stored at 10C and 45% RH during

the experiment, and no seed treatments were applied.

Bulk Conductivity Tests

Twenty seeds were soaked in 25 ml of distilled water at

15 (C13), 25 (C23), or 30C (C33). The electrical

conductivity of the leachate was measured each hour up to 24

h by a conductivity meter (Lecto Mho-meter, Lab-Line

Instruments Inc., Melrose Park, Ill.) and expressed as gmhos

g'1 of seed. Conductivity measured after 3 h soaking was used

in the correlation analysis.

Alternate Stress Temperature Conductivity Test

Electrical conductivity of the leachate was measured

and calculated, as described in the bulk conductivity test,

after the seeds (20) were soaked in 25 ml of distilled water

at 5 or 30C for 3 h, then transferred to 30 (C536) or 5C

(C356) respectively, for an additional 3 h. Also, a value

was derived which was the mean between the conductivity

after the initial 3 h of soaking and the final conductivity

after 6 h soaking. This was calculated for each soaking

treatment 5/30C (AC53) and 30/5C (AC35).

Embryo Conductivity

To remove intact embryos (axis plus scutellum) seeds

were incubated at 100% RH and 15C during 4 days to soften

the tissue and avoid embryo damage (Bruggink et al., 1991).

Embryos were dried at 30C for 3 days. Ten embryos were then








45

soaked in 25 ml distilled water at 25C and electrical

conductivity of leakage was recorded every hour for 24 h,

and expressed as gmhos g- of embryo. The conductivity of

the leachate after soaking for 3 (EC3) and 6 h (EC6) were

used for the correlation analysis.

Cold Germination Test

This test was performed according to standard AOSA

procedures (1983). Fifty seeds were sown in a plastic box

(25 x 25 x 10 cm), filled with 2.5 cm of Arredondo fine

sandy soil (loamy, silaceous, hyperthermic Grossarenic

Palenundult) (GCTS) or Terra-Lite vermiculite (Grace & Co.

Cambridge, MS) (GCTV). The soil or vermiculite were

compacted and another 2.5 cm layer of substrate was placed

on top of the seeds. The media were adjusted to 70% of their

water holding capacity. The containers were sealed and

incubated at 10C for 7 days, then transferred to 25C for 4

days. Total emergence percentage was calculated. Seedlings

with leaves 2 mm in length above the soil were considered

germinated.

Standard Germination Test

The test was performed according to AOSA procedures

(1983). Twenty five seeds were placed on 3 layers of moist,

non-toxic, germination paper (Anchor paper Co. St. Paul,

MN). The papers were rolled, placed in a plastic container

(21.5 x 32.5 x 5.5 cm), and incubated in a dark germinator

at 15 (GR15), 25 (GR25) or 30C (GR30) for 7 days. Dry weight








46

of the shoot and root (DW15, DW25, and DW30) was also

recorded for each test.

Complex Stress Vigor Test

Two hundred seeds were soaked 24 h in 250 ml of

distilled water at 25C then transferred at 5C for additional

24 h (Barla-Szabo and Dolinka, 1988). After soaking, the

rolled towel test was performed, where the seeds were placed

with the radicle pointing downwards in a germinator and

incubated at 15 (ST251), 25 (ST252), or 30C (ST253) for 96

h. Also, a 25 or 30C soaking temperature combination was

used with the same range of incubation temperatures (ST231,

ST232, and ST233). The conductivity of the leachate after

each soaking period was measured as described in the bulk

conductivity test. An index of combination between

conductivity and germination percentage was calculated as:


ICS= 1 G
AVC



where ICS is the index of conductivity and complex stressing

vigor test; AVC is the average conductivity between the two

soaking periods expressed in gmhos g' seed, and G is the

final germination percentage of the towel test. ICS was

calculated for each complex stressing test (ICS253, ICS252,

ICS251, ICS233, ICS232, and ICS231).

Glutamic Acid Decarboxvlase Activity Test (GADA)

The enzyme activity was measured with a Gilson








47

differential respirometer (Gilson Medical Electronics, Inc.

Middleton, WI) and expressed in glCO2 g-1 min" at 25C. Seeds

(5 g) were ground (Virtis, Virtis Co., Gardiner, NY) at high

speed for 2 min. In a reaction flask 1 g of the ground seed

was mixed with 2.5 ml solution of 0.1 M glutamic acid in

0.067 M phosphate buffer at 5.8 pH (Ram and Wiesner, 1988).

The flasks were incubated at 25C in a water bath and

agitated at 100 oscillations/min. After 10 min stabilization

the CO2 production was measured for 10 min.

Field Evaluation

Field trials were established on 28 March, 18 April, and

3 December, 1991 at the Institute of Food and Agricultural

Science (IFAS, UF) Horticultural Unit in Gainesville, FL on

an Arredondo fine sand soil (loamy, silaceous, hyperthermic

Grossarenic Palenundult); 4 April and 3 December, 1991 at

the IFAS Agricultural Research and Education Center, Fort

Pierce FL on a Oldmar fine sand soil (sandy, siliceous,

hyperthermic Alfic Arenisc Haplaquods); and 9 September and

18 December, 1991 at the Everglades Research and Education

Center, Belle Glade FL on a Pahokee Muck (evic, hyperthermic

Lithic Medisaprist). Fifty seeds were seeded 4 cm deep in

two rows in each plot. The plots size, fertilization,

irrigation system and mean temperature for each site are

described in Table 1. Emergence Rate Index (ERI) (Shmueli

and Goldberg, 1971) and percent emergence were calculated

for each plot.










Statistical Analyses

Each laboratory test and field emergence experiment was

conducted as a randomized complete block design, with four

replications. The SAS statistical package (SAS Institute,

Cary, NC) was used for data analysis. Percentage data were

arcsine transformed before analysis. Correlation

coefficients were determined between laboratory tests and

field emergence or ERI. Simple correlation analysis was used

to select the best single vigor test to predict field

emergence of the cultivars. Multivariate factor analysis was

used to separate independent, non-collinear vigor tests that

were subsequently used in multiple regression models

(Steiner et al., 1989). The variables were rotated using the

varimax orthogonal rotation (Delis and Adams, 1978).



Results and Discussion

High variability of seed emergence (7 to 92%) in the

field, occurred among cultivars, and locations (Table 3-1).

