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POOR FIELD EMERGENCE OF LATE-MATURING PEANUT CULTIVARS (Arachis
hypogaea L.) DERIVED FROM PI-203396
BARRY R. MORTON
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
02007 Barry R. Morton
This dissertation is the beneficiary of many contributors. In particular I wish to thank Dr.
Barry L. Tillman for providing a research assistantship and making it possible for me to be an
integral part of the Agronomy Department while studying for a doctorate degree. Dr. Tillman,
chair and Dr. Kenneth J. Boote, cochair have guided both my academic progress and the research
and editing of this dissertation. Their patience and contributions were invaluable. Dr. Daniel W.
Gorbet, Dr. Jerry M. Bennett, and Dr. Jerry A. Bartz have served as important committee
members and I appreciate being able to rely on their expertise. Dr. Susan Percival provided
advice and access to her laboratory to assay antioxidants in peanut seed. Her assistant, Meri
Nantz, directed me in the basic laboratory assay. Dr. Jean Thomas oriented me in the moist
towel germination procedure. George Person and Robert Kerns provided peanut analysis,
support, supplies, and suggestions.
I wish to thank all of these individuals for their support. I thank my wife, Paula, for
encouraging me to pursue the doctorate degree and supporting me with humor and good food.
I wish to thank the University of Florida faculty for quality instruction and the peanut
grower cooperatives for funding the research.
TABLE OF CONTENTS
ACKNOWLEDGMENT S .............. ...............3.....
LI ST OF T ABLE S .........._.... ...............6..._.........
LIST OF FIGURES .............. ...............8.....
AB S TRAC T ............._. .......... ..............._ 10...
1 INTRODUCTION ................. ...............12......... .....
Cultivars ................ ...............12......... ......
Poor Field Emergence ........_................. ........_._ .........1
Seed Handling and Storage ........_................. ............_........1
Hyp others e s................. ...............14......... ....
2 LITERATURE REVIEW ..........._.__........... ...............16.....
Effect of Temperature and Relative Humidity on Germination ..........._.._. ............. .......16
Germination, Seed Vigor, and Field Emergence .....___.....__.___ .......____ ...........1
Deterioration of Seed Quality ........._.. ..... ._ __ ...............18....
W ater in Seeds ............... ...............20....
Seed Protection Mechanisms ........._.._ ......___ ...............22....
Accelerated Aging ................. ...............23...
Mode of Action ofAspergillus spp ................. ......... ........ ........... ...............24
Effect of Aspergillus on Peanut Seed Germination ................ ...............24........... ..
Hypotheses and Obj ectives ....__. ................. ........__. ........2
3 MATERIALS AND METHODS .............. ...............27....
Cultivars and Storage Locations ....__. ................. ........__. ........2
Germination Tests for Seed Viability ...._.._ ................ ........._.._ ....... 2
Seedling Emergence Field Tests ....__. ................. ........__. ........3
Storage Pathogen Assays ................. ...............31...............
Seed Vigor Tests ....._ _. ................... ........_.._.........3
Electrolyte Conductivity Tests .............. ...............33....
Accelerated Ageing Tests ......_. ................ ........__. ........3
Antioxidant Capacity Assay .............. ...............35....
Experimental Design and Data Analysis .............. ...............36....
4 RE SULT S AND DI SCUS SSION ............_ ......_.._ ...............37....
Effect of Seed Storage Environment on Germination and Field Emergence .........................37
Prior Germination Tests of Cultivars at NFREC .............. ... ...._ ... ...............37
Germination Tests of Seeds from NFREC and FFSP Stored in Bags in Various
L locations .............. ... ... .... ......... .. ......... ... .. .. .. .......3
Field Emergence of Seeds from NFREC and FFSP Stored in Bags in Various
Locations........ ......... .. .. ............ ... ........._ ..............3
Towel Germination and Field Emergence of Bulk Stored Seed from Production
Y ear 2004 .............. .. .......... ... .. .._ .. ...... .. ..............4
Comparison of Treatment Effects on Bagged Seed Samples and Bulk Stored Seed
Sam ples .............. .. ... .....__ .. ......._ ... ...........4
Correlation of Towel Germination and Field Emergence .............. ...... ..............4
Summary of Results of Towel Germination Tests and Field Emergence Tests..........._...44
Storage Environment Characteristics as Possible Factors in Declining Seed Vigor.......45
Effect of Storage Pathogens............... ...............4
M measures of Seed Quality ................. ...............47.......... .....
Comparative Vigor Index Tests............... ...... ..............4
Electrolyte Conductivity and Comparative Vigor Tests .............. ....................4
Extended Accelerated Ageing Tests ................. ...............51................
Antioxidant Capacity As say ........._.___..... .__. ...............53....
5 CONCLUSIONS .............. ...............83....
Seed Deterioration and Poor Field Emergence .............. ...............83....
Scenario of Seed Quality Deterioration............... .............8
6 RECO MMENDAT IONS ........._.___......___ ...............8 8....
Recommendations for Continued Research............... ... ..............8
Recommendations for Improving Quality of Seed Peanut ....._.__._ ........___ ...............88
Improve Ventilation of Storage Facilities .............. ........ .. ..................8
Replace Towel Germination Tests with a Test that Measures Seed Vigor .....................89
APPENDIX. SEED VIGOR DIFFERENCES IN GERMINATING PEANUT SEED ...............90
LIST OF REFERENCES ................ ...............91........... ....
BIOGRAPHICAL SKETCH .............. ...............96....
LIST OF TABLES
1-1 Performance of runner market-type peanut cultivars in two or three Florida locations
over four years (2002-2005)............... ..............1
4-1 Analysis of Variance (ANOVA) for towel germination and April field emergence of
peanut seed as affected by year (Y), cultivar (C), origin of seed (0), and storage
environment/location (L) for crop production years 2004 and 2005 ................ ...............55
4-2 Means of towel germination and field emergence tests of peanut seed from crop
production years 2004 and 2005 .............. ...............55....
4-3 Means of cultivar and seed origin in towel germination tests and April field
emergence test of peanut seed produced in crop year 2004............... ..................5
4-4 ANOVA P-values for towel germination and field emergence of bulk stored peanut
as affected by cultivar and storage location for crop production year 2004. .....................58
4-5 Effect of cultivar and storage location on April field emergence of bagged seed
versus bulk stored seed of peanut for crop production year 2004. ........... ...................60
4-6 Comparison of April field emergence to October field emergence of bulk stored
peanut as affected by cultivar and storage location for crop production year 2004. S.....64
4-7 Comparison of towel germination to field emergence of bulk stored peanut as
affected by cultivar and storage location for crop production year 2004. .......................64
4-8 Correlation of towel germination, field emergence, comparative vigor index, and
leachate conductivity of peanut as affected by cultivar and storage location for
production years 2004 and 2005. ........... ...............65......
4-9 Incidence of fungi per 20 seeds per storage location in bulk stored peanut for crop
production in 2004 and 2005. ............. ...............73.....
4-10 ANOVA for vigor tests #1 and #2 of peanut germination as affected by cultivar and
storage environment/location for crop production in 2005............... ..................7
4-11 Effect of cultivar, storage environment/location and accelerated ageing (AA) on
seedling vigor (Vigor Test #1) for peanut seed from 2005 crop production year. ............74
4-12 Effect of cultivar and storage environment/location on seedling vigor (Vigor Test #2)
for peanut seed from 2005 crop production year. ............ ...............74.....
4-13 Electrolyte conductivity ofleachate, comparative vigor index (CVI), April field
emergence, and October field emergence as affected by cultivar and seed storage
environment/location of seed peanuts produced in crop year 2004. ............ .................75
4-14 Pearson correlation of electrolyte conductivity of leachate, comparative vigor index
(CVI), April field emergence, and October field emergence as affected by cultivar
and seed storage environment/location of seed peanuts produced in crop year 2004. ......75
4-15 Changes in seed moisture in 5-week accelerated ageing test of peanut cultivars stored
in 3 environments ................. ...............78......._.. ....
4-16 ANOVA for field emergence of peanut seed as affected by 3 treatments: 1) 130C and
67% relative humidity, 2) 320C/100% relative humidity, and 3) 320C <10% relative
humidity in 5-week accelerated ageing test ................. ...............78...............
4-17 Electrolyte conductivity of germinating peanut leachate, comparative vigor, towel
germination, and field emergence at 8 and 12 days after planting (DAP) as affected
by cultivar and by three simulated storage treatments for 5 weeks .............. ..................79
4-18 ANOVA for antioxidant capacity of peanut seed as affected by cultivar and date/year
of sam pl e. .............. ...............8 1....
4-19 Antioxidant capacity of peanut seed as affected by cultivar over three sampling
years. In 2006 seed of Hull was not produced. ................ ................. ..............81
4-20 Antioxidant capacity of peanut seed as affected by seed storage environment/location
and year of crop production. ........... ...............8 1.....
LIST OF FIGURES
4-1 Effect of cultivar on moist towel germination and field emergence at the end of
winter storage of seed peanut from crop production years 2004 and 2005. ....................56
4-2 Effect of environment/storage location on moist towel germination and field
emergence of seed peanut from crop production years 2004 and 2005. ..........................57
4-3 Moist towel germination of bagged seed produced in crop year 2004 as affected by
cultivar and storage environment/location. ...._. ......_._._ .......__. ............5
4-4 Effect of cultivars on moist towel germination and field emergence of bulk stored
peanut produced in crop year 2004. .............. ...............59....
4-5 Effect of storage environment/location on moist towel germination and field
emergence of bulk stored peanut produced in crop year 2004. ......... .....................60
4-6 Field emergence in April 2005 of bulk stored peanut as affected by cultivar and
storage environment/location of seed peanut produced in crop year 2004. .....................61
4-7 Field emergence in October 2005 of bulk stored peanut as affected by cultivar and
storage environment/location of seed peanut produced in crop year 2004 ................... .....62
4-8 Representation of differences in rate of deterioration of seed viability and vigor
showing decrease from 100% to 0% over time .............. ...............63....
4-9 Mean daily air temperature at 2 meters above ground level as measured at the Florida
Automated Weather Network (FAWN) substation, Marianna, Florida, September 16
to January 24 for crop years 2004 and 2005. ............. ...............66.....
4-10 Mean daily temperature within the bulk pile of peanut cultivar AP-3 stored in a
traditional storage bin at the Florida Foundation Seed Producers (FFSP) for October
15 to January 24 for crop years 2004 and 2005. ............. ...............67.....
4-11 Mean daily temperature and relative humidity within the bulk pile of peanut cultivar
DP-1 stored in a peanut wagon at the Florida Foundation Seed Producers (FFSP) for
the period October 15, 2004 to January 31, 2005. ............. ...............68.....
4-12 Mean temperature of the seed storage room located at the University of Florida
Research and Education Center (NFREC), Marianna, Florida for the periods October
15 to January 3 1, 2004 and 2005. ............. ...............69.....
4-13 Comparison of mean temperature in the seed storage room located at NFREC and the
mean daily temperature within the bulk pile of peanut cultivar AP-3 stored in a
traditional storage bin at FFSP for the periods October 17, 2004 to January 24, 2005.....70
4-14 Comparison of relative humidity (RH) in the seed storage room at the University of
Florida Research and Education Center (NFREC) for October 15 to January 31 for
crop years 2004 and 2005. ............. ...............71.....
4-15 Comparison of relative humidity (RH) within the bulk pile of peanut cultivar AP-3
stored in a traditional storage bin at the Florida Foundation Seed Producers (FFSP)
for October 15 to January 31 for crop years 2004 and 2005. ............. .....................7
4-16 Comparative vigor index (CVI) versus electrolyte conductivity of leachate (LCH)
from germinating peanut seed over all cultivars and storage environments .....................76
4-17 April field emergence versus electrolyte conductivity of leachate (LCH) from
germinating peanut seed over all cultivars and storage environments ............. ................77
4-18 Field emergence at 12 DAP of peanut cultivars from crop production year 2005 as
affected by treatment in an extended accelerated ageing test (EAA). ........... ................80
4-19 Antioxidant capacity of seed peanut measured in Clequivalents gl as affected by year
of production, cultivar, and sample date. ............ ...............82.....
A-1 An example of seed vigor differences as evident in variation of hypocotyl/radicle
length of seeds germinating in a moist towel test. .............. ...............90....
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
POOR FIELD EMERGENCE OF LATE-MATURING PEANUT CULTIVARS (ARACHIS
HYPOGAEA L.) DERIVED FROM PI-203396
Barry R. Morton
Chair: Barry L. Tillman
Cochair: Kenneth J. Boote
Late-maturing peanut cultivars DP-1, C-99R, Hull, and Florida MDR-98 (Arachis
hypogaea L.) have superior resistance to leafspot (Cercosporidium personatum, Berk & Curt.),
white mold (Sclerotium rolfsii, Sacc.), and tomato spotted wilt virus. The improved resistances
are primarily derived from PI 203396. The cultivars are high yielding. They provide the grower
the opportunity to reduce fungicide applications and variable costs without reducing yields.
Because of poor field emergence, commercial seed companies have stopped producing Florida
MDR-98, DP-1, and Hull. Official towel germination tests usually show acceptable seed quality.
Reduced field emergence seldom occurs when the seed peanuts have been grown, harvested, and
stored in small batches in research storage facilities. The poor field emergence occurs when seed
production is through commercial channels with large volumes being harvested, stored in bulk
bins, and treated with fungicides. The problem may be related to the commercial practice of
storing seed peanuts in large piles with inadequate ventilation.
Four cultivars from two different field origins were stored in four environments and then
tested for field emergence. Field origin did not affect field emergence, but storage environment
did. Peanuts stored in bulk in a traditional peanut warehouse at elevated temperatures and
relative humidity had reduced field emergence. There was a genotype by storage environment
interaction. Field emergence was maintained when seed was stored at < 160C and < 70% relative
humidity. Standard towel germination tests were not reliable indicators of field emergence.
Electrolyte conductivity tests and seed vigor tests were highly correlated with field emergence.
The increased electrolyte conductivity and decreased rate of growth of the hypocotyl-radicle
indicated that cellular membranes were damaged during storage at elevated temperatures and
relative humidity. The literature suggests that peroxidation of lipids occurred resulting in the
production of free radicals and autoxidation. The antioxidant capacity of seed varied by cultivar
and year of production.
Field emergence could be improved by reducing temperature and relative humidity in the
storage environment. Since standard towel germination tests were not reliable indicators of field
emergence for these late-maturing cultivars, an alternative method of evaluating peanut seed
quality should be adopted.
