Citation
Seed vigor and germination of shrunken-2 maize

Material Information

Title:
Seed vigor and germination of shrunken-2 maize
Creator:
Parera, Carlos Alberto, 1956-
Publication Date:
Language:
English
Physical Description:
xiii, 144 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
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).
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.
Statement of Responsibility:
by Carlos Alberto Parera.

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029829093 ( ALEPH )
29507146 ( OCLC )
AJV9069 ( NOTIS )

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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
Pace
ACKNOWLEDGEMENTS.......................................... iii
LI1ST OF TABL~ES............................................. vi
LIST OF FIGURIES ..... .. .. ....... .. ... ........ ix
ALBSTR.ACT....................................... xi
CHAPTERS
I INTRODUCTIION......................................... 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
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
v




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
vi




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 Pacre
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
ix




6-4. Sugar concentration in embryos(a) and
endosperms (b) of concordant and nonconcordant seeds. Shrunken endosperm/colored embryo (B), Colorless endosperm /colored embryo (0).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 (BE),colorless endosperm/colored embryo (I).Data represent the
mean of 4 measures (se). ......................... 115
x




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 cause(s) 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
xi




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
5




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 aleuronecontrolled 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 solidmatrix 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), K3P04 (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.




12
Table 2-1. Selected species and osmotica reported to be used for seed priming in osmotic solutions.
Crop OBMOtiCUM Reference
Asparagus PEG, NaNO3, Pill et al.,1991
Asparagus Saline Seawater
officials L.
Aubergine Mannitol Passam et al.,1989
Solanum melongena
Beet PEG, MgSO4 Taylor et al.,1985
Beta vulgaris L.
Beet (sugar) PEG, NaCl Osburn and Schroth,1989
Cabbage PEG Hill et al.,1989
Brassica oleracea L. Perkins-Veazie
var. capitata et al.,1989
Carrot PEG Brocklerhurst and
Daucus carota L. Dearman,1984
Gray et al.,1990
Hill et al.,1989
Murray,1989
Sodium
Polypropionate Zuo et al.,1988 ab
KH,P04, Glycerol BrocklerhurBt and
Dearman,1984
KNO3 + KH2PO4 Globerson and Feder,1987
KNO31 K2HP041 K3P04 Haigh and Barlow,1987 KN03+K3PO4I
KN03+K2HP04 Haigh et al.,1986
Celery PEG Brocklerhurst and
Apium graveolens L. Dearman,1984
Singh et al.,1985
Tanne and Cantliffe,1989 Sodium Polypropionate Zuo et al.,1988 ab KH2PO4, Glycerol Blocklerhurst and
Dearman,1984
KN03 + KH2PO4 + GA Globerson and Feder,1987
KNO3 + K3PO41 K3P04 Tanne and Cantliffe,1989
Field Corn PEG, KH2PO41 K2HP04 Basra et al.,1988 ab
Zea Mays L. KN03+KH2PO4
Corn cockle PEG De Klerk,1986
Agrostemma ghitago L.
Cucumber Mannitol PaBsam et al.,1989
Cucumis sativus L. NaCl, Milk Thanos and Georghiou, 1988
Dusty miller PEG Carpenter,1990
Senecio cineraria DC.




13
Continuation table 2-1.
crop Osmoticum Reference
Kale PEG Rao et al.,1987
Brassica oleracea L.
var. acephala
Leek PEG Bray et al.,1989
Allium porrum L. Gray et al.,1990
Parera and Cantlif f e, 1992 Mannitol Parera and Cantlif f e, 1992
Lettuce PEG Cantliffe,1981
Lactuca sativa L. Hill et al.,1989
Tarquis and Bradford, 1992 Valdez and Bradford,1987 K3Po' Cantliffe,1981
Cantliffe et al.,1984
Guedes and Cantlif f e, 1980 Perkins-Veazie and
Cantliffe,1984
Wurr and Fellows,1984
Muskmelon Mannitol Passam et al.,1989
Cucumis melo L. KNO3 Bradford et al.,1988
KH2PO4 + KNO3 Nerson and Govers,1986
onion PEG Ali et al.,1990
Allium cepa L. Dearman et al.,1986
Gray et al.,1990
Haigh and Barlow,1987 PEG + Enriched air Bujalsky et al.,1989
Mannitol, NaCl Furutani et al.,1986
Glycerol Broclehurst and
Dearman,1983 ab
KN03+K3P041
KN03+K2HP04 Haigh et al.,1986
K2HP04 Haigh and Barlow,1987
K3PO4, KNO3 Haigh et al.,1986
Pansy PEG Carpenter and
Viola x wittrockiana Boucher, 1991 ab
Gams.
Parsley PEG Akers et al.,1987
Petroselinum hortense Pill,1986
Hoff. Rabin and Berkowitz,1988
Parsnip PEG Gray et al. 1984
Pastinaca sativa L.