A wide range of environments was obtained when the

temperatures (sowing date) and soil types of the locations

were combined (Table 3-2). The seedling emergence percentage

varied significantly among field locations. Cultivar (C) x

location (L) interaction for field emergence percentage was

significant. Since the sums of square of the (CxL)

interaction represented only 6.9% of the total as compared

to 74.7% of cultivar differences (Table 3-1), all the












Table 3-1. Means and Anova table for seed field emergence of
the 7 sweet corn cultivars and 7 sowing locations and
planting date



Location

Gainesville Fort Pierce Belle Glade

Cultivar 28 Mar. 18 Apr. 3 Dec. 4 Apr. 3 Dec. 9 Sept. 18 Dec.

Emergence %
Sweet Belle 82 84 76 42 73 83 84
Even Sweeter 38 43 54 18 32 15 39
Dazzle 35 49 12 35 40 35 54
XPH-3009 26 42 12 27 22 24 41
Challenger 74 78 64 50 55 84 79
How Sweet It Is 25 46 7 38 33 16 36
Crisp N' Sweet 89 89 72 85 70 92 91

ANOVAW Source df MS F

Location (L)b 6 0.3555 8.05"
Error a 21 0.0441
Cultivar (C) 6 2.3207 346.33"
C x L 36 0.0397 53.06"
Error b 126 0.0067
Total 195

* Transformed data.
b Location and planting date
SSignificant at the P= 0.001.












Table 3-2. Sowing date, soil type, bed size, fertilization,
irrigation system and average temperature for the locations
used in the study.


Location

Gainesville Fort Pierce Belle Glade

28 Mar. 18 Apr. 3 Dec. 4 Apr. 3 Dec. 9 Sept. 18 Dec.

Sowing
date 3/28 4/18 12/3 4/4 12/3 9/9 12/18

Soil type Sandy Sandy Sandy Sandy Sandy Muck Muck

Bed size (m)
Lenght 7.6 7.6 7.6 7.6 7.6 7.6 7.6
Wide 1.2 1.2 1.2 2.1 2.1 0.7 0.7
Apart 0.7 0.7 0.7 1.1 1.1 0.5 0.5

Fertilizer
(Kg ha-')
Nitrogen (N) 180 180 180 153 153 0 0
Phosphorous (P) 60 60 60 67 67 97 97
Potassium (K) 180 180 180 127 127 0 0

Irrigation Overhead Sub- Sub-
sprinkler surface surface

Mean'
temp.(C) 21.3 23.5 13.1 23.8 19.7 26.1 21.6

* Average air temperature of the first 10 days after sowing









51

correlation coefficients, factor analysis and multiple

regression coefficients were performed on the pooled means

of the 7 planting date and place combinations.

The simple linear correlation coefficients between the

laboratory vigor tests and field emergence and ERI ranged

from 0.97 to 0.54 and 0.92 to 0.53, respectively (Table 3-

3). The index of conductivity and complex stressing vigor

test (ICS251) (25/5C soaking time 15C incubation) had the

highest correlation coefficient to field emergence (r=0.97")

and ERI (r=0.97"). The index of conductivity and complex

stressing vigor test calculated when the incubation

temperature was 25C (ICS252) was also a good predictor of

field emergence and rate of emergence (r=0.94").

The performance of the conductivity tests was consistent

with the results reported by Waters and Blanchette (1983)

and Tracy and Juvick (1988). A higher negative correlation

coefficient occurred when field emergence and ERI were

correlated with bulk conductivity tests at 25C (C23) or 30C

(C33) soaking temperature and alternate stress temperature

conductivity test (AC35). The bulk conductivity test is a

rapid and simple method to measure seed vigor by evaluating

damage to seed coats and cell membranes. Several reports

suggest that leakage electrical conductivity should be at

least one of several variables to assess seed vigor

(Loeffler et al., 1988; Tracy and Juvick, 1988).













Table 3-3. Simple linear correlation coefficients (r)
between laboratory vigor tests and seed field emergence and
emergence rate index (ERI) of 7 sh2 sweet corn.


Laboratory Field' Emergenceb
Test Emergence Rate Index

-----------r--------


ICS251
C23
GCTS
AC35
C33
GR30
ICS252
C13
C356
ICS253
C536
ST253
DW15
ICS232
AC53
ST251
ST232
ST252
GADA
ICS233
DW25
GR25
ICS231
ST233
ST231
EC3
EC6
GR15
GCTV
DW30


0.97"
-0.95"
0.95"
-0.94"
-0.94"
0.94"
0.94"
-0.93"
-0.93"
0.91"
-0.90"
0.89"
0.89"
0.88"
-0.88"
0.88"
0.87"
0.86"
0.85"
0.85"
0.84"
0.84"
0.83"
0.83"
0.82"
-0.82"
-0.81"
0.70"
0.57*
0.54*


0.97"
-0.95"
0.94"
-0.93"
-0.93"
0.93"
0.94"
-0.93"
-0.93"
0.92"
-0.90"
0.90"
0.89"
0.87"
-0.87"
0.88"
0.85"
0.87"
0.86"
0.83"
0.82"
0.83"
0.82"
0.81"
0.81"
-0.81"
-0.81"
0.70"
0.57"
0.53*


*, Significant at the P= 0.05 or 0.01 respectively.
ab Data pooled over locations and planting dates.








53

The cold test was a good predictor of seedling emergence

(r=0.95") and ERI (r=0.94") when soil (GCTS) was used as a

substrate. However, the cold test had the lowest correlation

coefficient (r=0.57') when vermiculite (GCTV) was the

substrate. Fiala (1987) reported that seed quality and

characteristics of the substrate (type, pH, moisture

content, and microorganism activity) highly influence the

results in the cold test. In our experiment, vermiculite

exhibited a poor correlation with field emergence and ERI

compared to soil. The results confirm the potential

difficulties with standardizing the cold test among

laboratories as previously reported by Burris and Navratil

(1979).

Multiple stepwise procedures have been used to combine

two or more vigor tests to predict field emergence (Abdul-

Baki and Anderson 1973; Hall and Wiesner, 1990). In wheat,

Steiner et al. (1989) reported a high collinearity among

laboratory vigor tests used to predict field emergence. The

prediction of field emergence was not improved by adding a

collinear test to the model, thus a limited number of

potential tests were available for use as a valid multiple

regression model. In this experiment, four factors resulted

after the factor analysis and the varimax rotation (Table 3-

4). The following independent (noncollinear) laboratory

vigor tests were selected to calculate multiple linear

models: AC53 and GADA (Factor 1); ICS233, ICS231, ST233,













Table 3-4. Factors originated after orthogonal rotation
(varimax) from laboratory vigor tests to predict field
emergence of sh2 sweet corn.