The University of Florida Agricultural Experiment Station (FAES) at the North Florida
Research and Education Center (NFREC), Marianna, Florida, has released several late-maturing
peanut cultivars, namely Florida MDR-98 (Gorbet and Shokes, 2003), C-99R (Gorbet and
Shokes, 2002), DP-1 (Gorbet, 2003), and Hull (Gorbet, 2003). These cultivars have superior
resistance to late leafspot (Cercosporidium personatum, Berk & Curt.), white mold (Sclerotium
rolfsii, Sacc.), and tomato spotted wilt virus (genus Tospovirus; family Bunyaviridae) (Table 1-
1). The improved pathogen resistances were derived primarily from lineage to PI 203396
through a common parent or grandparent UF81206. DP-1 has 50% genetics inherited from PI
203396, Florida MDR-98 and Hull have 38%, and C-99R has 25%. The cultivars are high
yielding with good grades and acceptable flavor and processing characteristics. They provide the
grower the opportunity to reduce fungicide applications during the growing season and,
therefore, to reduce variable field costs without reducing yields. Because of the indeterminant
flowering of peanut, late-maturing cultivars have the ability to fill pods longer and can
compensate for unfavorable weather or pathogen damage.
Poor Field Emergence
Florida MDR-98 was released for commercial production in 1998. Within two years, seed
production of Florida MDR-98 was terminated because of poor field emergence. DP-1 and Hull
were released in 2002. Field emergence was poor and seed is no longer commercially available
for either cultivar. Southern Runner, a cross of PI 203396 and Florunner, is a parent to both
Florida MDR-98 and DP-1 and, like Florida MDR-98 and DP-1; Southern Runner had
germination problems (Gorbet et al., 1987). Unpublished studies found that Southern Runner
immature seed was sensitive to cold soil temperatures. The University of Florida
recommendation was to delay planting Southern Runner as long as soils were cool, and to
increase screen size in grading to an 18/64 mesh to screen out small and immature seed (Gorbet,
2006. Per. Comm.). Southern Runner is a parent of Georgia Green, the current dominant variety
grown in the Southeast. Georgia Green is medium maturity and has no major field emergence
problems. C-99R with only 25% PI 203396 was released in 2000. Field emergence problems
for C-99R have been less frequent and less severe and seed is commercially produced. Prasad et
al. (2006) reported a genotype difference in field emergence response to cool soil temperatures.
Florida MDR-98 was the most sensitive, followed by Southern Runner and then C-99R. Georgia
Green was the least sensitive to cool soil temperature. This ranking of sensitivity to cool soils
coincides with the percent genetic lineage of these cultivars to PI 203396.
Official towel germination tests usually show acceptable seed quality for these late-
maturing cultivars (Tillman, 2004, Per. Comm.). Research data from NFREC show that reduced
field emergence of these cultivars seldom occurs when the seed has been grown, harvested, and
stored in research storage facilities (Tillman, 2004, Per. Comm.). The poor field emergence
occurs when seed is produced in commercial channels with large volumes being harvested,
stored in bulk in-shell, shelled, and treated with fungicides. The problem may be related to
commercial storage of in-shell seed peanuts in piles in large warehouses with no humidity
control, temperature control, or forced ventilation.
Seed deterioration is inexorable and not uniform (McDonald, 2004). Many factors
contribute to seed deterioration including genetic composition, seed moisture content,
mechanical and insect damage, pathogen attack, seed maturity, and relative humidity and
temperature of the storage environment. Of these factors relative humidity and temperature are
the most important (McDonald, 2004). Relative humidity directly influences seed moisture.
Increasing temperature increases the amount of moisture air can hold and the rate of cellular
metabolism. Harrington (1972) concluded that for seed storage the sum of the temperature in
degrees Fahrenheit and the percentage relative humidity should not exceed 100.
Seed Handling and Storage
Seed peanuts in the southeast USA are harvested starting in early September by digging
the pods and vines, inverting the biomass to air dry, and then 3-5 days after digging, machine
combining to separate pods from stems. With forced heated air, pods and seeds are dried in
wagons to approximately 9.5% moisture content (Stadskley, 2004, Per. Comm.). The peanut
pods are stored in large bulk bins at ambient atmospheric temperatures that may exceed 320C and
relative humidity that may exceed 95%. Dimensions of the bulk pile may be very large, often
exceeding 7,000m 3. Ventilation usually is accomplished by circulating surface air through
opened doors and exhausting through roof vents. Shelling of seed peanuts begins as early as
December. Shelled peanuts are stored in a warehouse at ambient temperature and relative
humidity in solid cardboard containers on pallets until treated with fungicides, bagged, and ready
for delivery to the farm producer. During storage in the bulk bins, the peanut pile surface
frequently shows substantial fungal growth and the hulls may become dusty from spores.
C-99R, DP-1, and Hull have important disease resistances for peanut production and PI
203396, the primary source of these resistances, appears in the pedigree of many lines in the
University of Florida peanut breeding program. This research was conducted to identify the
factors in production and storage of these peanut cultivars that contribute to poor field
emergence. The research examines the following four hypotheses:
* High temperature or high relative humidity in commercial storage conditions may reduce
* There is an interaction of cultivar by storage environment on seed germination and field
* Reduced field emergence may be caused by storage pathogens.
* Potential field emergence can be measured by testing for seed vigor and the electrolyte
conductivity of the leachate of germinating peanuts.
Table 1-1. Performance of runner market-type peanut cultivars in two or three Florida locations
over four years (2002-2005). Entries are sorted by maturity and the four year
average yield (in descending order) (Tillman, 2007).
YIELD TSMK TSWV***
(lbs./acre) (%) (1-10)
Name Maturity* 200_5 3-YRt 4-YRtt 2005 3-YR 4-YR 2005 3-YR 4-YR
AP-3 M 3177 4094 4162 71.6 73.0 73.9 3.3 2.1 2.2
C-99R L 4107 4458 4349 75.3 76.5 77.2 2.8 2.2 2.2
DP-1 L 3320 3983 3953 73.6 74.7 75.3 2.7 1.9 2.0
Hull** L 3134 3780 3579 73.6 74.9 75.6 4.2 2.9 2.8
C.V. 16 13 13 2.4 1.8 1.9 23.1 22.4 20.7
LSD 351 239 209 2.1 0.9 0.8 0.9 0.4 0.3
*E = early, M medium, L = late; **High oleic oil chemistry. t3 YR = average of 2003, 2004
and 2005; tt4 YR = average of 2002, 2003, 2004 and 2005. ***Tomato Spotted Wilt
Virus ratings (1-10, 1 = no disease).
Effect of Temperature and Relative Humidity on Germination
Ideal conditions for storage of peanut seed are 100C and 65% relative humidity (Ketring,
1992). The resulting seed moisture content is approximately 6%. Maintaining these conditions
is difficult when peanuts are stored in warehouses without climate control. Navarro et al. (1989)
studied the interaction of temperature and moisture concentration on the germination of peanut
seeds. Germination of peanuts stored in-shell at 15oC and 79-83% relative humidity (RH)
remained above 80% for 150+ days. In the same peanut cultivars stored at 15oC and 85-89%
RH, germination decreased dramatically to 30% in 80 days. If the storage temperature increased
to 200C and RH was at 79-83%, germination steadily decreased to 80% at 80 days and, if stored
at 200C and 85-89% RH, germination dropped to 20% in 80 days. Navarro et al. (1989) found
an interaction of the tested cultivars with the temperature and RH. The cultivar Congo (Valencia
type) tolerated higher storage temperatures and RH better than the cultivar Hanoch (Virginia
type). Ketring (1992) reported peanut seed tolerated temperatures of 44oC when relative
humidity was low, but that genotypes varied in tolerance of high temperatures within seasons
and across seasons, showing the effect of genetic variation and environmental conditions during
seed maturation. Hypocotyl-radicle length was more adversely affected than germination.
Germination, Seed Vigor, and Field Emergence
Germination tests are required for the commercial sale of peanut seed and are intended to
be an indicator of the seed' s potential for field emergence. Germination is variously defined.
For the producer, it is the appearance of the seedling at the soil surface. For the peanut seed
tester, it is the initial protrusion of the radicle from the seed and germination is reported as the
percent of normal seedlings at the conclusion of the test (Association of Official Seed Analysts,
1970). Germination is triphasic (Black and Bewley, 1994). In phase I when water is imbibed
rapidly, leakage of ions and soluble sugars occurs until the organelle membranes are repaired. In
phase II, the lag phase, organelle membranes are organized, mitochondria increase in size and
number, enzymes are produced de novo, and the seed becomes prepared for rapid growth. Phase
III begins when the radicle protrudes from the seed and continues until the plant has emerged
from the soil, produced leaves, and commenced photosynthesis. This is the phase when food
reserves are mobilized and cell elongation and cell division commence. The producer accessing
field emergence observes the conclusion of phase III; whereas, the seed tester observes the
commencement of phase III.
The Seed Vigor Testing Handbook (Association of Official Seed Analysts, 2002) states
"Seed vigor comprises those properties which determine the potential for rapid, uniform
emergence and development of normal seedlings under a wide range of field conditions." Vigor
testing quantifies the vitality of the seed and places a premium upon uniform emergence.
Ketring (1993) developed a Vigor Index for peanut that takes into account germination and rate
of seedling growth. Peanut seed exposed to short periods of elevated temperature had reduced
seedling vigor, but no reduction in germination. Repeated exposure to adverse temperatures
resulted in additional loss of vitality and eventual loss in germination. The Vigor Index provides
a numerical value for statistical analysis of seed vitality resulting from genotype interactions
with storage conditions. Field emergence was positively correlated with rapidly growing
seedlings. At 63 days after planting (DAP), plants originating from seeds with low vigor had
shorter main stems, narrower plant width, and reduced ground cover. They produced lower
yields than those plants from seeds with high seed vigor. Ketring concluded that Vigor Index is
a better indicator of potential field emergence and final plant stand than germination tests.
Deterioration of Seed Quality
Seed deterioration is the result of changes within the seed that decrease the ability of the
seed to survive. It is distinct from seed development and germination and it is cumulative
(McDonald, 2004). Peanut seed quality is highest just prior to physiological maturity, but can
deteriorate rapidly during storage (Perez and Arguello, 1995). McDonald (2004) points out that
a seed is a composite of tissues that differ in their chemistry and proximity to the external
environment and that seed deterioration does not occur uniformly throughout the seed. The
embryonic axis is more sensitive to aging than the cotyledons. In the axis, the radicle is more
sensitive to deterioration than the shoot. In soybean during imbibition, the radicle absorbs water
more rapidly than the cotyledons (McDonald et al., 1988). In maize, water uptake begins in the
radicle followed by the scutellum and then the shoot axis and coleoptile (McDonald et al., 1994).
McDonald suggested that water present in the atmosphere may be attracted by the same matric
forces as soil water resulting in higher water content in the radicle compared to the storage
reserves (McDonald, 1998). The higher moisture content could selectively accelerate seed
deterioration in the axis. He concluded that studies of seed deterioration should focus on the
seed part that deteriorates first.
Wilson and McDonald (1986) reviewed the literature concerning lipid peroxidation in
plants, animals, and in vitro and proposed a model of ageing mechanisms that explains seed
deterioration, especially the tendency of oilseeds to deteriorate rapidly. Lipid peroxidation is the
result of either autoxidation or the action of lipoxgenases. In either process, fatty acid chains
become oxidized producing highly reactive free radical intermediates termed hydroperoxides. In
the divinyl methane structure of the polyunsaturated fatty acids, the hydrogen atoms are easily
removed and hydroperoxides are formed (Mead, 1976). Once a free radical is produced, usually
involving oxygen attack, a chain reaction is initiated producing additional free radicals. The
susceptibility of fatty acids to peroxidation increases exponentially with increasing unsaturation.
Antioxidants, such as ot-tocopherol, reduce autoxidation by scavenging free radicals thereby
breaking the chain reaction cycle (Kaloyereas et al., 1961 in Wilson and McDonald, 1986).
The effects of peroxidation are extensive and include biomembrane degradation, protein
denaturation, interference with protein and DNA synthesis, accumulation of toxic materials, and
destruction of the electron transport system of oxidative phosphorylation (Wilson and
McDonald, 1986). Membrane degradation is evident as increased electrolyte leakage from the
hydrated seed and can be measured with conductivity tests (Dey et al., 1999). In addition,
hydroperoxides may decompose into volatile aldehydes such as malondialdehyde (MDA). The
volatile aldehydes produce a wide array of cytotoxic effects including reaction with sulfhydryl
groups causing inactivation of proteins. Harman et al. (1982) demonstrated an association
between volatile aldehyde production during early germination and low soybean seed vigor.
Harman and Mattick (1976) studied the effect of free radicals on biomembranes. The
phospholipids of membranes, especially the inner membranes of mitochondria, have a larger
surface area, are oxygen sinks, and are usually more unsaturated than storage lipids (Moreau,
1978). Lipid peroxidation within the membrane results in polar bridges across the hydrophobic
barrier of the membrane leading to an increase in permeability and a decrease in respiratory
competence (Simon, 1974). The changes in membrane properties result in increased leakage of
sugars, amino acids, and inorganic salts; reduced phosphorylative capacity; reduced activity of
enzymes such as cytochrome oxidase and malic and alcohol dehydrogenases; and reduction in
protein and carbohydrate synthesis (Abdul-Baki and Baker, 1973).
Perez and Arguello (1995) found in aging tests that germination tests by International Seed
Testing Association Rules (ISTA) were not a sensitive assay for detecting the degree of
deterioration in peanut. To analyze the poor membrane structure that results in leaky cells and
subsequent low seed vigor, they used electrical conductivity tests to measure the amount of
electrolyte leakage. Their study analyzed leakage from the whole seed, the cotyledons, and the
embryonic axes of the seeds. The conductivity tests using accelerated aging showed that the axis
was the structure most sensitive to deterioration. In addition, using MDA as an indicator of
hydroperoxides and lipid peroxidation in peanut, they determined that MDA increase was most
pronounced in the axis. Perez and Arguello (1995) concluded that biochemical changes which
take place in the membranes of peanut seed as they age may be detected best in the embryonic
axes, either through changes in the leakage of electrolytes or in MDA content and that the
embryonic axis may be the active center in relation to vigor.