14
Continuation table 2-1.
Crop Osmoticum Reference
Pea
Pisum sativum L. PEG DellAquila and
var.sativum Bewley,1989
Sodium Polypropionate Zuo et al.,1988 ab
Peanut PEG Fu et al.,1988
Arachis hypogea L.
Pepper PEG Cantliffe and
Capsicum anuum L. Watkins, 1983
Aljaro and Wyneken,1985 O'Sullivan and Bow,1984 Rivas et al.,1984
Stofella et al.,1992
Mannitol Georghiou et al.,1987
Passam et al.,1989
Sodium polypropionate Zuo et al.,1988 a, b KN03 Bradford et al.,1990
Globerson and Feder, 1987 Rivas et al.,1984
Smith and Cobb.,1991
Sundstrom and
Edwards,1988-89
KH2Po, Globerson and Feder,1987
KNO3+K2HPO4 Jones and Sanders,1987
NaCl, MgSO4 Aljaro and Wyneken,1985
Smith and Cobb,1991
KC1, K2SO4, Na,S041
CaCl, Na2HP04, K2HPO4,
NaCl+CaCl. Smith and Cobb,1991
Purple Coneflower KNO3 Samfield et al.,1990-91
Echinacea purpurea (L.)
Moench.
Ryegrass PEG Dannenberg et al., 1992
Lolium perenne L.
Sorghum PEG, KN031 K2HPO41
Sorghum bicolor (L.) K3PO41 KNO3+K3Po4I Moench. KNO3+K2HPO4 Haigh and Barlow,1987
Soybean PEG, Mannitol Helsel et al.,1986
Glycine max (L.) Merr. PEG + Ga3 + Kinetin Lorenz et al.,1988 Sodium polypropionate Zuo et al.,1988 ab
Spinach Sodium polypropionate Zuo et al.,1988 ab
Spinacea olereacea L.
Sweet Corn PEG Murray,1990
Zea mays L.
Tabasco pepper PEG, KNO3 Rivas et al.,1984
Capsicum frustecens L. KN03+Ga SundBtrom et al.,1987




15
Continuation table 2-1.
Crop Osmoticum Reference
Tickseed
Coreopsis lanceolate L. KN03 Samfield et al.,1990-91
Tomato PEG Ali et al.,1990
Lycopersicon esculentum Avarado et al., 1987
Mill Alvarado and
Bradford,1988 ab Bino et al.11992 Haigh and Barlow,1987 Hill et al.,1989 Mannitol, NaCl, Milk, Sucrose Thanos and
Georghiou,1988 Sodium polypropionate Zuo et al.,1988 ab KNO3 Ali et al.,1990
Alvarado et al.,1987 Alvarado and Bradford,1988 ab Argerich and Bradford,1989 Haigh and Barlow, 1987 K2HPO4, K3PO4 Haigh and Barlow, 1987
KN03+K3PO4I KN03+K2HPOI Haigh and Barlow, 1987 KN03+K2HP04 Argerich et al.,1989
Argerich and Bradford,1989 Haigh et al.,1986 Haigh and Barlow,1987 KNO3+K3PO4 Haigh et al.,1986
Haigh and Barlow,1987 Odell and Cantliffe,1986
KN03+K3PO4+
Uniconazole Davis et al.,1990
Turnip PEG Rao et al.,1987
Brassica rapa L. var. rapa
Watermelon KN03 Elmstrom,1985
Citrullus lanatus KN03+K3PO4 Elmstrom,1985
(Thumb.) Matsumi&Nakai 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 K3P04 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 NaCl 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 K3P04 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 solidmatrix 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 Microcell 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