Lab. Vigor Test FACTOR FACTOR FACTOR FACTOR


AC53
GADA
C536
C356
AC35
C13
GCTS
ICS253
ST253
ICS252
ICS232
ST232
C33
ICS251
C23
ST251
GR30
DW15
EC6
EC3
DW25
ICS233
ICS231
ST233
ST231
ST252
GR15
GCTV
GR25
DW30


0.78598
-0.75394
0.78681
0.78037
0.77238
0.72944
-0.64931
-0.56658
-0.54300
-0.51171
-0.44635
-0.43615
0.68593
-0.62031
0.61599
-0.50956
-0.46855
-0.61807
0.50860
0.48517
-0.39829


-0.36709
-0.44873
-0.44557
-0.42072
0.51921
0.67988
0.60307
0.73505
0.76838
0.72570
-0.40919
0.62875
-0.44418
0.48088
0.46327




0.82848
0.80694
0.78331
0.76731
0.66831


-0.36821
0.37319
-0.41942
0.53403
0.47295
0.36612
-0.72481
-0.74535





0.48017
0.85717
0.83199
0.72549


0.36701



0.66902








0.83605


Var. Explained (%) 34.95


33.77


22.44 8.84








55

ST231 (Factor 2); GR15, GCTV, and GR25 (Factor 3); and DW30

(Factor 4). Multiple stepwise procedure was used to

determine the regression equations to predict field

emergence. The best two-variable combination (test) included

average alternate stress temperature conductivity test

(AC53) and standard (towel) germination test incubated at

25C (GR25) (Table 3-5). However the R2 was lower than the

best single regression model. The highest three variable

(test) model equation incorporated AC53, GR25 and GADA. The

correlation coefficient (0.93"') was equal to the best

simple linear predictor. GADA has been reported as a good

indicator of seed performance in corn (Grabe, 1964; Bautista

and Linko, 1962); beans (Phaseolus vulgaris L.) (James,

1968), and wheat (Steiner et al., 1989).

The seed is a complex biological system where several

factors such as genetic characteristics, seed and soil borne

diseases, environmental conditions, and seed aging can

affect vigor. The complex mechanism of germination includes

a sequence of physical, biological, and biochemical

components which coexist and interact. This complexity is

very difficult to evaluate with a single laboratory test.

One test is not enough to predict the behavior of the seed

under variable environmental conditions after sowing in the

field. The combination of biochemical and physiological

vigor tests has been proposed as a valid alternative to

predict seed emergence (Ram and Wiesner, 1990; Hampton and










56

Table 3-5. Best (higher R2) one- or two-, and three- non
collinear factor models for predicting field emergence by
laboratory vigor tests in sh2 sweet corn.



Regression model R2 "

20.133 + 12.350 ICS251 0.94
117.542 0.684 C23 0.90
25.144 + 0.825 GCTS 0.90
105.305 0.439 C33 0.88
15.970 + 0.744 GR30 0.88
23.219 + 12.610 ICS252 0.88
59.087 0.375 AC53 + 0.396 GR25 0.89
28.127 0.242 AC53 + 0.344 GR25 + 0.1513 GADA 0.93

* Significant at P= 0.001









57

Coolbear, 1990). The best single predictor of field

emergence for sh2 sweet corn in this experiment was an index

where the conductivity of the leachate and germination under

stress conditions were combined (ICS). Also, the best two

non-collinear test included the conductivity of the leachate

at stress temperatures (AC53) and a standard (towel)

germination test at 25C (GR25).

The ICS proposed to estimate field emergence combines two

main factors affecting seed emergence and seedling vigor of

sh2 sweet corn: the conditions of the membrane system of the

seed and potential susceptibility to seed pathogens. The

electrical conductivity of the leachate considers the first

factor. The germination percentage after the towel test at

stress temperature may evaluate the tolerance of the seed

and seedling to environmental stress and potential

susceptibility to seed and soil borne pathogens.

The advantages of the index of conductivity and

germination of the complex stressing vigor test herein

proposed compared to other vigor tests recommended to

evaluate field emergence of sh2 sweet corn are: non-

subjective, simple, and easy to standardize among

laboratories.



Summary

Poor emergence and seedling vigor are common

characteristics of many sweet corn (Zea mays L.) cultivars








58

with the shrunken-2 (sh2) mutant endosperm. A rapid and

reliable predictor of sweet corn seed field emergence is

required to produce high quality uniform crops. Field

emergence of seven sh2 sweet corn cultivars grown at 3

geographic locations in Florida over 2 planting periods

(fall and spring) were correlated with laboratory vigor

tests. Factor analysis was used to separate non-collinear

vigor tests for subsequent multiple regression models. The

best single predictor test (R2=0.93"') was an index based on

conductivity of the leachate and germination percentage

after complex stressing vigor test incubated at 15C. Leakage

conductivity after 3 h soaking at 25 or 30C (R2=0.90"*), cold

test in soil (R2=0.90**), mean alternate temperature stress

conductivity test(R2=0.88""), standard germination test

incubated at 30C (R2=0.88"'), and the index incubated at 25C

(R2=0.88"') were also good predictors of field emergence.

Non-collinear tests including the towel germination test at

25C and an alternate temperature stress conductivity test

generated the highest most significant two factor predictor

(R2=0.89"*), and with glutamic acid decarboxylase activity

(GADA) the best three factor predictor (R2=0.93*'). The index

of conductivity and complex stressing vigor test (ICS)

proposed as a predictor of seed emergence considered two

main factor affecting emergence in sh2 sweet corn: the

condition of the membrane of the seeds and potential

pathogen infection.














CHAPTER IV
IMPROVEMENT OF EMERGENCE AND SEEDLING VIGOR IN
shrunken-2 SWEETCORN VIA SEED DISINFECTION
AND SOLID MATRIX PRIMING


Seed priming is used to increase germination rate,

improve stand establishment, uniformity, and increase yield

(Khan et al., 1980). Seed priming consists of imbibing seeds

in an osmotic solution that allows seeds to imbibe water and

go through the initial germination stages, but prevents

radicle protrusion through the seedcoat (Cantliffe, 1981).