Water in Seeds
Although lipid peroxidation occurs in all cells, in fully imbibed cells water acts as a buffer
between the autoxidatively generated free radicals and target macromolecules, thereby reducing
damage from the free radicals (McDonald, 2004). Between 6% and 14% moisture, lipid
peroxidation may be minimal because sufficient water is available to serve as a buffer against
autoxidation, but is insufficient to activate lipoxygenase-mediated free radical production
(McDonald, 2004). Below 6% moisture, lipid autoxidation may be the prime factor in seed
deterioration as water is unavailable to buffer the free radicals. Above 14% moisture, the
increasing water content increases the activity of oxidative enzymes, such as lipoxygenase, and
the production of free radicals. As seed moisture increases, autoxidation increases and is further
accelerated if temperature increases. During imbibition seed moisture increases dramatically and
lipoxygenase-mediated free radical production increases, thus creating additional damage.
Antioxidants can suppress additional free radical damage. Concurrently, hydration increases
anabolic enzyme activity and cellular repair of the damage created by free radicals. Seed vigor
and ultimately seed field emergence depends upon the extent of cellular damage and the seed's
ability to repair the damaged membranes, enzymes, and DNA.
Water concentration is a measure of water in seeds but does not measure the
thermodynamic properties of seed water. Water in a system exists in a continuum of energy
states. Walters (1998) reviewed the mechanisms and kinetics of seed ageing. Her central
hypothesis is that the nature of chemical reactions and/or the kinetics of these reactions change at
critical water concentrations. Such critical water concentrations may be related to the viscosity
of the aqueous milieu, for example, a glass versus a rubber amorphous state. Drying of seeds
reduces the viscosity of seed water to a glass state. The high intracellular viscosity typical of
glasses slows molecular diffusion and decreases the probability of chemical diffusion (Krishnan
et al., 2004). At a certain temperature (Tg), the glass undergoes a state change to an amorphous
rubber. Rubbers differ from glasses by greater fluidity and free volume changing the nature of
chemical reactions and making the seed more susceptible to ageing reactions. For each seed and
its genetics and maturation environment, optimum moisture concentration will vary and may
correspond to the point of saturation of strong binding sites (Walters, 1998). With an increase in
temperature the amorphous structure becomes increasingly fluid allowing reactions to occur
more rapidly. Thus, temperature above Tg may have a disproportionate effect on the stability of
A dry seed is metabolically incompetent and chemical aberrations go unrepaired. Over
time, damage from a suite of degrading reactions will accumulate. A change occurs from strong
viability to a weaker seed to a non-viable seed. Walters (1998) developed a "kinetic model of
chemical degradation in seeds" to predict seed ageing time-lines. The model demonstrates that
different reactions are involved in seed deterioration, and that, if the kinetics of these reactions
are differentially controlled by temperature and water concentration, the relative importance of
each reaction in the overall loss of seed viability is likely to vary among different storage
regimes. Comparing soybean seed, 20% oil content, with wheat seed, 1-5% oil content,
Krishnan et al. (2004) reported a sudden change in thermodynamic properties of water for
soybean at 1 and 8 days of storage at temperatures of 450 and 350C, respectively, and for wheat
at 4 and 11 days at 450 and 350C, respectively. They concluded that soybean seeds have a higher
water activity and are more sensitive to changes in water status compared to wheat seeds.
Seed Protection Mechanisms
Antioxidants suppress autoxidation and limit the damage from lipid peroxidation.
Enzymatic antioxidants, such as superoxide dismutase, catalase, and glutathione peroxidase, act
to neutralize activated oxygen species (McDonald, 1998). Although enzyme activity is limited
in the quiescent seed, these enzymes are vital during imbibition as autoxidation increases with
the increasing fluidity of the cytoplasm. The nonenzymatic antioxidants include glutathione,
vitamin E (tocopherol), and vitamin C (ascorbic acid). These antioxidants function as free
radical scavengers and react with the free radicals to block the propagation of free radical chain
reactions. One tocopherol molecule can protect several thousand fatty acid molecules (Bewley,
1986). Soybean seed subjected to ageing had reduced content of tocopherol, suggesting that the
tocopherol was consumed while protecting the seed from free radical attack. The seed
antioxidant content may reduce the extent of cellular damage resulting from free radical attack
during seed storage (McDonald, 2004). Talcott et at. (2005b) found that antioxidant capacity
varied with genotype and oleic acid concentration and Talcott et at. (2005a), in analysis of
oxidative stability of polyphenolics, reported that, in dry roasted peanut, rates of lipid oxidation
are directly proportional to the degree of fatty acid unsaturation. Hashim et at. (1993) suggested
that natural antioxidants should always be considered as a parameter when predicting peanut
Some seed degradation is inevitable (McDonald, 2004). In addition to the protective
action of antioxidants, repair enzymes and repair pathways exist specifically to fix damage
caused by free radicals. During imbibition the pathways can repair DNA by removing damaged
bases and oxidative lesions and correct misincorporation of nucleotides during DNA replication.
If the damage is not severe, the cellular mechanisms are quickly repaired and the seed germinates
into a vigorous seedling.
Accelerated aging (AA) tests expose seeds for short time periods to elevated temperature
and relative humidity, the two environmental factors most influential in seed quality loss
(TeKrony, 2005). The AA test is used to evaluate seed vigor in crops and has been successfully
correlated to field emergence and stand establishment in soybean (Egli and TeKrony, 1995). The
AOSA Seed Vigor Testing Handbook 2002 (AOSA, 2002) provides standards for using AA to
test peanut seed vigor. Accelerated-aged peanuts had increased MDA in the axes and cotyledons
and the axes had greater lipid peroxidation and accumulated more peroxides than the cotyledons
(Jeng and Sung, 1994). The AA peanuts tended to have less soluble protein and reduced activity
of peroxide-scavenging enzymes. Germination decreased and the number of weak seedlings
increased compared to the control. These effects are consistent with increased lipid peroxidation
in the axes and cotyledons. The AA test is easy to conduct and relatively fast. However,
researchers debate whether AA produces the same biochemical events as occur in natural aging
(McDonald, 1999). Liklatchey et al. (1984) concluded that biochemical changes during
accelerated aging were the same as those in natural aging; the only difference was the rate at
which they occur.
Mode of Action ofAspergillus spp.
Aspergillus spp. are facultative saprophytes capable of producing a large quantity of extra
cellular enzymes, which probably enhance their ability to utilize a broad assortment of organic
resources to produce mycotoxins (van der Hondel et al., 1994). Mycotoxins are a sub-set of
secondary metabolites that can be synthesized from primary metabolites, such as proteins, fatty
acids, and sterols (Moss, 1994). Synthesis involves several precursor steps resulting in the
production of inducible enzymes, in particular cytochrome oxygenases that may be involved in
hydroxylations, oxidative cleavages, and rearrangements leading to a remarkable diversity of
Effect of Aspergillus on Peanut Seed Germination
Neergaard (1977) summarized the categories of storage fungi and their temperature and
relative humidity required for active growth. Most of the storage flora are species ofAspergillus
and Penicillium, which are active at humidity ranging from 70 to 90%. Aspergillus spp. each
have a characteristic temperature range and minimum and optimum water activity (Lacey, 1994).
A. halophihts Sartory & Mey is active at 70 to 73% relative humidity; A. flavus Link is active at
85-95 percent relative humidity. Damage to the pod predisposes the seed to invasion. Invasion
can occur at seed moisture concentration levels as low as 13.2 percent. Seed water concentration
determines the species of Aspergillus and a small change in the water concentration may result in
colonization by a different species. Neergaard (1977) pointed out that there is great variation of
the moisture level within a bulk seed storage unit. The problem of unequal distribution of
moisture in a bulk storage mass occurs where no forced aeration system is available. For seed
susceptibility to storage fungi, Neergaard prefers the term water activity (aw). The development
of storage fungi depends on the water activity of the seed rather than its relative moisture
concentration. Water activity differs for species and, assuming that lipid is non-miscible, the
critical moisture concentration should be computed on the non-lipid portion of the seed. For
example, in soybean (Glycine max L.), which has lipid concentration of =18%, fungi will grow at
a moisture concentration 1.5 percentage points lower than in cereal grains, which have lipid
concentration of < 5%. Seed may be affected internally without showing any external evidence.
Infected seed can lose ability to germinate within a few weeks or months. Aspergillus spp.
eventually kills peanut seeds or seedlings (Lopez in Neergaard 1977).
Dhingra et al. (200 1) studied the effect of Aspergillus ruber Thom & Church on soybean
seed stored at 250C and water activity varying from 0.66 to 0.86 (moisture concentration 11.3%
to 17%). Seedling emergence rate in sand began to decline for all treatments within 20 days of
storage and continued to decline significantly with increased storage time. The decline differed
with water activity (aw) and was slower in the samples of lower aw and increased as aw increased.
Concurrently, free fatty acids (FFA) increased significantly. At aw of 0.66 (1 1.3% moisture),
emergence decreased to zero by 140 days while free fatty acids increased approximately 0.5
units. The proportion of normal seedlings decreased. All abnormal seedlings exhibited some
degree of negative geotropism and the radicle tips were necrotic. They concluded that loss of
seed viability during storage was directly dependent upon the amount of fungal growth as
measured by FFA content. Storage fungi lipases in maize incite production of free fatty acids
(Neergaard, 1977). Trawatha et al. (1995) concluded that increased FFA disrupts membranes
resulting in seed deterioration that is evident in high electric conductivity of seed leachates.
During germination of oilseeds, fats are converted to sucrose, which is the energy molecule
transported to growing sites for biosynthetic processes. Singh et al. (1974) reported that the rate
of formation of sucrose, at all stages of germination, decreased with increases in the
concentration of aflatoxins. Aflatoxin B has been reported to affect the osmotic behavior of
mitochondria and inhibit tissue respiration (Bassir and Bababunmi 1972). Ratnavathi and
Sashidhar (2000) looked at the effect of aflatoxin on enzyme activity within germinating seed of
sorghum (Sorghum bicolor L.). Although sorghum is not a high oil content seed, increasing
aflatoxin reduced the activity of lipase. The normally very high 8-amylase and 8-amylase
activity in germinating seeds was also significantly less in infected grains. However, protease
activity was observed to be higher in the infected grains. They hypothesized that the increase in
proteases may be attributed to new fungal proteins. Christensen (1957) observed that fungal
infection associated with stored grain caused under-development of the plumule and radicle.
Singh et al. (1974) found lower sucrose concentration and lower percent germination as result of
Aspergilhts infection of seed.
Hypotheses and Objectives
Poor field emergence of late maturing, disease resistant peanut cultivars occurs when seeds
are produced in commercial channels with large volumes being harvested, stored in bulk in-shell,
shelled, processed, and treated with fungicides. The problem may be related to commercial
storage of in-shell seed peanuts in large piles with no humidity control, temperature control, or
forced ventilation. Based on the review of literature, the materials and methods of this study
were designed to test the following four hypotheses:
* The high temperatures and high relative humidity in commercial storage conditions may
reduce field emergence of peanut.
* There is an interaction between storage environment and cultivar relative to seed
germination and field emergence.
* Reduced field emergence may be caused by storage pathogens.
* Potential field emergence can be measured by testing for seed vigor and the electrolyte
conductivity of the leachate of germinating peanuts.
MATERIALS AND METHODS
Cultivars and Storage Locations
To study the interaction of cultivars and storage environment, four cultivars were selected:
C-99R, DP-1, Hull, and AP-3. C-99R, DP-1, and Hull are late-maturing cultivars with parentage
tracing to PI 203396, the primary source of their resistance to tomato spotted wilt virus (genus
Tospovirus; family Bunyaviridae), late leafspot (Cercosporidium personatum, Berk & Curt.), and
white mold (Sclerotium rolfsii, Sacc.). These cultivars consistently produce high yields and good
grades (Table 1-1). However, field emergence has been unreliable after storage in commercial
bulk peanut bins. AP-3 was chosen as the control, mainly because AP-3 has no lineage of PI
203396 and has good field emergence after storage in commercial bulk bins. AP-3 is medium
maturity and yields well, has resistance to tomato spotted wilt virus and white mold, but is
susceptible to leafspot and rust. For seed origin comparison, seed was obtained in October 2004
from two different field-growing environments: Florida Foundation Seed (FFSP), Marianna,
Florida, and the University of Florida North Florida Research and Education Center (NFREC)
near Marianna, Florida. Pods were dried with forced heated air (-330C) to a seed moisture
content of 8 to 10%. The pods of each cultivar within a seed source were thoroughly mixed to
minimize variation and bagged in burlap sacks for placement in four storage locations. Samples
of each seed origin and cultivar were frozen to preserve seed condition prior to the storage
treatment. All peanuts were stored in-shell, unless otherwise stated. Bags of in-shell peanuts of
each cultivar from each seed origin in each year were placed into storage treatment locations as
* NFREC temperature and relative humidity controlled storage facility (UNICOOL),
* Approximately 1.5 meters into the base of the bulk in-shell peanut pile within the FFSP
commercial peanut storage barn (STACKS),
* In bags on pallets in the FFSP warehouse (WHSE),
* In the center of bulk peanuts being stored in wagons housed in open-sided sheds at FFSP
A HOBO" Pro Series temperature/relative humidity recorder manufactured by Onset
Computer Corp., Bourne, Mass., was placed adj acent to the stored samples at each treatment
location. UNICOOL location provided uniform air temperatures of 12-150C and relative
humidity at 60-70% throughout October to December. In the STACKS piles varied in height to
15 meters and had a volume of 7,000m3. Ventilation was accomplished through open bin doors
that allowed ambient air to move across the face of the pile and exhaust by fans through roof
openings. No forced air or intra-pile ventilation was provided. Peanut bags in WHSE were
exposed to daily fluctuating temperatures and relative humidity without being deeply buried
under other in-shell peanuts. The WAGON storage site was in drier wagons under open sheds
and provided a fourth storage condition. Temperature and relative humidity data were recorded
at 15 minute intervals in each storage location. Ambient temperatures and relative humidity
outside the storage facilities were recorded by the Florida Automated Weather Network (FAWN)
substation located at Latitude 30.850 and Longitude -85.165 approximately 50m from the FFSP
storage facilities. In December, FFSP began shelling peanuts. When a bin became empty, the
treatments were ended; the peanut bags were removed from their storage places and placed into
controlled atmospheric conditions at 12-130C and 66-68% relative humidity to hold condition
until tested for germination or field emergence (April and October).