22
Table 2-2. Selected species and solid-matrix reported to be
used for solid matrix priming.
specie Solid Reference
Beet Microcell E.+Fungicide Khan et al.,1992a
Beta vulgaris L. Agro-lig, Vermiculite #5
Carrot Microcel and Vermiculite#5 Khan et al.,1992b
Daucus carota L. Leonardite Shale Taylor et al., 1988
Celery Calcined Clay + NaOCl Parera et al.,1992
Apiurn graveolens L.
Cucumber Leonardite shale+Trichoderma Harman and
Cucumis sativus L. Taylor, 1988
Bituminous coal, Sphagnum moss Harman and +Enterobacter cloacae+Trichoderma Taylor, 1989
Peas Leonardite shale+Trichoderma Harman et al.,1989
Pisum sativun L.
var. sativun
onion Leonardite shale Taylor et al., 1988
Allium cepa L.
Pepper Microcell E, Vermiculite #5 Khan et al.,1992b
Capsicum anuum L. Calcined clay+Fungicide Kubick et al.,1988
Snap bean Leonardite shale+Trichoderma Harmanet al.,1989
Phaseolus vulgaris L.
Sweet Corn Calcined clay + NaOCl Parera and
Zea mays L. Cantliffe, 1990b
Leonardite shale+Trichoderma Harman and
Taylor, 1988
Parera and
Cantliffe, 1990a
SMP+Bacteria SMP+Fungicide Cantliffe and
Bieniek, 1988
Tomato Leonardite shale Taylor et al., 1988
Lycopersican Sphagnum moss, soft coal
sculentun Mill Calcined clay+Fungicide Kubicket al.,1988
Microcell, Vermiculite #5 Khan et al.,1992b
Leonardite shale+Trichoderma Harman and
Taylor, 1988
Bituminous charcoal+Trichoderma Harman and Taylor, 1988
Sphagnum moss+Trichoderma 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 solidmatrix 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 K3PO4 (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 K3P04 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 Na2HP04 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 IOC 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 (Seneclo cineraria) seed performance after 16 weeks of storage at 50 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 Primingr 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 'tabasco' 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 'osmotic 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 41




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 IOC 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 g1 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 IOC 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 (GRI5), 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 Decarboxylase Activity Test (GADA)
The enzyme activity was measured with a Gilson




47
differential respirometer (Gilson Medical Electronics, Inc. Middleton, WI) and expressed in plCO2 g1 mi7 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.




48
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




49
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
ANOVA Source df MS F
Location (L)b 6 0.3555 8.05w
Error a 21 0.0441
Cultivar (C) 6 2.3207 346.33
C x L 36 0.0397 53.06w
Error b 126 0.0067
Total 195
a Transformed data.
b Location and planting date
Significant at the P= 0.001.




50
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 (in)
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- Subsprinkler 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 33). 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).




52
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 0.97" 0.97"
C23 -0.95" -0.95"
GCTS 0.95" 0.94"
AC35 -0.94" -0.93"
C33 -0.94" -0.93"
GR30 0.94" 0.93"
ICS252 0.94" 0.94"
C13 -0.93" -0.93"
C356 -0.93" -0.93"
ICS253 0.91" 0.92"
C536 -0.90" -0.90"
ST253 0.89" 0.90"
DW15 0.89" 0.89"
ICS232 0.88" 0.87"
AC53 -0.88" -0.87"
ST251 0.88" 0.88"
ST232 0.87" 0.85"
ST252 0.86" 0.87"
GADA 0.85" 0.86"
ICS233 0.85" 0.83"
DW25 0.84" 0.82"
GR25 0.84" 0.83"
ICS231 0.83" 0.82"
ST233 0.83" 0.81"
ST231 0.82" 0.81"
EC3 -0.82" -0.81"
EC6 -0.81" -0.81"
GR15 0.70" 0.70"
GCTV 0.57* 0.57*
DW30 0.54* 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=o.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 (AbdulBaki 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 34). The following independent (noncollinear) laboratory vigor tests were selected to calculate multiple linear models: AC53 and GADA (Factor 1); ICS233, ICS231, ST233,