Priming treatments have been reported as successful

presowing treatments for many species (Bradford, 1986).

Priming corn seed has yielded variable results. The

emergence rate of corn germinated at cool temperatures was

improved by priming in a polyethylene glycol (PEG) solution

(Bodsworth and Bewley, 1981). Seed of sugary (su) and sh2

sweet corn genotypes primed with PEG 8000 had lower field

emergence than nontreated seeds (Bennett and Waters, 1987).

Also, the aeration of the solution, the large volume of

solution needed per seed, and the large amount of seeds

required for commercial use has restricted osmotic priming

for large seeded species.

Solid matrix priming (SPM) is a priming method wherein

seeds are moistened for a given time at constant temperature








60

in an organic or inorganic solid matrix carrier to which

water has been added (Harman and Taylor, 1988). The SMP uses

the osmotic and physical characteristics of the solid

carrier to restrict water absorption (Kubik et al., 1988).

Early results with SMP on sh2 sweet corn were not always

favorable. Rate of emergence and stand uniformity of sh2

sweet corn sown in the field were not improved by SMP

compared to nonprimed seeds (Cantliffe and Bieniek, 1988).

Seedling emergence was enhanced by SMP in 'Jubilee' sweet

corn, but was reduced in 'Florida Staysweet' compared.to

nontreated seeds (Harman et al., 1989). Fungicide seed

treatments have been reported to improve stand establishment

and uniformity in supersweet corn cultivars (Cantliffe et

al., 1975; Parera and Cantliffe, 1990 a).

The poor seedling emergence in sh2 sweet corn cultivars

has been attributed to several factors such as low seed

vigor (Styer and Cantliffe, 1983), high susceptibility to

seed and soil-borne diseases (Berger and Wolf, 1974), and

imbibitional damage (Parera and Cantliffe, 1991). Solid

matrix priming combined with sodium hypochlorite successfully

improved laboratory seed germination of sh2 sweet corn

(Parera and Cantliffe, 1991). After priming, the sh2 seeds

had low water uptake and leakage conductivity.

The objective of this study was to evaluate a SMP

treatment which would consistently improve emergence rate

and total emergence of sh2 sweet corn cultivars under








61

varying field conditions. To effectively prime sh2 sweet

corn, SMP has to control seed borne pathogens.



Materials and Methods

Plant Material

Seeds of two sh2 sweet corn cultivars, yellow kernel

Crisp N'Sweet 711 (CNS-711) and white kernel How Sweet It Is

(HSII) (Crookham Seed Co., Caldwell, Idaho) were used in

this study.

Seed Treatments

Surface disinfection was accomplished by soaking 200

seeds for 15 min in a 0.05% solution (v/v) of sodium

hypochlorite (SH). Solid matrix priming consisted of placing

3 g of seed, 6 g of calcined clay, and 2.5 ml (HSII) or 2 ml

(CNS-711) of distilled water in a closed container

continuously rotated at 5C for 6 h, then transferring the

sample to 25C and rotating it for 24 h. After 30 h of

incubation, 2 ml (HSII) or 1.5 ml (CNS-711) of water (SMP)

or sodium hypochlorite solution (0.05%) (SMPsh) was added

and incubated an additional 15 h (Parera and Cantliffe,

1991). The fungicide combination seed treatment consisted of

soaking 200 g of seed for 2 min in 1 liter solution of

imazalil [(1-(2-(2,4-dichlorophenyl)-2-(2-

propenyloxy)ethyl)-lH imidazole)] at 0.653 ml/kg seed,

captain [N-[(trichloromethyl)thio]-4-cyclohexene-1,2-

dicarboximide] at 1.958 ml/kg seed, apron [N-(2,6-








62

dimethylphenyl)-N-(methoxyacetyl) alanine methyl ester] at

0.488 ml/kg seed, and thiram [tetramethylthiuram disulfide]

at 3.264 ml/kg seed (Parera and Cantliffe, 1990 a). After

treatment, the seeds were dried back to their initial

moisture content (6%) in a incubator at 25 1C and 45%

relative humidity (RH). The seeds were stored at 10C and 45%

RH before and after treatment.

Cold Germination Test

Twenty seeds were sown in a plastic box (18.7 x 12.5 x

9 cm) on top of 2.5 cm of soil (Arredondo fine sand, loamy,

silaceous, hyperthermic Grossarenic Palenundult) taken from

a field where corn was grown continuously for 2 years. The

soil was compacted and another 2.5 cm of soil was placed on

top of the seeds. The soil was adjusted to 70% of its water

holding capacity. The containers were sealed and incubated

at 10C for 7 days, then transferred to 25C for 4 days. Total

germination percentage was calculated. Seedlings with leaves

2 mm in length above the soil were considered germinated.

Field Studies

Field plots were established on 26 October, 1989 and 7

March, 27 April, and 8 November 1990 at the Institute of

Food and Agricultural Science (IFAS) Horticultural Unit in

Gainesville, Florida on an Arredondo fine sand soil (loamy,

silaceous, hyperthermic Grossarenic Palenundult). The plots

were 7.60 m long on beds 1.22 m apart, with each bed 0.70 m

wide and 0.20 m in height. Two seeds were sown 4 cm deep,








63

every 30 cm in each plot (50 seeds/plot). Overhead

sprinkler irrigation was applied as needed. Emergence rate

index (ERI) (Shmueli and Goldberg, 1971) and percent

emergence were calculated. Five seedlings were cut at the

soil level 22 (DAS), weighed, and then dried at 75C for 72 h

and reweighed. In the March and April sowings, the central 6

m of each plot were harvested. The ears were classified

according to US Dept. of Agriculture (1954) quality

standards, then counted and weighed. Daily maximum and

minimum soil temperatures at the 5 cm depth were recorded

for each planting.

Statistical Analyses

The experiments were conducted as a randomized complete

block design, with treatments replicated 4 times. Percentage

emergence data were analyzed after square root arc sine

transformations. Main effects of treatments were partitioned

into orthogonal contrasts.



Results and Discussion

Since the interaction of treatment x cultivar and

treatment x sowing date were significant, main effects were

partitioned and analyzed for each cultivar and sowing time.