In crop production year 2004, placement of bagged in-shell peanut samples was delayed
and bagged peanuts could not be placed within the center of STACKS or WAGON. For
comparison of storage effects deeper within the STACKS and WAGON, a second set of samples
designated Bulk Stored Seed was collected at the end of the storage period from pods deep
within the bulk piles of STACKS and the center of WAGON. This sample set is similar to the
bagged peanut samples in all aspects except that the peanuts during storage were located deeper
within the bulk piles of the STACKS or the bulk peanuts of the WAGON. In the analysis of
towel germination and Hield emergence tests of bulk stored seed, data for the controlled
environment treatment was the same data as UNICOOL in the bagged seed analysis.
All seed was stored in-shell. Bagged seed was shelled in small lots using a pod separator,
a standard Federal/State grade sheller, a seed sizer, and sized, keeping sound mature kernels
(SMK) that rode a 16/64 screen. Bulk seed was shelled by FFSP as follows: A front-end
payloader scoops the peanuts out of the bin, loads the peanuts into a bulk wagon, and peanuts are
dumped into an elevator, lifted to the top of the elevator leg, dropped to the sheller and then sized
using a 16/64 screen. The seed was stored in large cardboard boxes in the warehouse.
Germination Tests for Seed Viability
Germination tests were conducted according to the Rules for Testing Seeds published by
the Association of Official Seed Analysts (2004). Seeds were treated with Vitavax" PC for
control of fungi. Active ingredients in Vitavax" by weight are Captan 45.0%, pentochloronitro
benzene 15%, and Carboxin 10%. Samples of 50 seeds of each treatment were evenly spaced on
double germination towels, covered with a third towel, the lower edge of the towels was folded
to retain seeds, and the towels were rolled into a cylinder shape and set on end in sealed plastic
containers. Unless otherwise noted, germination tests were conducted in a Model I-35LVL
germination chamber manufactured by Percival Mfg. Co., Boone, Iowa. Temperature was set at
250C and seedlings with > 1-cm radicle were counted 10 days after imbibition. Each year towel
germination tests were run just prior to sowing Hield emergence tests. In April 2005 the towel
germination test of seed from crop production year 2004 included four replications of 50 seeds
each of four cultivars, C-99R, DP-1, Hull, and AP-3, from two Hield origins, NFREC and FFSP,
stored in the four treatment locations. In April 2006 the towel germination test of seed from crop
production year 2005 included the same four cultivars plus three new cultivars McCloud,
Florida-07, and York. For comparison, at the conclusion of the storage period, seed from these
same cultivars was tested for germination by Tallahassee Seed Testing, LLC, located at 1510,
Capital Circle SE Suite El, Tallahassee, Florida, 32301.
Seedling Emergence Field Tests
Four replications of 50 seeds each of the treatments derived either from the bagged peanuts
or the bulk stored peanuts were sown on successive days in a Randomized Block Design (RBD)
in Millhopper Eine sand in Hield research plots located at the University of Florida, Gainesville,
Florida. Soil temperatures exceeded 15.5oC at the sowing depth of 5 cm. All seeds were treated
with Vitavax Seeds were placed in twin rows spaced 15 cm apart with in-row spacing between
seeds of 8 cm. The plots were watered by overhead irrigation and all emerged plants were
counted 14 days after sowing (DAP). No fertilizer was applied. All subsequent seedling
emergence field tests followed this procedure unless otherwise noted. For production year 2004,
sown seed in April came from both the bagged seed (FFSP and NFREC origins) and bulk stored
seed (FFSP origin only). In the October seedling emergence test, the seed sown came from the
bulk stored seed (FFSP origin) that had been stored from the time of the April sowing until
October in UNICOOL at 12-130C and 66-68% relative humidity. The additional storage time
was 5.5 months. Samples sown consisted of the four cultivars selected at the following time
periods from the specified storage locations: UNICOOL-Jan, STACKS-Jan, WAGON-Jan,
UNICOOL-Mar, STACKS-Mar, WHSE-shelled-Mar, and WAGON-Mar. Treatments were
sown October 4, 2005, in a polyethylene-covered hoop greenhouse located at the University
Research Plots in Gainesville. Replications were sown at Hyve-day intervals.
For the 2005 production year seed, a seedling emergence field test was sown May 5, 2006.
Three additional cultivars, McCloud, Florida-07, and York, were added to the previously
selected four cultivars. Seed origin was FFSP only; since prior year tests showed no effect of
seed origin. Storage treatment locations were: 1) UNICOOL, 2) STACKS, and 3) WHSE. Four
replications were sown on successive days in the same field site as the prior year' s field
Storage Pathogen Assays
Pathogen assays were conducted using Difco Sabouraud Maltose Agar mixed thoroughly
at the rate of 65 grams of agar suspended in one liter of de-ionized water. Suspension of the agar
was accomplished with constant agitation and the suspension was dissolved by boiling the water
for one minute. The solute was autoclaved at 1210C for 15 minutes and poured into Petri dishes
for cooling. The approximate formula per liter of solute (Tuite, 1969) was 10g of enzymatic
digest of casein, 40g of maltose, and 15g of agar resulting in a final pH of 5.6 + 0.2. Peanut
seeds were disinfected with 1% Clorox brand of sodium hypochlorite for 2 minutes, rinsed in
sterile water for 2 minutes, and then, using aseptic techniques, the seeds were split in half and the
cotyledon containing the embryo was placed flat-side down on the maltose agar. The specimens
were incubated at 320C for 84 hours and then pathogens were identified. The assay consisted of
20 seeds for each of the four cultivars, AP-3, C-99R, DP-1, and Hull, from three storage
treatments in years 2004 and 2005. The samples from the 2004 crop production year were 1)
seed frozen at harvest, 2) seed stored in UNICOOL, and 3) seed stored in the STACKS. The
samples from the 2005 crop production year were: 1) seed frozen at harvest, 2) seed stored in
UNICOOL, and 3) seed stored in the STACKS.
Seed Vigor Tests
Seed vigor tests were conducted in accordance with the guidelines presented in the Seed
Vigor Testing Handbook published by the Association of Official Seed Analysts (2002) and a
"vigor index" developed by D.L. Ketring (1993). Seed vigor is difficult to standardize from test
to test. Moisture and temperature have profound effects upon rate of growth and the
measurement technique may vary among seed analysts. Vigor tests in this research were
conducted to test cultivar effect and the interaction between cultivar and storage location. The
results of the vigor tests are reported as "comparative vigor index" (CVI) for the specific purpose
of comparing seedling growth within a specified towel germination test and differs from the
Ketring (1993) Vigor Index, in which he adds a factor of total percent germination. CVI tests
were conducted using the standard towel germination test procedure for peanut (Association of
Official Seed Analysts, 2004). Seedling growth rate was measured as linear growth of the
hypocotyl/radicle axis and seedlings were separated into four classes: no growth, radicle < 3cm,
radicle 3-6cm, and radicle > 6cm. Numerical values were assigned to the classes to reflect
relative value of the seedlings: 0 for no growth, 1 for growth < 3cm, 2 for growth 3-6cm, and 3
for growth > 6cm. The CVI for the treatment equaled the sum of the products of number of
seedlings per class times the value assigned to the class. Percent germination was computed by
counting all seeds with protruding radicles >1cm.
For the Seed Vigor Tests #1 and #2, the seed source was from production year 2005. For
Vigor Test #1, the treatments were four cultivars, AP-3, C-99R, DP-1, and Hull, from each of
four storage locations. Two replications of 25 seeds were held for 44 hours at 400C and 100%
relative humidity in an accelerated ageing chamber. Two replications of 25 seeds were placed
into the controlled storage at 130C and 67% relative humidity for 44 hours. All four replications
of 25 seeds were germinated at 30/200 for 7 days in the University of Florida Seed Laboratory
germination chamber. In Vigor Test #2 the treatments were seven cultivars, AP-3, C-99R, DP-1,
Hull, McCloud, Florida-07, and York, each stored in three locations, UNICOOL, STACKS, and
WHSE. Four replications of 25 seeds were germinated at 250C for 7 days in the University of
Florida Seed Laboratory germination chamber.
Electrolyte Conductivity Tests
The procedure for determining the electrolyte conductivity of leachate of germinating
peanut seed followed the guidelines suggested by McDonald and Copeland (1989). Sample size
was 50 seeds. Each sample was weighed and then placed into 200mL de-ionized water in
beakers in a controlled temperature chamber at 30/200C for 24 hours. After 24 hours, 10 mL of
leachate per treatment were withdrawn from the leachate beakers to test for electrolyte
conductivity. The leachate was agitated by swirling gently and electrolyte conductivity was
determined using a Fisher Scientific Digital Conductivity Meter (Fisher Scientific, Pittsburgh,
PA). Data were expressed as Clmhos and were divided by the seed g weight to obtain Clmhos g .
Unless otherwise stated, electrolyte conductivity tests followed this procedure.
All electrolyte conductivity tests were accompanied with a companion CVI test. There
was a leachate conductivity test of seed from production year 2004 and a second leachate
conductivity test as part of the accelerating aging test on seed from production year 2005. To
determine CVI and percent germination, the seeds, after the 24 hours of imbibition, were
wrapped in moist towels and incubated at 30/200C for an additional 5 days. In the leachate
conductivity tests, each year the seed source was from excess seed stored at temperature < 00C
since the April field emergence tests. The cultivars were AP-3, C-99R, DP-1, and Hull. Storage
locations were UNICOOL, STACKS, WHSE, and WAGON.
Accelerated Ageing Tests
Accelerated ageing treatments were used to simulate the storage environment of the
STACKS. The seeds were first subjected to ageing treatments and then evaluated for electrolyte
conductivity, seedling vigor, germination, and field emergence. The seed for this test came from
seed of production year 2005 stored in WHSE until March 2006 and then stored in UNICOOL at
12-130C and 66-68% relative humidity. Total sample size per treatment was 250 seeds of each
of the seven cultivars: AP-3, C99, DP-1, Hull, McCloud, Florida-07, and York. The treatment
time period was 5 weeks. The treatments were (1) control, in the UNICOOL at 130C and 67%
relative humidity, (2) accelerated ageing chamber at 320C and 100% RH (EAA~wks), and (3) the
University germination chamber at 320C and relative humidity < 10% (HT/LRH). All seeds
were treated with Vitavax". For the EAA~wks treatment, seeds were placed in a single layer on
a copper screen above a water surface in small plastic germination boxes sealed with lids
(Association of Seed Analysts, 2002). The boxes were placed in an accelerated ageing chamber
containing water and a submerged electrical heating element. For the HT/LRH treatment, the
seeds were placed in plastic cups without lids in a larger sealed plastic box containing desiccants
and held in the germination chamber. The 250 seeds of each cultivar of each treatment were
divided into two lots, one of 200 seeds for subsequent field planting and one of 50 seeds to be
used for percent moisture measurements, electrolyte conductivity tests, seedling vigor tests, and
germination tests. Upon completion of the 5 week test period, samples of 20 seeds from each
treatment were placed in 100 mL de-ionized water at 300C for 24 hours. The electrolyte
conductivity of the resulting leachates was recorded. After 24 hours of imbibition, the samples
of 20 seeds were wrapped in moist towels and returned to the germination chamber at 300C for
96 hours for determination of comparative seed vigor and percent germination. The lots of 200
seeds selected for the seedling field emergence test were divided into four replications of 50
seeds each and sown into the field plots at the University of Florida beginning September 20,
2006. Soil temperatures exceeded 220C at the sowing depth of 5 cm. The number of vigorous
plants was counted at 8 DAP and 12 DAP. A plant was considered vigorous if the trifoliate
leaves were open, horizontal, and the plant was green and healthy. The 8 DAP counts indicated
relative vigor of the treatment sample and the 12 DAP counts provided final percent field
Antioxidant Capacity Assay
Antioxidant capacity of seeds was evaluated using a method designed to assay antioxidants
in animal tissue (Glavind, 1963). The cultivars were AP-3, C-99R, DP-1, and Hull. The seed
sample sources were seed from the 2004 harvest, the 2005 harvest, the 2006 harvest and seed
from 2004 production year stored in STACKS. Peanuts from 2004 harvest were stored in
UNICOOL at 12-130C until January 31 and then stored at temperature < 00C. Seed from 2005
harvest and 2006 came from samples stored at temperature < 00C since harvest. Seed from
STACKS was stored at temperature < 00C upon removal from STACKS. There were three
replications. For this assay, the testa was removed and the seeds were trimmed to approximately
uniform weights of 0.400 g. Peanuts were ground individually with mortar and pedestal and
soaked in 5mL of methanol for 15 minutes. The mixture was centrifuged at 2500 rpm for 10
minutes at room temperature. A sample of 150C1L of methanol extract was drawn from each
sample and placed into a small test tube containing 850C1L of DPPH at 0.25mM. DPPH is 90%
1-Diphenyl-2-picryl -hydrazyl (C18H12N5O6). DPPH at 0.25mM is prepared from 0.0098 g DPPH
in 100 mL methanol. This procedure was replicated three times per seed. The combination of
peanut methanol extract and DPPH was allowed to react for a minimum of 15 minutes.
Standards for comparison were prepared using Trolox, a Vitamin E equivalent. Trolox is 1-6-
hydroxy 2,5,7,8-tetra-methychromane-2-carboxylic acid A-6-hydroxy-2, 5,7,8-tetra-methyl-
chroman-2- carbonsaure. Trolox at 1.0mM was prepared from 0.0048g Trolox placed into 20
mL of methanol and then diluted with additional methanol to create one mL standards containing
5C1M, 10CIM, 25C1M, 50C1M, 75C1M, and 100C1M of Trolox. A blank standard consisted of zero
Trolox made from 100C1L of methanol and 900C1L DPPH. The resulting total number of samples
was three replicates x four cultivars x four storage treatments with each sample assayed three
times. Samples were pipetted into 96-well Corning Costar plates and assayed for antioxidant
capacity using a Spectra Max 340 PC manufactured by Molecular Devices Corporation, Union
City, CA. Spectra Max 340 PC is a visual range spectrophotometer applicable for evaluating
ELISA assays. The software program was Softmax pro 4.8. The assay was conducted at a wave
length of 517 nm.
Experimental Design and Data Analysis
Laboratory tests and field emergence trials were set up as Randomized Block Designs
(RBD). Analyses of Variance (ANOVA) was accomplished by the procedures in SAS System
Release #9.1 (SAS Institute). Least Squares Means and Duncan's Multiple Range Tests were
generated using the general linear model (PROC GLM). Pearson correlation coefficients were
generated using the correlation procedures (PROC CORR). Regression analyses were conducted
using PROC REG. Unless stated otherwise, differences reported were significant at alpha of less
than or equal to 0.05.