54
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 FACTOR2 FACTOR3 FACTOR4
AC53 0.78598
GADA -0.75394
C536 0.78681 -0.36709
C356 0.78037 -0.44873
AC35 0.77238 -0.44557
C13 0.72944 -0.42072
GCTS -0.64931 0.51921
ICS253 -0.56658 0.67988
ST253 -0.54300 0.60307
ICS252 -0.51171 0.73505
ICS232 -0.44635 0.76838
ST232 -0.43615 0.72570
C33 0.68593 -0.40919 -0.36821
ICS251 -0.62031 0.62875 0.37319
C23 0.61599 -0.44418 -0.41942
ST251 -0.50956 0.48088 0.53403
GR30 -0.46855 0.46327 0.47295 0.36701
DW15 -0.61807 0.36612
EC6 0.50860 -0.72481
EC3 0.48517 -0.74535
DW25 -0.39829 0.66902
ICS233 0.82848
ICS231 0.80694
ST233 0.78331
ST231 0.76731
ST252 0.66831 0.48017
GRI5 0.85717
GCTV 0.83199
GR25 0.72549
DW30 0.83605
Var. Explained (%) 34.95 33.77 22.44 8.84




55
ST231 (Factor 2); GRl5, 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 R
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
a 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: nonsubjective, 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 (R=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
59




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 succesfully 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-(2propenyloxy)ethyl)-lH imidazole)] at 0.653 ml/kg seed, captan [N-[(trichloromethyl)thio]-4-cyclohexene-l,2dicarboximide] 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




64
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 42). 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




66
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 treatmentb (%) (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 ** ** n n 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.




67
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 treatmentb 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
A', Nonsignificant or significant at P= 0.05 or 0.01, respectively.




68
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 n ns ns ns
SMPsh vs F ns ns no 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
m" Nonsignificant or significant at P= 0.05 or 0.01, respectively.




69
32
30 -* ~ - U,,,
30"
28
26
L_ \
U ,A
I
/
22
2o /
,/
20 /
SOctober 89 March 90 April 90 November 90
18 I I I I I
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.




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




71
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
,n 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
74




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 PriminQ 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.




78
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 mim4 in dark. Oxygen depletion was calculated at 15 min, 4, 16, and 32 h after imbibition and expressed as a AL 02 mi-'.
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 Am 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 51). 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 51).
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
60so
15 C
A -*
20 C 50 W
30 C 40 C 40
30
20
10
0 1' f -. .
0 10 20 30 40
60
B
50
40
30
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.




83
Table 5-1. Dehydration rate of CNS-711 and HSII sh2 sweet corn seeds redried at different temperatures after priming
Dehydration rate (mg h1)
Temperature (C) CNS-711 HSII
15 48 241
20 67 254
30 97 337
40 361 620
LSDys 11 47
Table 5-2. Imbibition rate and leakage conductivity of CNS711 and HSII sh2 sweet corn seeds either redried at different temperatures after priming and non primed.
Imbibition a 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(Op 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




85
50
NP
15 a
40 ,,A20
30
N_ a
O b -ab
-U
30 a bc
It -- - - - - -
aa
10 b
ns b
0 I 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.




86
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-' miW-)
Temperature (C) CNS-711 HSII
15 170 105
20 178 84
30 183 101
40 288 136
No primed 165 98
LSD(0 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 gEmbryo 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
LSDmn 0.5 0.2 0.1 0.1




87
of the HSII cultivar, which has a smaller and more severely shrunken seed, contained more sucrose than did CNS-711 counterparts at all redrying temperature tested. Amounts of sucrose in both the endosperm and embryo of primed CNS-711 seeds were significantly lower than the same tissue in nonprimed seeds. The reverse was evident in primed HSII seeds, where significantly more sucrose was present in the endosperm and embryo compared to nonprimed seeds regardless of the drying temperature,.
When germination and seed vigor were evaluated, the
cultivars also had differential responses to the redrying temperatures (Table 5-5). Final germination and seed vigor were evaluated through the SCI and germination percentage after the complex stressing vigor test, and were significantly greater regardless the seed treatment in CNS711 compared to HSII. When seeds of CNS-711 seeds were dried at the lower temperatures (15 and 20C), they had significantly lower rates of final laboratory germination, percentage of germination in the complex stressing vigor test and lower SCI compared to the seeds redried at higher temperatures (30 or 40C), or nonprimed. There were no significant differences between these last thre treatments. The HSII seeds dried at 15C had the lowest rates of germination in both tests. Primed seeds redried at 30C had a greater germination percentage in the LGE test compared to nonprimed seeds or primed seeds dried at 20C. Seeds dried at




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