Only 4% of the nontreated HSII and 31% of CNS-711 seeds

germinated in a cold test experiment (Table 4-1). The SMPsh

treatment significantly improved germination in both

cultivars (up to 33% in HSII and 46% in CNS-711) compared to












Table 4-1. Effect of seed treatments on germination of two
sh2 sweet corn cultivars in a cold germination test.


Cultivar

HSII CNS-711

Germination Germination
Seed treatment (%) (%)

SMPsh 33 46
SMP 2 13
SH 0 2
F 20 57
C 4 31
Orthogonal Contrast
SMPsh vs C ** *
SMP vs SH ns **
SMPsh vs SMP ** **
SMPsh vs F ns ns

SSMPsh: Solid Matrix Priming + Sodium Hypochlorite.
SMP: Solid Matrix Priming
SH: Sodium Hypochlorite
F: Fungicide
C: Control
."" Nonsignificant or significant at P= 0.05 or 0.01,
respectively.








65

non-treated seeds. In both cultivars, the germination of

seed treated with fungicide did not significantly differ

from the SMPsh treated seeds.

The cultivar CNS-711 had earlier seedling emergence

(higher ERI), higher emergence percentage and seedling vigor

as compared to HSII in the field test in fall 1989 (Table 4-

2). The seed treatments did not significantly improve the

emergence percentage and seedling vigor of CNS-711. In HSII

cultivar, SMPsh treatment significantly improved emergence

percentage, ERI and dry weight of the seedlings compared to

nontreated seeds (Table 4-2). The SMPsh and fungicides

treatments produced the highest and faster emergence, but

were not significantly different from each other.

Similar to fall 1989 trials, CNS-711 had more rapid

emergence and greater emergence and seedling vigor than HSII

in both spring 1990 sowings (Tables 4-3 and 4-4). The SMPsh

treatment significantly improved the emergence rate compared

to nontreated seeds in both cultivars at both plantings

(March and April). The SMPsh and fungicide treatments both

increased final emergence and ERI of HSII compared to the

other treatments. Under more stressful conditions (higher

soil temperature) in April (Figure 4-1) the SMPsh treatment

also significantly enhanced HSII seedlings dry weight over

the control, and rate of emergence compared to SMP treatment

alone (Table 4-4). There were significant differences in

yield among treatments in March and April 1990 trials (data












Table 4-2. Effect of seed treatments on emergence rate index
(ERI), emergence percentage, and dry weight (DW) of two sh2
sweet corn cultivars in a field experiment planted in 26
October, 1989 at Gainesville, FL.




Cultivar

How Sweet It Is Crisp N'Sweet 711

ERI Emer DW a ERI Emer DW
Seed treatment (%) (mg) (%) (mg)

SMPsh 76 63 71 122 90 165
SMP 70 50 69 121 81 175
SH 30 27 55 105 89 153
F 79 74 73 116 94 168
C 38 44 32 107 89 135
Orthogonal Contrast
SMPsh vs C ** ** ns ns ns
SMP vs SH ** ** ns ns ns ns
SMPsh vs SMP ns ns ns ns ns ns
SMPsh vs F ns ns ns ns ns ns

' Values are means of 20 plants 19 DAS.
b SMPsh: Solid Matrix Priming + Sodium Hypochlorite.
SMP: Solid Matrix Priming
SH: Sodium Hypochlorite
F: Fungicide
C: Control
"'* Nonsignificant or significant at P= 0.05 or 0.01, respectively.












Table 4-3. Effect of seed treatments on emergence rate index
(ERI), emergence percentage, and dry weight (DW) of two sh2
sweet corn cultivars in a field experiment planted in 7
March, 1990 at Gainesville, FL.



Cultivar

How Sweet It Is Crisp N'Sweet 711

ERI Emer DWa ERI Emer DW
Seed treatment b (%) (cm) (%) (mg)

SMPsh 217 66 2240 370 98 1117
SMP 100 34 645 343 93 1110
SH 118 42 715 364 93 1475
F 275 80 1125 386 95 1115
C 124 42 955 323 91 1136
Orthogonal Contrast
SMPsh vs C ** ** ns ** ns ns
SMP vs SH ns ns ns ns ns ns
SMPsh vs SMP ** ** ns ns ns ns
SMPsh vs F ns ns ns ns ns ns

I Values are means of 20 plants 27 DAS.
b SMPsh: Solid Matrix Priming + Sodium Hypochlorite.
SMP: Solid Matrix Priming
SH: Sodium Hypochlorite
F: Fungicide
C: Control
"'-* Nonsignificant or significant at P= 0.05 or 0.01, respectively.












Table 4-4. Effect of seed treatments on emergence rate index
(ERI), emergence percentage, and dry weight (DW) of two sh2
sweet corn cultivars in a field experiment planted in 23
April, 1990 at Gainesville, FL.




Cultivar

How Sweet It Is Crisp N'Sweet 711

ERI Emer DW ERI Emer DW
Seed treatment (%) (mg) (%) (mg)

SMPsh 145 50 1327 230 83 2403
SMP 98 32 1550 208 79 2480
SH 76 32 1300 187 72 2073
F 132 58 668 192 79 2120
C 56 22 393 160 66 1901
Orthogonal Contrast
SMPsh vs C ** ** ** ** ns ns
SMP vs SH ns ns ns ns ns ns
SMPsh vs SMP ns ns ns ns ns
SMPsh vs F ns ns ns ns ns ns

SValues are means of 20 plants 27 DAS.
b SMPsh: Solid Matrix Priming + Sodium Hypochlorite.
SMP: Solid Matrix Priming
SH: Sodium Hypochlorite
F: Fungicide
C: Control
'"* Nonsignificant or significant at P= 0.05 or 0.01, respectively.





















a----

// S
-- -*. /-













/

/


October 89 March 90 April 90 November 90

1 2 3 4 5 6 7
1 2 3 4 5 6 7


DAP




Figure 4-1. Average daily soil temperature (5 cm deep) for
first 7 days after sowing in Fall 1989, Spring 1990 and Fall
1990 field experiments.


26

L.
E 24
a-








70

not shown), where the final marketable yields were directly

related to the final stand. Under cooler conditions of

November 1990 (Figure 4-1), HSII seeds treated by SMPsh

emerged more rapidly, and had significantly higher final

emergence and dry weight compared to the other treated and

nontreated seeds (Table 4-5). Significantly higher ERI was

also shown in CNS-711 seeds primed via SMPsh.