RESULTS AND DISCUSSION
Effect of Seed Storage Environment on Germination and Field Emergence
Prior Germination Tests of Cultivars at NFREC
Peanut cultivars Florida MDR-98, C-99R, DP-1, and Hull were released by the University
of Florida for commercial production in 1999 2002. Poor Hield emergence and unacceptable
stands occurred (Tilllman, 2004, Per. Comm.). The 2002 and 2003 NFREC Hield emergence
trials included limited entries of seeds produced by Florida Foundation Seed Producers (FFSP)
(Tillman and Gorbet, 2004). A comparison of the entries showed that FFSP seed had lower
germination than NFREC seed and that the cultivars were not equally affected. Germination of
AP-3, C-99R, DP-1, and Hull seed produced by FFSP was 10.7%, 13.4%, 33.1%, and 32.4%
lower than germination of seed produced and stored by the NFREC peanut breeding program.
Germination Tests of Seeds from NFREC and FFSP Stored in Bags in Various Locations
These towel germination and Hield emergence tests were designed to identify (1) the
process and factors in commercial seed production that affected seed vigor and Hield emergence,
(2) the reliability of germination tests in predicting Hield emergence, and (3) year to year
variation in Hield emergence.
At the conclusion of the storage seasons, mean towel germination for crop production year
2004 was 93.6% and was superior to the mean towel germination of 87. 1% for production year
2005 (P <0.0001) (Tables 4-1 and 4-2).
Mean towel germination for seed from FFSP origin was 94.4% and was superior to the
mean germination of 92.8% of the NFREC origin (P=0.0321) (Tables 4-1 and 4-3). The lower
mean germination of seed from NFREC resulted from the weaker germination of NFREC seed of
C-99R and DP-1. C-99R and DP-1 of NFREC origin germinated at 88.4% and 85.8% compared
to 95.3% and 93.1% for C-99R and DP-1 from FFSP origin. The low towel germination was not
expected and may have resulted from residual dormancy or cultural conditions during seed
The main effects of cultivar and storage location for germination tests and field emergence
tests of peanuts stored in bags in various locations for crop years 2004 and 2005 are presented in
Figures 4-1 and 4-2. In both 2004 and 2005, cultivar differences were evident in towel
germination tests (2004, P<0.0001; 2005, P=0.0442; combined, P=0.0239) (Table 4-1). In 2004,
towel germination of AP-3 and Hull was superior to C-99R and DP-1; C-99R was superior to
DP-1 (Figure 4-1). In 2005, towel germination of AP3 and C-99R was similar and greater than
that of Hull; towel germination of DP-1 was intermediate. Combined across both years, towel
germination of C-99R was 92.6%, which was greater than both DP-1 and Hull. Towel
germination of AP-3 was intermediate.
Storage environment/location affected towel germination (2004, P=0.0493; 2005,
P=0.0498; combined, P=0.0847 (Table 4-1). For the 2004 crop, towel germination of seed from
the warehouse (WHSE) and wagon (WAGON) storage locations was superior to the University
of Florida controlled storage facility (UNICOOL); germination of seed stored in the bulk bin
facilities of FFSP (STACKS) was intermediate (Figure 4-2). For the 2005 crop, towel
germination of seed stored in STACKS was superior to UNICOOL; germination of seed from
WHSE and WAGON was intermediate. In the combined analysis, towel germination of seed
stored in the WAGON was superior to UNICOOL; germination of seed from STACKS and
WAGON was intermediate.
The towel germination tests showed interaction of cultivars with storage locations for crop
production year 2004 (P=0.0021), but not for crop production year 2005 (P =0.6526) (Table 4-1).
Germination of AP-3 and Hull was consistent across storage environments, but germination of
DP-1 and C-99R was not consistent across storage environments (Figure 4-3). Germination of
C-99R stored in UNICOOL was inferior to C-99R stored in STACKS (P=0.0003), WHSE
(P=0.0474), and WAGON (P=0.0191). Germination of DP-1 stored in UNICOOL was inferior
to DP-1 stored in WAGON (P=0.0627) and WHSE (P=0.0007).
In summary, the towel germination tests indicate that seed quality in crop year 2004 was
superior to seed quality in crop year 2005 (Table 4-2). Contrary to expectations, FFSP seed
origin was not inferior to NFREC seed origin (Table 4-3). UNICOOL storage location was not
superior to other storage locations. Germination of cultivars was differentially affected by
storage location (Table 4-1).
Field Emergence of Seeds from NFREC and FFSP Stored in Bags in Various Locations
In 2004 seed origin did not affect field emergence (P=0.9104) (Table 4-1). Based on this
finding and the fact that in the towel germination tests, seed of FF SP origin was not inferior to
seed of NFREC origin, and the fact that poor seed emergence occurred frequently with peanut
produced and stored by FFSP and only infrequently with peanut produced and stored by NFREC,
the decision was made to limit all subsequent seed tests to seed of FFSP origin.
Year of production affected field emergence (P=0.0082) (Table 4-1). Mean emergence for
crop production year 2004 was 88.3% and was superior to the mean emergence of 85.1% for
crop year 2005 (Table 4-2).
For both 2004 and 2005 crop years, cultivar affected field emergence (2004, P<0.0001;
2005, P=0.0043; combined P<0.0001) (Table 4-1). For the 2004 crop year, field emergence of
AP-3 was superior to DP-1 and Hull, and similar to C-99R (Figure 4-1). Field emergence of DP-
1 was less than AP-3, C-99R, and Hull. For the 2005 crop year, field emergence of AP-3 and C-
99R was similar and greater than that of DP-1 and Hull. Combined across both years, Hield
emergence of AP-3 and C-99R was about 89%, which was greater than both DP-1 and Hull.
Storage location affected Hield emergence (2004, P=0.0208; 2005, P=0.0957; combined,
P=0.0063) (Table 4-1). From the 2004 crop, Hield emergence of seeds stored in the STACKS
was lower than that from UNICOOL and WAGON, but similar to that from WHSE (Figure 4-2).
For the 2005 crop year, Hield emergence of seeds stored in the STACKS was less than those
stored in the WHSE and emergence of seeds stored in UNICOOL and WAGON was
intermediate. In the combined analysis, Hield emergence of seeds stored in the STACKS was less
than all other locations (P=0.0711).
In summary, Hield emergence tests confirmed that seed origin was not a significant factor
in poor field emergence; that the seed quality of crop production year 2004 was superior to the
seed quality of crop production year 2005; and that cultivars AP-3 and C-99R had superior field
emergence, compared to DP-1 and Hull. In contrast to towel germination tests, field emergence
of seed stored in STACKS had lower field emergence than all other storage locations.
Towel Germination and Field Emergence of Bulk Stored Seed from Production Year 2004
Bagged seed in-shell samples were derived by hand mixing peanuts from several wagons
and placing the bags as deep as possible into STACKS and WAGON. This depth was
approximately 1.5m. In contrast to the bagged samples, the bulk stored seed samples were
collected from the same STACKS and WAGONS either by probing into the bulk pile or as the
peanuts were being emptied at the conclusion of the storage season. Since the bulk stored seed
samples came from deeper within the piles than the bagged seed, they should be more
representative of the effects of interaction of cultivar and storage environment and provide a
comparison to the towel germination and field emergence of the shallower buried bagged
The main effects of cultivar and storage location for towel germination tests and field
emergence of bulk stored in-shell peanuts sampled from various locations for production year
2004 are presented in Figures 4-4 and 4-5. Seed origin was FFSP. Cultivar differences were
evident in towel germination tests, and in field emergence tests in April and October (towel,
P=0.0002; April field emergence, P<0.0001; October field emergence P<0.0001) (Table 4-4). In
towel germination and the April field emergence, cultivars AP-3 and C-99R were superior to
DP-1 and Hull and in the April field emergence, Hull was superior to DP-1 (Figure 4-4). In
October field emergence, AP-3 was superior to C-99R, DP-1, and Hull; C-99R was superior to
DP-1 and Hull.
Storage location also affected towel germination and April and October field emergence
(towel, P=0.0309; April field emergence, P<0.0001; October field emergence, P<0.0001) (Table
4-4). Towel germination of seeds stored in UNICOOL and WAGON was superior to those
stored in STACKS (Figure 4-5). In April field emergence, UNICOOL was superior to STACKS
and WAGON, and WAGON was superior to STACKS. In October field emergence, UNICOOL
was superior to both STACKS and WAGON.
The interaction of cultivar and storage location is presented in Figures 4-6 and 4-7. The P-
values for interaction of cultivar and storage location for towel germination, April field
emergence, and October field emergence were P=0.5447, P<0.0001, and P<0.0001 respectively
(Table 4-4). Field emergence of AP-3, C-99R and DP-1 was similar in the UNICOOL, but when
stored in either the WAGON or STACKS, field emergence of DP-1 was inferior to that of AP-3
or C-99R (Figure 4-6). In October field emergence, the interaction of cultivar and storage
environment was more pronounced (Figure 4-7). For all cultivars, storage in UNICOOL was
superior to storage in STACKS. Comparing UNICOOL to STACKS, October field emergence
of AP-3 decreased from 89% in UNICOOL to 69%, C-99R from 75% to 57%, DP-1 from 79%
to 26%, and Hull from 71% to 37%. AP-3 stored as well in the WAGON as in the UNICOOL,
but C-99R, DP-1, and Hull had greatly reduced field emergence when stored in WAGON as
compared to UNICOOL. Note that field emergence in October of AP-3 stored in STACKS was
less than field emergence of AP-3 stored in UNICOOL (P=0.0003), indicating that seed vigor of
AP-3 while stored in STACKS decreased. The implication is that STACKS storage may reduce
vigor for all cultivars and, therefore, in conditions of stress, reduce seedling populations in the
Comparison of Treatment Effects on Bagged Seed Samples and Bulk Stored Seed Samples
In comparing the April field emergence of bagged seed and the April field emergence of
bulk stored seed from production year 2004, there was no interaction between cultivar and
storage location among the bagged seed (P=0.1355). However, among the bulk stored seed the
P-value for interaction of cultivar and storage location was <0.0001 (Tables 4-1 and 4-4). A
comparison of field emergence of bagged seed and bulk stored seed (both in STACKS) is
presented in Table 4-5. Bulk stored seed had reduced field emergence when compared to the
field emergence of bagged seed. The extreme example is the decrease of 37.5% in field
emergence of bulk stored seed of DP-1.
The effect of interaction between cultivar and storage environment/location was very
pronounced for the field emergence test in October (Figure 4-7). Field emergence was greatly
reduced except when cultivars were stored in UNICOOL. Seed deterioration is a result of
changes within the seed that decrease the vigor of the seed (McDonald, 2004). Over time,
damage from a suite of degrading reactions accumulates and a change occurs from strong
viability to a weaker seed to a non-viable seed (Walters, 1998). Seed vigor declines faster than
germinability (Figure 4-8). Although the towel germination tests indicated acceptable seed
quality in April, the interaction of cultivar and storage environment/location may have greatly
reduced seed vigor, as seen in the differences in April field emergence of bagged seed versus the
April field emergence of bulk stored seed (Table 4-5). The storage period for all treatments from
April field emergence to October field emergence was 5.5 months in UNICOOL at 12-130C and
66-68% relative humidity. The additional stress was uniform and minimal for all samples, and,
yet, in October field emergence, the number of emerged seedlings was dramatically lower (Table
4-6). The greatly reduced field emergence in October indicates that vigor of seed not stored in
UNICOOL was marginal in April and that seed germinability and seed vigor had reached the
points of steep descent indicated by the Ys on the viability and vigor curves of the seed
deterioration graph (Figure 4-8).
Correlation of Towel Germination and Field Emergence
Towel germination tests were completed one week preceding sowing peanut seed in April.
A comparison of towel germination data, the germination tests conducted by Tallahassee Seed
Testing, LLC, and the field emergence in April of bulk stored seed is presented in Table 4-7.
Germination in the Tallahassee Seed Tests and the germination in the towel germination tests of
this research are similar. Field emergence of AP-3, C-99R, and DP-1 stored in UNICOOL had
final seedling stands similar to the towel germination tests. However, field emergence of seed of
AP-3, C-99R, and DP-1 not stored in UNICOOL had diminished final seedling stands (Figure 4-
6). For example, the difference for DP-1 was -38.5% for seed stored in STACKS and -33.5% for
seed stored in WAGON. Towel germination was not correlated with April field emergence (P=
0.6763) or with October field emergence (P=0.4507) (Table 4-8). Similarily, April field
emergence was not correlated to October field emergence (P=0.1519). The insignificant P-
values for correlation of towel germination tests and field emergence support the concept that
vigor decreases at a rate different from the rate of decrease of seed viability and that towel
germination tests do not reflect seed vigor and are not reliable for estimating field emergence.
Summary of Results of Towel Germination Tests and Field Emergence Tests
The data from the crop year 2004 and 2005 towel germination and field emergence tests is
in accord with the antidotal reports that poor field emergence and stand failures vary from year to
year and that failures are more frequent with DP-1 and Hull. Researchers and growers have
depended upon standard towel germination tests to evaluate the effect of winter storage and to
estimate seedling population stands. Progress in peanut breeding programs has been hampered
by the poor correlation of towel germination tests to seed vigor and the failure of towel
germination tests to identify loss of seed vigor when peanuts are stored in unventilated bulk bins
at elevated temperatures and relative humidity. The results of these studies support the
* Seed production origin is not a significant factor in eventual field emergence (Tables 4-1
* There may be an effect of season upon seed vigor and field emergence (Table 4-1).
* Across years the field emergence of AP-3 and C-99R cultivars was superior to DP-1 and
Hull (Figure 4-1).
* Field emergence of seed was maintained when seed was stored in UNICOOL at
temperatures < 160C and relative humidity < 70% (Figures 4-2, 4-6 and 4-7).
* Field emergence of seed may decrease when seed is stored in large bulk bins (STACKS) or
drying wagons (WAGON) (Figures 4-2, 4-6 and 4-7).