The SMP presowing treatment provides ideal conditions

to deliver other products, such as biocontrol agents, to the

seed (Harman and Taylor, 1988). Sodium hypochlorite has been

used successfully as a seed disinfectant in su sweet corn to

control Fusarium moniliforme (Anderegg and Guthrie, 1981).

The SH and SMP treatments alone were not effective in both

cold test and field experiments. The addition of sodium

hypochlorite to the SMP treatment significantly enhanced

seed germination and emergence compared to seeds only

disinfected with sodium hypochlorite or primed alone. The

results indicated that SMP is an excellent delivery system

to include sodium hypochlorite as a seed disinfectant.

Greater differences in seed and seedling performance

between the nontreated seeds and primed seeds via SMPsh were

measured under high (April sowing) or low soil temperature

(cold test and Fall 1990). Rapid imbibition, increased seed

leakage and pathogen growth and development may contribute

to rapid deterioration of the seeds under these stressful

conditions. Lower imbibitional rate and seed leakage had












Table 4-5. Effect of seed treatments on emergence rate index
(ERI), emergence percentage, and dry weight (DW) of two sh2
sweet corn cultivars in a field experiment planted in 8
November, 1990 at Gainesville, FL.



Cultivar

How Sweet It Is Crisp N'Sweet 711

ERI Emer DW' ERI Emer DW
Seed treatment b (%) (mg) (%) (mg)

SMPsh 167 84 85 178 85 83
SMP 67 45 24 167 79 87
SH 25 19 25 160 82 65
F 116 76 42 179 89 86
C 26 19 16 136 81 57
Orthogonal Contrast
SMPsh vs C ** ** ** ** ns ns
SMP vs SH ** ** ns ns ns ns
SMPsh vs SMP ** ** ** ns ns ns
SMPsh vs F ** ** ns ns ns

" Values are means of 20 plants 22 DAS.
b SMPsh: Solid Matrix Priming + Sodium Hypochlorite.
SMP: Solid Matrix Priming
SH: Sodium Hypochlorite
F: Fungicide
C: Control
""* Nonsignificant or significant at P= 0.05 or 0.01, respectively.








72

been observed in primed than in nonprimed sweet corn seeds

(Parera and Cantliffe, 1991). The disinfectant treatment of

sodium hypochlorite added after partial seed hydration in

the SMP process may have contributed to a more effective

control of seed-borne pathogen growth and development. The

fungicide combination treatment was also effective in

increasing germination in the laboratory and field stand in

supersweet corn as previously reported (Cantliffe and

Bieniek, 1988; Parera and Cantliffe, 1990 a). However, it

was necessary to combine four fungicides to achieve the same

germination, rate and field emergence reached with the SMPsh

treatment.

From the laboratory and field results presented, SMPsh

improved seed germination, emergence rate, final field

stand, and seedling vigor in CNS-711 and HSII sh2 sweet corn

cultivars compared with nontreated seeds, especially when

sh2 cultivar has inherently poor seed quality and under

stressful conditions. The treatment may be a practical

replacement for fungicide seed treatments on sh2 sweet corn

cultivars.



Summary

Presowing seed treatments were devised to improve

emergence and crop uniformity of two sweet corn (Zea mays

L.) cultivars Crisp N' Sweet 711 (CNS-711) and How Sweet It

Is (HSII), which both carry shrunken-2 (sh2) mutant








73

endosperm. The treatments included a fungicide combination

(F), sodium hypochlorite (SH), solid matrix priming (SMP),

and SMP combined with sodium hypochlorite during treatment

(SMPsh). Seed germination was tested in a laboratory cold

test. Emergence percentage, emergence rate index (ERI) and

seedling dry weight were calculated from field trials.

'Crisp N'Sweet 711', in the cold test and field trials, had

a higher germination rate, ERI, final emergence, and

seedling dry weight than HSII. In both cultivars, SMPsh

treatment significantly improved germination in the cold

test and final emergence and ERI in the field trials for

HSII compared to nontreated seeds. There was no significant

difference between the fungicide and SMPsh treatments

regardless of cultivar. These results suggest that the

combination of SMP and disinfection with sodium hypochlorite

can be an alternative seed treatment to fungicides to

improve uniformity and stand establishment in sh2 sweet

corns.














CHAPTER V
DEHYDRATION RATE AFTER SOLID MATRIX PRIMING ALTERS
SEED PERFORMANCE OF shrunken-2 MAIZE



In maize (Zea mays L.), the shrunken-2 (sh2) endosperm

mutation increases endosperm sugar retention and

concentration, improving postharvest storage properties and

fresh eating quality (Laughnam, 1953). Unfortunately, poor

germination and seedling vigor is a common characteristic of

sh2 cultivars (Wann, 1980). The poor seed and seedling vigor

in sh2 sweet corn could be an interaction of several factors

previously reported: deficient mobilization of reserves by

the embryo (Styer and Cantliffe, 1984 a), potential

imbibition damage (Chern and Sung, 1991; Parera and

Cantliffe, 1991), reduced seed maturity at harvest (Borowski

et al., 1991), and greater susceptibility to seed and soil

borne diseases (Berger and Wolf, 1974).

Seed priming is a presowing treatment used to increase

germination rate and synchronize germination in many species

(Bradford, 1986). Germination and emergence have generally

not been improved in sweet corn seeds primed with osmotic

solutions (Bennet and Waters, 1987). Solid matrix priming

(SMP) is another priming technique, where the seeds are

mixed and incubated with water and a solid matrix such as








75

calcined clay (Kubick et al., 1988) or leonardite shale

(Harman et al., 1989) in place of a liquid osmoticum. SMP

treatments with leonardite shale as solid-matrix had

successfully increased germination rate of tomato, onion and

carrot (Taylor et al., 1988). In sh2 sweet corn, SMP

treatments combined with sodium hypochlorite have increased

germination, field emergence and seedling vigor (Parera and

Cantliffe, 1990 b). After priming treatment, the seeds must

be dried to storage or manipulation. However, effects of

conditions for redrying the seeds after priming have not

been studied extensively regardless of the method of

priming.

Corn ears harvested for seed production are usually

picked while relative moisture content of kernels remain

high (40 to 50%) to avoid deterioration in the field and to

decrease damage during harvest (Herter and Burris, 1989).

Corn seeds are subsequent dried in hot air to 11-12%

moisture (Seyedin et al., 1984). The temperature and rate of

dehydration can affect germination percentage and seedling

vigor of corn seeds (Herter and Burris, 1989).

During corn seed drying, membranes and pericarp are more

susceptible to damage (Wann, 1980). Cell membrane injury in

soybean cotyledons has been detected through leakage

conductivity (Schoettle and Leopold, 1984). In sweet corn

hybrids carrying the sh2 mutant endosperm, high leakage

conductivity and imbibition rate have been negatively









76

correlated with laboratory germination (Parera and

Cantliffe, 1991) and field emergence (Water and Blanchette,

1983). Several tests have been developed to relate damage to

membranes and poor seed quality. In the first stage of

germination the seed respiration rate is a good indicator of

corn seed quality and seedling vigor (Woodstock and Grabe,

1967). Seed deterioration has been correlated with low

activity of the glutamic acid decarboxylase enzyme in the

seeds (Linko, 1961). Low metabolic activity has been

reported in seeds injured by chilling temperatures (Herner,

1986). As shown in chapter III, the complex stress vigor

test and the index of conductivity (ICS) are both, alone or

in combination, reliable indicators of seed vigor for sh2

corn.

In chapter IV, the advantages of SMP treatment were

clearly demonstrated. However, the response of sh2 sweet

corn to a different redrying conditions after SMP are not

understood. The objective of the present study was to

investigate the effects of drying rate on seed quality after

solid matrix priming (Parera and Cantliffe, 1991) on two sh2

sweet corn cultivars. Laboratory and field tests were

conducted to determine changes in seed vigor of sh2 primed

seeds redried at various temperatures.









77

Materials and Methods

Plant Material and Solid Matrix Priming Treatment

Two cultivars of sweet corn with sh2 mutant endosperm:

'How Sweet It Is' (HSII) and 'Crisp N' Sweet 711' (CNS-711)

(Crookham Seed Co. Caldwell, Idaho) were used in this study.

Before and after treatment, the seeds were stored at 15C in

45% RH. Seeds were not generally retained for more than 1

month after treatment.

Seeds were primed via SMP combined with sodium

hypochlorite (Parera and Cantliffe, 1991), where 9 g of

seeds were mixed in a container with 27 g of calcined clay

(Emathlite, Mid-Florida Mining, Lowell, FL) and 14 ml of

0.1% solution (v/v) of sodium hypochlorite, incubated at 5C

for 6 h, then moved to 25C for another 66 h under continuous

rotation (20 rpm). The seeds were dried in a single layer in

a controlled temperature incubator to their original fresh

weight (6-7% moisture) at either 15, 20, 30 or 40C and 25%

RH. The rate of dehydration was calculated every hour on 25

seeds and expressed as a percentage of fresh weight.

Leachate Conductivity

Twenty seeds were soaked in 25 ml of distilled water at

25C. The electrical conductivity of the leachate was

measured each hour up to 6h by a conductivity meter (Lecto

Mho-meter, Lab-Line Instruments Inc., Melrose Park, Ill.)

and expressed as Amhos g1 of seed.










Seed Imbibition

Seeds (25) were soaked in 25 ml of water at 25C.

Imbibition was calculated at 2, 4, 8 and 16 h. and expressed

as percentage increase in fresh weight.

Seed Respiration

Respiration of the seeds was monitored in a Gilson

differential respirometer (Gilson Medical Electronics, Inc.,

Middleton, WI). Five seeds were placed in a 15 ml vessel

with 0.2 ml of 10% KOH on the center well and incubated at

25C and 85 oscillations minm in dark. Oxygen depletion was

calculated at 15 min, 4, 16, and 32 h after imbibition and

expressed as a jL 02 min'.

Glutamic Acid Decarboxylase

Evolution of CO2 was measured in a Gilson differential

respirometer. In a 15 ml reaction flask, 1 g of ground seed

was mixed with 2.5 ml solution of 0.1 M glutamic acid in

0.067 M phosphate buffer at 5.8 pH (Ram and Wiesner, 1988)

and incubated as previously described for seed respiration.

After 10 min stabilization CO2 produced was measured in 10

min periods and expressed as AL CO2 g' min'.

Sugar Determination

Embryos (25 axis plus scutellum) were separated from

the endosperm. Both seed fractions were homogenized (Virtis

homogenizer, Virtis Co., Gardiner, NY) with 10 ml of 80%

ethanol. The homogenate was centrifugated at 20000 x g for

10 min. The supernatant was separated and the pellet washed








79

with 5 ml of 80% ethanol. The supernatants were combined and

recentrifugated (Chen and Burris, 1990). The ethanol was

removed by forced air evaporation at 30C. Five milliliters

of water was added to the extract and filtered through 0.2

jm poresized nylon filter. The sugars were analyzed in a

BioRad HPLC (Bio Rad Chemicals, Richmond, CA). The sugars

were separated on an Aminex carbohydrate HPX-87C column

with water as a mobile phase at a flow rate of 0.6 ml min-'.

The quantification of sugars was done by comparing areas

under peaks of interest with those of comparable standards

and expressed as a mg g' of fresh weight.

Germination Test

Seeds (25) were placed in a 15 mm Petri dish on blue

blotter germination paper (Anchor paper Co. St. Paul, MN)

and incubated in the dark at 25C. After 7 days, germination

percentage was calculated. Pathogen growth and development

were visually evaluated in each seed treatment.

Complex Stress Vigor Test

Two hundred seeds were soaked 24 h in 250 ml of

distilled water at 25C then transferred to 5C for an

additional 24 h (Barla-Szabo and Dolinka, 1988). After

soaking, 25 seeds were placed with the radicle pointing

downward on 3 layers of moist, non-toxic, germination paper

(Anchor paper Co. St. Paul, MN). The papers were rolled,

placed in a plastic container (21.5 x 32.5 x 5.5 cm), and

incubated in the dark at 25C for 4 days. Conductivity of the








80

leachate after each soaking period was measured. Germination

percentage and an index (index of conductivity and stress

test (ICS)), which combines the conductivity after each

soaking period and the germination percentage, was

calculated.