* There was a cultivar by storage environment/location interaction for field and laboratory
germination, based on the two year analysis of bagged seed and the 2004 bulk stored seed
(Tables 4-1, 4-4 and Figures 4-6 and 4-7). Compared to storage in UNICOOL, field
emergence of DP-1 and Hull stored in STACKS and WAGON declined more than that of
* Towel germination tests are not a reliable measure of field emergence for peanut (Tables 4-
7 and 4-8).
Storage Environment Characteristics as Possible Factors in Declining Seed Vigor
External atmospheric temperature and relative humidity data at 2m height for 2004 and
2005 were recorded at the Florida Automated Weather Network (FAWN) adj acent to the storage
facilities. Daily mean temperatures for the period September 16 to January 24 are presented in
Figure 4-9. The mean of the daily temperatures was 15.80C in 2004 and 15.70C in 2005.
Although there is no meaningful difference in the mean daily temperatures of the external air for
ventilation, the temperatures within the center of STACKS October 11 to January 24 for the
2004 crop year averaged 22.80C compared to 15.80C for the 2005 crop, a difference of 7.00C
(Figure 4-10). Conceivably the timing of warm and cool weather fronts for ventilation will
affect the relative rate of cooling of peanuts in STACKS. In 2005 from October 23 to November
11, external air temperatures averaged 13.4oC, which was 8.60C cooler than the average of
22.00C for the same period in 2004. During that time interval, temperature in STACKS in 2005
decreased SoC and temperature in STACKS in 2004 increased 30C. However, the 11i-day period
of higher temperatures in 2004 by itself does not account for more than a part of the higher
temperatures throughout the storage period in 2004. Although external air temperature may
change rapidly, sometimes as much as 100C within 24 hours, the temperatures at the center of the
STACKS changed slowly, usually less than 20C per day. The temperature and relative humidity
for the DP-1 in WAGON storage fluctuated with the external atmospheric conditions and that
temperature was cooler than in STACKS for the same time period (Figure 4-11). A comparison
of temperature in STACKS to the outside temperatures recorded at FAWN and by sensors in
WAGON under sheds suggests that the temperature in STACKS in 2004 is unexpectedly higher,
possibly associated with heating in STACKS by peanut respiration resulting from insufficient
drying or a climate event in 2004 that may have affected maturity of the seed prior to drying.
Temperatures in UNICOOL varied between 10 and 15oC in 2004 and fluctuated from 7 to
210C in 2005 (Figure 4-12). The UNICOOL 2005 seed was stored in an inadequately insulated
building and temperatures could not be held constant. Mean temperature in UNICOOL was
12.30C in 2004 and 14.4oC in 2005. Compared to STACKS, in 2004 temperature in the center of
STACKS exceeded that in UNICOOL by 5-150C until the end of December (Figure 4-13). By
contrast, in 2005 the mean temperature of 15.80C in the center of STACKS exceeded the
UNICOOL mean temperature of 14.4oC by only 1.4oC. For 2005, STACKS environment was
cooler and UNICOOL environment was warmer; a fact that may explain the differences in effect
of year in the towel germination and field emergence tests.
Harrington (1972) and McDonald (2004) stated that relative humidity and temperature
were the two most important factors affecting the rate of seed deterioration and that seed
moisture and temperature interact. Relative humidity in all storage conditions varied throughout
the storage period (Figures 4-11, 4-14, and 4-15). In the UNICOOL the relative humidity
fluctuated between 58% and 79% in 2004 and between 44% and 77% in 2005. In the STACKS
the relative humidity decreased from 86% to a range fluctuating between 52% and 62% in 2004
and relative humidity decreased from 86% to 73% in 2005. The differences in relative humidity
across years and storage locations is minor, thus indicating that relative humidity may have been
a contributing factor to loss of seed vigor, but was not the single factor causing loss in seed
The temperature and relative humidity data support the conclusions:
* In 2004, temperatures in STACKS averaged 7.00C higher than temperatures in STACKS in
2005 and 10.5oC higher than temperatures in UNICOOL in 2004 (Figures 4-10 and 4-13).
* Temperatures at the center of the STACKS during the first months of storage may range
from 200C to >300C (Figure 4-13).
* The internal temperature in STACKS changes much slower than external air temperature
(Figures 4-9 and 4-10).
* Temperature in STACKS may differ from year to year (Figure 4-10).
* Differences in relative humidity were present but not enough to be the only factor in poor
field emergence of peanut (Figure 4-15).
* The literature states that relative humidity and temperature are the two most important
factors affecting the rate of seed deterioration and that seed moisture and temperature
interact. In the STACKS in 2004 relative humidity and temperatures were elevated and
probably interacted to reduce the seed vigor during the storage of the 2004 seed.
Effect of Storage Pathogens
Five storage fungi were isolated; Aspergillus flavus Leek, Aspergillus niger Thom &
Raper, Rhizopus spp., Fusarium spp. and Penicillium spp. (Table 4-9). In 480 seeds assayed,
contamination by A. Flavus was 0.4%, A niger was 2.5%, Rhizopus spp. was 1.5%, and
Fusarium spp. was 2.7%. Penicillium spp. were the most numerous fungi isolated and were
found on 5.8% of the seed assayed. Seed stored in the stacks had more incidences of fungi than
seed at harvest or seed stored in UNICOOL. The highest incidence of storage fungi was
Penicillium spp. present in seed stored in the stacks during the winter of 2005. The very low
incidence of storage fungi and the random distribution of fungal contamination support the
* Storage fungi were not an important factor contributing to poor field emergence in 2004
* Storage environmental conditions did not cause excessive growth of Aspergillus spp.
Measures of Seed Quality
Comparative Vigor Index Tests
Seed vigor is reported here as "comparative vigor index" (CVI). The hypocotyl-radicles of
germinating seeds were measured and placed into classes with numerical values. The CVI for a
seed sample equals the sum of the products of number of seedlings per class times the value
assigned to the class. A photograph presented in the appendix demonstrates the variation in rate
of growth of germinating peanuts. There are 11 seedlings with excellent vigor (value 3), 10
seedlings with medium vigor (value 2), 3 seedlings with low vigor (value 1), and one seed
counted as zero vigor (value 0). The CVI computes to 56.
In vigor tests #1 and #2, the seed source was from crop production year 2005. Vigor Test
#1 consisted of cultivars AP-3, C-99R, DP-1, and Hull representing the four storage environment
locations; half of the seed was subj ected to accelerated ageing (AA) at 400C and 100% humidity
for 44 hours and the other half was the control. Vigor Test #2 consisted of the same four
cultivars plus McCloud, Florida-07, and York, each stored in three locations, UNICOOL,
STACKS, and WHSE.
Cultivar had a significant effect on CVI Test #1 (P=0.0058) and in CVI Test #2 (P=0.0083)
(Table 4-10). In Vigor Test #1, AP-3 and C-99R were more vigorous than DP-1 (Table 4-11).
In Vigor Test #2, AP-3, C-99R, Florida-07, and York were more vigorous than Hull and
McCloud; but only York was superior to DP-1 (Table 4-12). The relative ranking of cultivars by
seed vigor in these two vigor tests correlates with the April and October field emergence (Table
4-8). P-values for correlation of Vigor Test #1 with field emergence were 0. 1084 for April and
0.0003 for October. In Vigor Test #2, P-values for correlation were 0.0027 for April and 0.0220
for October. The data support the conclusion that seed vigor tests can be used as reliable
indicators of potential final field population stands. This is in agreement with the conclusions of
Location effect was significant (0.0003) in CVI #1 (Table 4-10). UNICOOL was inferior
to all other storage locations (Table 4-11). The low vigor of seeds in this vigor test reflects the
low towel germination tests of seed stored in UNICOOL (Figure 4-2). In contrast, field
emergence of seed stored in UNICOOL was similar to all storage locations and superior to
STACKS (Figure 4-2). Surprisingly, seeds of the accelerated ageing treatments were
intermediate in vigor (Table 4-11). It was expected that peanut seed subj ected to accelerated
ageing temperatures of 400C and 100% relative humidity for 44 hours would have greatly
reduced vigor and that the mean vigor index would be low. Instead, the mean vigor index for
accelerated-aged seed was 54.7 compared to 39.5 for the control treatment (Table 4-11). In a
physical examination of the accelerated-aged seed, it was obvious that the seeds had absorbed
water. The unintended consequence of this accelerated ageing was seed priming. The increased
moisture concentration of the seed enabled the seed to commence germination earlier than seeds
in the control treatment. With a head start in germination, hypocotyl/radicle growth was
measured as increased comparative vigor index.
In Vigor Test #2 at day 7, storage environment/location had no effect upon seed vigor
(P=0.5031) (Table 4-10). Since storage location had no effect, then Vigor Test #2 may reflect
genotype differences in rate of germination of the cultivars. AP-3, C-99R, Florida-07, and York
may inherently germinate faster and in field plantings, seedling emergence would be faster than
seedling emergence of Hull and McCloud, but final population stands may be similar.
Electrolyte Conductivity and Comparative Vigor Tests
An electrolyte conductivity test measures the electrical conductivity of the leachate of
germinating seed. A higher electrolyte conductivity value indicates greater leakage of
electrolytes through the cellular membranes and decreased seed vigor (McDonald, 1998).
The seed samples were AP-3, C-99R, DP-1, and Hull from crop production year 2004
stored in UNICOOL, STACKS, WHSE, and WAGON. The data for leachate conductivity and
seed vigor along with the means from April and October field emergence are presented in Table
4-13. Seed stored in STACKS had higher electrolyte conductivity than seed stored in
UNICOOL, WHSE or WAGON. The mean electrolyte conductivity by storage location was
1.39 for STACKS, 0.88 for WAGON, 0.63 for UNICOOL, and 0.59 for WHSE. In the
subsequent seed vigor test, the comparative mean seed vigor for STACKS was 1.5; much lower
than the CVI of 34.5 for UNICOOL, 47.0 for WHSE, and 27.8 for WAGON. In the April field
emergence, the corresponding means for field emergence were 69.6% for STACKS, 91% for
UNICOOL, 88.5% for WHSE, and 77.7% for WAGON. In the October field emergence, the
corresponding means were 47.0% for STACK, 78.3% for UNICOOL, 48.5% for WHSE, and
44.5% for WAGON.
The Pearson Correlation Coefficient comparing electrolyte conductivity to comparative
seed vigor was -0.7638 (P=0.0009), as shown in Table 4-14. Correlation of electrolyte
conductivity and April field emergence was -0.8150 (P= 0.0002) and with October field
emergence was -0.5022 (P=0.0564) (Table 4-14). Comparative vigor index (CVI) correlated
with the April field emergence. During the intervening 5.5 months, vigor declined so rapidly
that by October the minimal residual vigor only correlated marginally at alpha = 0.05 with the
CVI test conducted in early April. Figure 4-16 presents a regression graph demonstrating the
relationship between electrolyte conductivity and seed vigor. As the electrolyte conductivity of
the leachate increases, seed vigor decreases as represented by the slower rate of
hypocotyl/radicle elongation. Figure 4-17 shows the negative regression of April field
emergence as electrolyte conductivity increases.
The electrolyte conductivity test and the subsequent vigor test confirm that, for seed stored
in STACKS in 2004, seed quality deteriorated. Seed quality of DP-1 and Hull deteriorated more
than the seed quality of AP-3 and C-99R. The significant correlations of electrolyte
conductivity, seed vigor test, and April field emergence confirm that both the electrolyte
conductivity test and the seed vigor tests are reliable indicators of potential field emergence.
Extended Accelerated Ageing Tests
In the extended accelerated ageing test (EAA), the treatments of 5-week duration for seed
from 2005 were: (1) the UNICOOL at 130C and 67% relative humidity, (2) the accelerated
ageing chamber (EAA~wks) at 320C and 100% RH, and (3) the laboratory germination chamber
(HT/LRH) at 320C and relative humidity < 10%. Seeds were evaluated for electrolyte
conductivity, seedling vigor, towel germination, and field emergence at 8 DAP and 12 DAP.
Seed moisture changed with seed treatment (Table 4-15). Seed moisture in UNICOOL remained
constant. Seed moisture in the EAA~wks increased from an average of 5.6% to a final moisture
average of 38.2%. In HT/LRH, moisture of seeds decreased from 5.6% to the final moisture
average of 1.4%.
P-values for effect of cultivar on seedling field emergence at 8 DAP and 12 DAP were
<0.0001 (Table 4-16). More seedlings of AP-3 and C-99R emerged at 8 DAP than in Florida-07,
DP-1, York, and Hull (Table 4-17). Hull had the lowest percent emerged seedlings. AP-3, C-
99R, Florida-07, and York at 12 DAP were similar in percent emerged seedlings, and exceeded
the percent emerged seedling of DP-1 and Hull. Hull had the lowest percent emerged seedlings.
The differences between seedlings emerged at 8 DAP and 12 DAP may indicate that AP-3 and
C-99R have the potential for more rapid initial establishment than Florida-07, and York (Table
Seedling field emergence at 8 DAP and 12 DAP of UNICOOL and HT/LRH treatments
was superior to EAA~wks (P<0.0001) (Tables 4-16 and 4-17). The EAA~wks treatment of 320C
and 100% relative humidity resulted in high average seed moisture of 38.2%, low electrolyte
conductivity, high vigor index, and high towel germination, but low seedling emergence in the
field (Table 4-17). The EAA test was designed as an attempt to duplicate possible post-harvest
storage conditions in bulk bins. The temperature was 2-30C above the expected initial high
storage temperatures. The increased temperature and 100% relative humidity increased moisture
absorption by the radicle (Table 4-15). The increased moisture would have allowed cellular
metabolic rates to increase sufficiently for the seed to begin phases I & II of germination and
repair DNA, enzyme and membrane damage, causing the seed to be primed. In the seed vigor
test, the EAA~wks treatment had a head start and the hypocotyl-radicle elongated faster than
seeds from the other treatments. At the time of sowing, the EAA~wks treatments appeared fully
imbibed with radicles protruding. The extent of priming varied by cultivar and within cultivar.
The seed was fragile and field emergence of EAA~wks was reduced possibly by damage to the
radicle during sowing, or possibly by direct ageing effects.
For interaction of treatment and cultivar, P-values at 8 DAP and 12 DAP were <0.0001 for
field emergence. Field emergence of C-99R, Hull, and York from the EAA~wks treatment 12
DAP was low compared to the treatments of UNICOOL and HT/LRH (Figure 4-18). Seed
priming was an unintended consequence in the EAA~wks treatment. Both the vigor test and the
field emergence results are not representative here and should be discounted, because vigor index
was inflated by priming effect of EAA~wks but was reduced by HT/LRH treatment, while field
emergence was reduced by EAA~wks as expected and not reduced by warm, dry treatment
(HT/LRH). Table 4-17 summarizes the mean values of the EAA tests sorted by both treatment
and cultivar. As Clmhos gl increased, vigor index, towel germination, and field emergence
decreased. This pattern is uniform throughout the table, except for the field emergence of
EAA~wks, which was confounded by the unintended priming of EAA~wks seed samples. The
pattern is in agreement with Table 4-8, which shows the strong negative correlation of electrolyte
conductivity of leachate with seed vigor and Hield emergence.
The data from electrolyte conductivity tests, seed vigor tests, and field trials support the
* The cultivars were affected differentially by temperature and relative humidity in the bulk
storage bins (Figures 4-4, 4-16, and 4-17).
* Electrolyte conductivity is negatively correlated to seed vigor and Hield emergence (Table
* Electrolyte conductivity tests and seed vigor tests correlate with Hield emergence and are
reliable indicators of seed quality (Table 4-8).
* In the accelerated ageing tests, the elevated temperature and relative humidity primed the
accelerated aged seed, resulting in good towel germination but in poor Hield emergence
Antioxidant Capacity Assay
A preliminary test was conducted to compare the seed antioxidant capacity of AP-3, C-
99R, DP-1, and Hull. The seed sources were from the 2004 harvest, the 2005 harvest, the 2006
harvest, and seed from the 2004 production year after storage for 4 months in STACKS. Peanut
seed from production years 2005 and 2006 after harvest were placed in storage at temperature <
00C. Peanuts from production year 2004 at harvest were stored in UNICOOL at 12-130C until
January 3 1 and then stored at temperature < 00C. Seed from the STACKS of 2004 production
seed year was placed in < 00C in April 2005 at the conclusion of the 4 month storage period.
The antioxidant capacity in peanut seed differed by cultivar (P=0.0016) and by
date/environmental storage conditions (P=0.0003) (Table 4-18). Antioxidant capacity of Hull
was superior to C-99R and DP-1; AP-3 was superior to DP-1; and for C-99R and DP-1
antioxidant was similar (Table 4-19). Antioxidant capacity at harvest for all years was greater
than antioxidant capacity of seed stored in STACKS in 2004 for four months (Table 4-20).
Listed by year of sampling, the mean Clequivalents/g peanut was 73.7 at harvest 2006, 63.6 at
harvest 2005, and 58.4 at harvest 2004. After storage in STACKS in 2004, the mean
Clequivalents/g peanut was 43.5. The variation in antioxidant capacity by year may reflect
different growing conditions during the crop year, or, for harvest 2004, the lower antioxidant
capacity of seed may reflect loss of antioxidant capacity during the four month period preceding
the freezing of the peanuts.
In 2004 antioxidant capacity of AP-3 was 68.4 at harvest and decreased to 32.9 during
storage in the STACKS (Figure 4-19). For DP-1 and Hull, the antioxidant capacity decreased
only minimally; DP-1 from 38.3 to 36.2 and Hull, a high oleic cultivar, from 76.8 to 71.9. The
decrease in antioxidant capacity of AP-3 may indicate that antioxidants were used to protect the
seed from peroxidation during storage; whereas, the poor field emergence of DP-1 and Hull may
have resulted from low antioxidant activity, and thus, low protection from autoxidation by the
antioxidants. The minimal antioxidant activity and the elevated temperatures of the stacks may
have allowed the production of free radicals resulting in increased cellular membrane and
enzyme damage, causing the loss of seed vigor which was evident in the comparative vigor,
leachate conductivity, and field emergence tests of DP-1 and Hull.
This antioxidant data is preliminary. Subsequent assays of antioxidant capacity may
support the observations that:
* Antioxidant capacity varies by cultivar and by year of production (Table 4-19).
* Antioxidant capacity of peanut seed may decrease during storage in bulk bins which are
similar to STACKS (Figure 4-19).
* Antioxidant capacity is an important factor for preserving seed vigor and cultivar
antioxidant capacity should be evaluated in peanut breeding programs.
Table 4-1. ANOVA for towel germination and April field emergence of peanut seed as affected
by year (Y), cultivar (C), origin of seed (0), and storage environment/location (L) for
crop production years 2004 and 2005
2004 2005 2004-05
Towel April Field Towel April Field Towel April Field
Source df Germination Emergence Germination Emergence Germination Emergence
Year (Y) 1 <.0001 0.0082
Rep 3 0.3340 0.0370 0.8065 0.0005 0.8931 0.0013
Cultivar (C) 3 <.0001 <.0001 0.0442 0.0043 0.0239 <.0001
Origin (0) 1 0.0321 0.9104
Location (L) 3 0.0493 0.0208 0.0498 0.0957 0.0847 0.0063
Y*C 3 0.2213 0.1370
Y*L 3 0.0002 0.4231
C*L 9 0.0021 0.1355 0.6526 0.0814 0.0188 0.0711
C*O 3 <.0001 <.0001
O*L 3 0. 1792 0.2222
C*O*L 9 0.0601 0.1917
Table 4-2. Means of towel germination and field emergence tests of peanut seed from crop
production years 2004 and 2005
Germination Field Emergence Number of
Crop Year (%) (%) Samples
2004 93.6 88.3 128
2005 87.1 85.1 64
Table 4-3. Means of cultivar and seed origin in towel germination tests and April field
emergence test of peanut seed produced in crop year 2004
Towel April Field
Germination Standard Emergence Standard
Cultivar Seed Origin Means Deviation Means Deviation
AP-3 FF SP 94.8 4.7 91.3 4.9
AP-3 NFREC 99.0 1.8 93.4 6.4
C-99R FF SP 95.3 3.6 91.1 9.5
C-99R NFREC 88.4 6.3 88.8 6.5
DP-1 FF SP 93.1 5.5 88.9 6.4
DP-1 NFREC 85.8 5.8 78.6 5.4
Hull FF SP 94.3 4.5 8 1.9 9.0
Hull NFREC 98.1 1.5 92.9 5.0
Mean FF SP 94.4 88.3
Mean NFREC 92.8 88.4
2004 Towel 2004 Field 2005- 2005 Field Mean Mean Field
5 AP-3 ITC-99R O DP-1 H Hull
Figure 4-1. Effect of cultivar on moist towel germination and field emergence at the end of
winter storage of seed peanut from crop production years 2004 and 2005. Within a
grouping, means with the same letter are not significantly different.
B ^r AB pA
AE A nR B.
BI~8 HCBI HI H
70 -- *- *
MM I M
60 --*'. .
2004 Towel 2004 Field 2005- Towel 2005 Field Mean Towel MeanField
2 UNICOOL 5 STACKS O WHSE H WAGON
Figure 4-2. Effect of environment/storage location on moist towel germination and field
emergence of seed peanut from crop production years 2004 and 2005. Within a
grouping, means with the same letter are not significantly different.
Table 4-4. ANOVA P-values for towel germination and field emergence of bulk stored peanut
as affected by cultivar and storage location for crop production year 2004.
Towel April Field Oct Field
Source df Germination Emergence Emergence
Rep 3 0.0915 0.4016 0.0078
Cultivar (C) 3 0.0002 <0.0001 <0.0001
Location (L) 2 0.0309 <0.0001 <0.0001
C*L 6 0.5447 <0.0001 <0.0001
Figure 4-3. Moist towel germination of bagged seed produced in crop year 2004 as affected by
cultivar and storage environment/location.
o B A
FC ii C-99R
0 40 -t~ -rrrre Orrr~ Drrr P-1
30 -~ 5 Hull
Towel Germination April Field October Field
Figure 4-4. Effect of cultivars on moist towel germination and field emergence of bulk stored
peanut produced in crop year 2004. Within a grouping, means with the same letter
are not significantly different.
Figure 4-5. Effect of storage environment/location on moist towel germination and field
emergence of bulk stored peanut produced in crop year 2004. Seed from UNICOOL
is a second sample from bagged seed and not truly bulk seed. Within a grouping,
means with the same letter are not significantly different.
Table 4-5. Effect of cultivar and storage location on April field emergence of bagged seed
versus bulk stored seed of peanut for crop production year 2004. Seed from
UNICOOL is a second sample from bagged seed and not truly bulk seed.
Cultivar Location Bagged Seed (%) Bulk Stored Seed (%) Difference (%)
AP-3 UNICOOL 92.0 94.0 2.0
AP-3 STACKS 90.3 81.5 8.8
AP-3 WAGON 94.5 97.0 2.5
C-99R UNICOOL 93.3 85.5 2.2
C-99R STACKS 85.5 76.0 9.5
C-99R WAGON 92.3 85.0 7.3
DP-1 UNICOOL 85.5 94.5 9.0
DP-1 STACKS 84.0 46.5 37.5
DP-1 WAGON 85.5 56.5 29.0
Hull UNICOOL 86.8 80.0 6.8
Hull STACKS 82.0 74.5 7.5
Hull WAGON 88.5 69.5 19.0
Figure 4-6. Fil emrec in ~ Y L Api 200 ofbl trdpenta fetd yclia n
strae nvromet/octin f ee pant roucd n ro yar2004 edfo
UNCOO sascn sml rmbgedse n o tuybl ed
< 40 W
Figure 4-7. Field emergence in October 2005 of bulk stored peanut as affected by cultivar and
storage environment/location of seed peanut produced in crop year 2004. Seed from
UNICOOL is a second sample from bagged seed and not truly bulk seed.
D ET ER I O RAT ION <
Figure 4-8. Representation of differences in rate of deterioration of seed viability and vigor
showing decrease from 100% to 0% over time from J.C. Delouche and W.P.
Caldwell, (1960) Seed vigor and vigor tests, Proceedings of the AOSA 50(1): 13 6.
Table 4-6. Comparison of April field emergence to October field emergence of bulk stored
peanut as affected by cultivar and storage location for crop production year 2004.
Seed from UNICOOL is a second sample from bagged seed and not truly bulk seed.
April Field October Field
Cultivar Location Emergence(%/) Emergence(%/) Difference (%)
AP-3 UNICOOL 94.0 89.0 -5.0
AP-3 STACKS 81.5 69.0 -12.5
AP-3 WAGON 97.0 87.5 -9.5
C-99R UNICOOL 95.5 74.5 -21.0
C-99R STACKS 76.0 57.0 -19.0
C-99R WAGON 85.0 38.5 -46.5
DP-1 UNICOOL 94.5 78.5 -16.0
DP-1 STACKS 46.5 25.5 -21.0
DP-1 WAGON 56.5 24.0 -32.5
Hull UNICOOL 80.0 71.0 -9.0
Hull STACKS 74.5 36.5 -38.0
Hull WAGON 69.5 28.0 -41.5
Table 4-7. Comparison of towel germination to field emergence of bulk stored peanut as
affected by cultivar and storage location for crop production year 2004. Seed from
UNICOOL is a second sample from bagged seed and not truly bulk seed.
Towel Seed Lab April Field Towel Germination
Germination Germination Emergence Minus Field
Cultivar Location (%) Test (%) (%) Emergence (%)
AP-3 UNICOOL 97.5 94.0 -3.5
AP-3 STACKS 96.5 91.0 81.5 -15.0
AP-3 WAGON 96.5 97.0 0.5
C-99R UNICOOL 94.5 95.5 1.0
C-99R STACKS 92.5 88.0 76.0 -16.5
C-99R WAGON 98.0 85.0 -13.0
DP-1 UNICOOL 87.5 94.5 -7.0
DP-1 STACKS 85.0 86.0 46.5 -38.5
DP-1 WAGON 90.0 56.5 -33.5
Hull UNICOOL 95.0 80.0 -15.0
Hull STACKS 86.5 89.0 74.5 -12.0
Hull WAGON 89.5 69.5 -20.0
Table 4-8. Correlation of towel germination, field emergence, comparative vigor index, and
leachate conductivity of peanut as affected by cultivar and storage location for
production years 2004 and 2005.
Pearson Correlation Coefficients
Prob > |r| under HO: Rho=0
Number of Observations
Towel April Field October Field Vigor Test #1 Leachate
Germination Emergence Emergence Conductivity
April Field 0.0532
October Field -0.13819 0.25924
Emergence 0.4507 0.1519
Vigor Index Test -0.28554 0.23467 0.60126
#1 0.0491 0.1084 0.0003
48 48 32
Leachate 0. 12429 -0.22936 -0.78774 -0.77338
Conductivity Test 0.4 0.1168 <.0001 <.0001
48 48 32 48
Vigor Index Test -0.00246 0.69661 0.56697 0.55237 -0.68839
#2 0.9928 0.0027 0.0220 0.0265 0.0032
16 16 16 16 16
9/ 1 6 011161/611
- Cro Yer20 -Co er20
Fiue49 endiyartmprtr t2mtr bv r oudlvla maurdath
Flrd Auoae ete ewr F W )sbttoMranFoida
September ~ ~ ~ ~ ~ ~ ~ I 6t aur 4frcopyas20 n 05
10/11 10/25 11/8 11/22 12/6 12/20 1/3 1/17
2004 Crop Year 2005 Crop Year
Figure 4-10. Mean daily temperature within the bulk pile of peanut cultivar AP-3 stored in a
traditional storage bin at the Florida Foundation Seed Producers (FFSP) for October
15 to January 24 for crop years 2004 and 2005.
Te prt re ( C) Reatv Hu iit %
Figur 4-1 Mea dal teprtr n rltv u iiy ihnteblkpl fpau
cultivar~ ~ DP1soe napau ao tte lrd onainSe rdcr
(FS)fr h eio coer1,204t au ry 31205
15 to Jaur 31 204nd205
10/1 103 111 112 212/61912
Fiur 413 Coprio ofma al eprtr ntese trg omlctda h
Unvriy fFoid eerh n dcainCntr(FE) n h ma al
teprtr wihntebl ieo entcliarA trdi rdtoa trg
bi tteFoiaFudto edPoues(FP o th peio Ocoer 1720
to~* Jaur 4 05
5 0 na*
10/1 103 114 1/8 1212261912
Fiur 414 Cmprionofreatvehuidty(R ) n hesed toag romatth Uivrstyo
Flrd eerchadEuainCne NRC frOtbr1 oJnay3 o
crop~I yer 200 an 05
10/12 10/26 11/9 11/23 12/7 12/21 1/4 1/18
-AP-3 STACKS 2004 AP-3 STACKS 2005
Figure 4-15. Comparison of relative humidity (RH) within the bulk pile of peanut cultivar AP-3
stored in a traditional storage bin at the Florida Foundation Seed Producers (FFSP)
for October 15 to January 31 for crop years 2004 and 2005.