Field Studies

Treatments were tested in two field trials established

on 3 December 1991 and 13 April 1992 at the Institute of

Food and Agricultural Science (IFAS, UF) Horticultural

Research Unit (Gainesville, FL) on an Arredondo fine sand

soil (loamy, silaceous, hyperthermic Grossarenic

Palenundult). Fifty seeds were sown 4 cm deep every 30 cm in

two rows in each plot. The plot length was 7.6 m on beds

with 1.2 m centers. Emergence Rate Index (ERI) (Shmueli and

Goldberg, 1971) and percent emergence were calculated for

each plot. Fresh and dry weight of five seedlings were

recorded 14 days after sowing. Each was cut at the soil

level and oven dried 4 days at 75C.

Statistical Analysis

All the experiments were conducted as a randomized

complete block design with four replications. Percentage

data were arsine transformed before analysis. The

respiration, imbibition and leachate conductivity

experiments were each analyzed as split blocks considering

time as the main block. Main effects of the treatments were








81

partitioned using a single degree freedom orthogonal

contrast.



Results

In both cultivars the increase in the 6-7% of the

original moisture content of the seed after priming

treatments varied between 45 to 54% (Figure 5-1). There were

significant differences between the two cultivars when the

rate of dehydration after priming was calculated (Table 5-

1). The HSII seeds desiccated more rapidly after priming

compared to CNS-711, regardless of the drying temperature.

The dehydration rate was also significantly higher at 40C in

both cultivars. In HSII, the rate of dehydration in seeds

redried at 15 or 20C did not differ significantly (Table 5-

1).

Significant interaction between cultivar and treatment

occurred when leakage conductivity and imbibition rate were

evaluated, thus the main effects were analyzed separately.

Seeds of CNS-711 had reduced leakage conductivity and

imbibition rate compared to HSII regardless the dehydration

treatment. As previously reported (Parera and Cantliffe,

1991), SMP treatment significantly reduced seed leakage and

imbibition in sh2 sweet corn compared to nonprimed seeds

(Table 5-2). More importantly in the present

work, the seeds dried at 30 or 40C had significant by lower

leakage conductivity compared to the other treatments and










82


60
so-

15 C
A -
20 C
50 -
30 C
Li
440C

40 -



30 I



20



10 -


0 1 f ,' ,- '' t I I T _
0 10 20 30 40


60

B
50


40


S30


20




10

0 1 "1 I I I ,I ,I ,, ,
0 10 20 30 40
Time (h)


Figure 5-1. Moisture content decrease, calculated as
percentage of the original fresh weight (7-6%) of CNS-711
(A) and HSII (B) sh2 sweet corn seeds redried at different
temperatures after priming.












Table 5-1. Dehydration rate of CNS-711 and HSII sh2 sweet
corn seeds redried at different temperatures after priming


Dehydration rate (mg h")

Temperature (C) CNS-711 HSII

15 48 241
20 67 254
30 97 337
40 361 620
LSD(o 11 47


Table 5-2. Imbibition rate and leakage conductivity of CNS-
711 and HSII sh2 sweet corn seeds either redried at
different temperatures after priming and non primed.



Imbibition Conductivityb

(%FW) (pmho g-)

Temperature (C) CNS-711 HSII CNS-711 HSII

15 72.5 90.7 57.7 128.4
20 64.0 76.3 73.5 150.3
30 73.9 73.6 52.1 59.0
40 71.4 87.5 53.3 83.5
No primed 94.3 110.8 89.3 220.1
LSD(o. 7.9 9.4 3.3 23.6

* Data pooled over 16 h imbibition.
b Data pooled over 6 h imbibition








84

nonprimed seeds regardless of cultivar. When HSII seeds were

primed and dried at 15 or 40C, they imbibed significantly

more water compared to seeds dried at 20 or 30C.

There was no significant interaction between cultivar and

dehydration rate when seed respiration was analyzed. Primed

and nonprimed seeds had the same respiration rate after 15

min of stabilization (Figure 5-2), however after 4 h of

imbibition 02 uptake was significantly greater in primed

seeds redried at 30C, 40C and 20C relative to those redried

at 15C or not primed at all. After 15 h of imbibition seeds

redried at 30C had the highest respiration rate compared to

the other treatments and the nonprimed seeds. Rates of 02

exchange 36 h after imbibition remained higher in seeds

redried at high temperatures (30 and 40C). In contrast those

redried at 15C had the lowest respiration rate during the

first 36 h of imbibition.

The activity of the enzyme glutamic acid decarboxylase

in the seeds was higher in CNS-711 compared to HSII. In both

cultivars, the activity of the enzyme was significantly

greater in seeds dried at 40 C (Table 5-3).

Sucrose, raffinose, glucose and fructose concentration in

the embryo and endosperm were analyzed. Only the sucrose

concentrations in both endosperm and embryo were

significantly different for cultivar and treatment (data not

shown). Cultivars differed in sucrose content in both

endosperm and embryo (Table 5-4). Both embryo and endosperm





















50

NP
-6----
15 a
40 ,,A
20
30
40 / a
30 _a
NI/ a
Sb -- ab

20 a ---- bc -
S ------------ ---
a bc b

10 b


ns b

0 1 I I I I I I I I I I ,I I I I ,
5 15 25
Time (h)








Figure 5-2. Respiration of CNS-711 and HSII sh2 sweet corn
seeds either redried at different temperatures after priming
and non primed (Data pooled over the 2 cultivars). Values
followed by the same letter are not significantly different
at 5% probability level by LSD test.












Table 5-3. Glutamic acid decarboxylase activity (GADA) of
CNS-711 and HSII sh2 sweet corn seeds either redried at
different temperatures after priming and non primed.


GADA (pL g-' minr1)

Temperature (C) CNS-711 HSII

15 170 105
20 178 84
30 183 101
40 288 136
No primed 165 98
LSD(. 36 22


Table 5-4. Sucrose concentration in endosperm and embryo of
CNS-711 and HSII sh2 sweet corn seeds either redried at
different temperatures after priming and non primed.



Sucrose mg g-

Embryo Endosperm

Temperature (C) CNS-711 HSII CNS-711 HSII

15 1.7 2.2 0.5 1.0
20 1.8 2.8 0.5 1.2
30 1.7 2.3 0.4 1.7
40 1.4 2.2 0.4 0.8
No primed 2.3 2.0 0.6 0.5
LSDO. 0.5 0.2 0.1 0.1




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