Percent seeds with pathogen present
Table 4-10. ANOVA for vigor tests #1 and #2 of peanut germination as affected by cultivar and
storage environment/location for crop production in 2005.
Vigor Test #1 Vigor Test #2
Source df P-value df P-value
Rep 1 0.9453 3 <.0001
Cultivar (C) 7 0.0058 6 0.0083
Location (L) 3 0.0003 2 0.5031
C*L 21 0.3248 12 0.2930
Table 4-9. Incidence of fungi per 20 seeds per storage location in bulk stored peanut for crop
production in 2004 and 2005.
Treatment m A. flavus A. niger Rhizopu
Total in 480 seeds
Table 4-11. Effect of cultivar, storage environment/location and accelerated ageing (AA) on
seedling vigor (Vigor Test #1) for peanut seed from 2005 crop production year.
Within a grouping, means with the same letter are not significantly different.
Cultivar Means Grouping N
AP-3 58.3 A 8
C-99R 56.6 A 8
AP-3 AA 55.1 A 8
C-99R AA 55.1 A 8
DP-1 AA 54.5 A 8
Hull AA 54.0 A 8
Hull 50.1 AB 8
DP-1 42.8 B 8
Location Means Grouping N
WAGON 56.9 A 16
STACKS 56.6 A 16
WHSE 54.1 A 16
UNICOOL 45.7 B 16
Test Means 53.3 64
Control no AA 39.5 32
AA Means 54.7 32
Table 4-12. Effect of cultivar and storage environment/location on seedling vigor (Vigor Test
#2) for peanut seed from 2005 crop production year. Within a grouping, means with
the same letter are not significantly different.
Cultivar Means Grouping N
York 61.0 A 12
Florida-07 59.9 AB 12
AP-3 59.8 AB 12
C-99R 59.7 AB 12
DP-1 55.3 BC 12
McCloud 54.4 C 12
Hull 54.3 C 12
Location Means Grouping N
STACKS 58.6 A 28
WHSE 57.9 A 28
UNICOOL 56.8 A 28
Table 4-14. Pearson correlation of electrolyte conductivity of leachate, comparative vigor index
(CVI), April field emergence, and October Hield emergence as affected by cultivar
and seed storage environment/location of seed peanuts produced in crop year 2004.
Pearson Correlation Coefficients, N=15
Prob > |r| under HO: Rho=0
(Clmhos g-1) CVI April Field Emergence
Comparative Vigor -0.76378
Index (CVI) 0.0009
April Field -0.81504 0.63036
Emergence 0.0002 0.0118
October Field -0.5022 0.32572 0.67772
Emergence 0.0564 0.2361 0.0055
Table 4-13. Electrolyte conductivity of leachate, comparative vigor index (CVI), April Hield
emergence, and October Hield emergence as affected by cultivar and seed storage
environment/location of seed peanuts produced in crop year 2004.
3rd Order polynominal
80 CVI= 366.9 -935.2*LCH + 776.7*LCH2 209.5*LCH3
70 R2 = 0.8855, P-value <0.0001
0 50 Linear with LCH <0.1
CVI= 179.7 -227.0*LCH
S40 R2= 0.7974, P-value = 0.0005
-1004 0.6 0.8 1.0 1.2 1.4 1.6
Leachate Conductivity (LCH)
+ CVI -*-3rd order polynomial -5-Linear
Figure 4-16. Comparative vigor index (CVI) versus electrolyte conductivity of leachate (LCH)
from germinating peanut seed over all cultivars and storage environments for seed
from crop production year 2004. Seed with a LCH > 0.1 were considered to be non-
CVI= -30.036*(LCH) +107.89
R2 = 0.6852, P-value = 0.0002
8 80 *
0.400 0.500 0.600 0.700 0.800 0.900 1.000 1.100 1.200 1.300
Leachate Conductivity (LCH)
+ April Field Emerge -Linear (April Field Emerge)
Figure 4-17. April field emergence versus electrolyte conductivity of leachate (LCH) from
germinating peanut seed over all cultivars and storage environments for seed from
crop production year 2004.
Table 4-15. Changes in seed moisture in 5-week accelerated ageing test of peanut cultivars
stored in 3 environments: 1) 130C and 67% relative humidity (UNICOOL), 2) 320C
and 100% relative humidity (EAA5wk), and 3) 320C and <10% relative humidity
(HT/LRH). Seed source was crop year 2005 seed that was stored in WHSE location.
Initial Five Ave. Ave. Change
Seed Week Change Initial Moisture Final in
Weight Weight in Weight Moisture Range Moisture Moisture
Treatment Cultivar (g) (g) (g) (%) (%) (%) (%)
UNICOOL AP-3 30.39 30.41 0.02 5.5 4.6-6. 5 5.9 0.4
Table 4-16. ANOVA for field emergence of peanut seed as affected by 3 treatments: 1) 130C
and 67% relative humidity, 2) 320C/100% relative humidity, and 3) 320C <10%
relative humidity in 5-week accelerated ageing test Seed source was crop year 2005
seed that was stored in WHSE location.
Days after Planting 8 Days 12 Days
Source df Pr > F Pr > F
Rep 3 <.0001 0.6505
Treatment (T) 2 <.0001 <.0001
Cultivar (C) 5 <.0001 <.0001
T*C 10 <.0001 <.0001
32.28 43.54 11.26
extensive fungal growth
Table 4-17. Electrolyte conductivity of germinating peanut leachate, comparative vigor, towel
germination, and field emergence at 8 and 12 days after planting (DAP) as affected
by cultivar and by three simulated storage treatments for 5 weeks: 1) 130C and 67%
relative humidity (UNICOOL), 2) 320C and 100% relative humidity (EAA~wk), and
3) 320C and <10% relative humidity (HT/LRH). Within a grouping means with the
same letter are not significantly different.
Towel 8 DAP Field 12 DAP Field
Vigor Germination Emergence Emergence
Treatment umhos g-1 Index (%) (%) (%)
EAA~wks 0.278 53.2 97.5 43.8 B 66.2 B
UNICOOL 0.393 49.5 89.2 68.2 A 84.3 A
HT/LRH 0.658 25.0 61.7 63.0 A 82.0 A
FL-07 0.240 50.0 95.0 57.2 B 82.3 A
C-99R 0.332 45.1 Error 67.4 A 79.2 A
York 0.356 45.0 83.3 50.2 BC 82.8 A
AP-3 0.463 44.3 81.7 72.8 A 85.2 A
DP-1 0.573 40.0 75.0 56.4 B 72.5 B
Hull 0.769 23.0 56.7 46.4 C 63.3 C
AA A .A
80 -- *
o 60 -
AP-3 C-99R DP-1 FL-07 Hull York
O EAA~wks 2 HT/LRH UNICOOL
Figure 4-18. Field emergence at 12 DAP of peanut cultivars from crop production year 2005 as
affected by treatment in an extended accelerated ageing test (EAA). The treatments
of five week duration were: 1) seed stored in a University of Florida seed storage
room (UNICOOL) at 130C and 67% relative humidity, 2) seed placed in an
accelerated ageing chamber (EAA~wks) at 320C and 100% relative humidity, and 3)
seed placed in a University of Florida germination chamber (HT/LRH) at 320C and
relative humidity < 10%. Within a grouping, means with the same letter are not
Table 4-18. ANOVA for antioxidant capacity of peanut seed as affected by cultivar and
date/year of sample.
Source df F value P-value
Rep 2 11.63 0.0002
Cultivar (C) 3 6.63 0.0016
Date (D) 3 8.95 0.0003
C*D 8 1.41 0.2365
Table 4-19. Antioxidant capacity of peanut seed as affected by cultivar over three sampling
years. In 2006 seed of Hull was not produced. Means with the same letter are not
Cultivar Means Grouping N
Hull 73.3 A 9
AP-3 66.0 AB 12
C-99R 54.6 BC 12
DP-1 45.2 C 12
Table 4-20. Antioxidant capacity of peanut seed as affected by seed storage
environment/location and year of crop production. In 2006 seed of Hull was not
produced. Means with the same letter are not significantly different.
Year Location Means Grouping N
2006 Harvest 73.7 A 9
2005 Harvest 63.6 AB 12
2004 Harvest 58.4 B 12
2004 Stacks 43.5 C 12
E B BB
HAR'o6 HAR'05 HAR'O4 STACK
BAP-3 H C-99R O DP-1 O1Hull
Figure 4-19. Antioxidant capacity of seed peanut measured in Clequivalents gl as affected by
year of production, cultivar, and sample date. Sample dates were harvest 2006
(HAR'o6), harvest 2005 (HAR'05), harvest 2004 (HAR'O4), and March 15, 2005 of
peanuts stored in stacks (STACKS) from crop production year 2004. Within a
grouping, means with the same letter are not significantly different.
Seed Deterioration and Poor Field Emergence
Late-maturing peanut cultivars with genetics related to PI 203396 frequently have poor
Hield emergence after storage in commercial bulk peanut bins. The cultivars with PI 203396
lineage include Florida MDR-98, C-99R, DP-1, Hull, and Southern Runner. Because of their
pathogen resistance and yield potential, these cultivars are important for peanut production. This
research looked at effects of storage environment on three of these cultivars compared to the AP-
3 check cultivar, as revealed by seed vigor and field emergence. Analysis of data from the 2004
and 2005 crop storage treatments show:
* The Hield of origin in crop 2004 year was not a factor in Hield emergence.
* The seed storage environment/location was a factor affecting leachate conductivity,
seedling vigor, and field emergence in 2004 and 2005.
* The bulk storage environment differed in 2004 and 2005. Temperatures within the peanut
stack piles from October 11 to January 24 in 2004 averaged 7.00C higher than temperatures
within the stack piles for the same period in 2005.
* Temperature within the peanut stack piles from October 11 to January 24 for crop
production year 2004 averaged 10.5oC higher than temperatures within the University
climate controlled storage room for the same period.
* There was a cultivar by seed storage environment (location) interaction affecting seedling
vigor and field emergence.
* Cultivars stored in the bulk bin location had reduced seed vigor and reduced field
* When stored in elevated temperatures and relative humidity, field emergence of DP-1 and
Hull was less than field emergence of AP-3 and C-99R.
* Seed vigor and field emergence were maintained when seeds were stored at < 160C and <
70% relative humidity.
* Storage fungi were not an important contributing cause to poor field emergence in 2004
* Standard towel germination tests were not reliable indicators of seed vigor or field
* Electrolyte conductivity tests and seed vigor tests were highly correlated with seed quality
as indicated by field emergence.
* At harvest the antioxidant capacity of peanut seed varied by cultivar and year of
The Seed Vigor Testing Handbook (2002) states "Seed vigor comprises those properties
which determine the potential for rapid, uniform emergence and development of normal
seedlings under a wide range of field conditions." The genetic constitution of seed establishes its
maximum physiological potential. Cultural and climatic factors during seed maturation may
limit the seed from developing maximum vigor potential. Regardless of the level of initial vigor,
at maturity seed vigor begins to deteriorate. Seed deterioration is the result of changes within the
seed that decrease the ability of the seed to survive. It is distinct from seed development and
germination and it is cumulative (McDonald, 2004). Of the many factors that can reduce seed
quality, elevated temperature and relative humidity are the most important (McDonald, 2004).
Tests for vigor of bulk stored seed showed increased electrolyte conductivity of peanut
leachate and decreased rate of seedling growth for seed stored in the bulk bin (stack) and wagon
locations, but not for seed stored in the controlled environment location. Although the towel
germination tests indicated acceptable seed quality, DP-1 emerged poorly in the April 2005 field
emergence test. After the seed from the same samples was stored for an additional 5.5 months at
< 160C and < 70% relative humidity and sown in October, seedling emergence was poor for seed
which had been stored during the winter months in the stack and wagon locations. Field
emergence generally was good for seed stored in the University of Florida cool storage location.
Seed vigor declines faster than germinability (Figure 4-8). As indicated by conductivity and
seed vigor tests, seed vigor by April had deteriorated. An additional 5.5 months of cool storage,
despite being only a minor additional stress, resulted in greatly reduced field emergence in the
October 2005 field test. The field emergence test in October 2005 demonstrated that seed vigor
in April 2005 was marginal and that the seed vigor had reached the point of steep descent
indicated by 'Y' on the seed deterioration graph of Figure 4-8.
Scenario of Seed Quality Deterioration
The data, in conjunction with the literature review, supports the following possible
scenario of seed quality deterioration:
* The elevated temperature and increased relative humidity during a warm storage period
may allow the exposed peanut radicle to absorb moisture (McDonald, 1998).
* Because of the high lipid content (>45%) of peanut, seed moisture is concentrated within
the embryo axis. With the increased moisture and temperature, the glass state of water can
reach Tg and water changes to an amorphorous state (Walters, 1998).
* The consequence is an increase in the rate of molecular diffusion within the cytoplasm, so
that reactive oxygen species are able to attack the unsaturated lipids of cellular membranes,
especially the lipids of mitochondrial membranes. The free radicals produced create a
chain reaction of autoxidation. The cellular membranes become porous and, at imbibition,
there will be increased leakage of solutes. In addition, the roaming free radicals damage
proteins, DNA, and the electron transport system (Wilson and McDonald, 1986).
* Although the seed water exists in an amorphorous state and autoxidation may occur, the
seed is still quiescent and there is insufficient water for the metabolic reactions necessary
for repair of the cellular machinery (Walter, 1998).
* Damage accumulates and seed quality deteriorates. In the initial phase of imbibition, the
cell rapidly repairs damaged membranes, proteins and DNA. The greater the damage, the
longer the repair phase, as is evident in the reduced rate of seedling growth and increased
number of non-viable seeds in the seed vigor tests. If damage is too severe, the seed
becomes non-viable (McDonald, 1998).
* Since germination tests are based on radicle protuberance and do not evaluate differences
in elongation of the hypocotyle-radicle, germination tests may over-estimate seed vigor
and field emergence.
The difference in field emergence of seed from production years 2004 and 2005 may be
related to the 7.00C higher temperature within the peanut piles (STACKS) during the common
period of October 1 1 to January 24, 2005 compared to the 2005 production year. The cause for
this temperature difference has not been determined, but, is speculated to be associated with
heating inside the pile; outside ambient temperatures during this period were similar over years.
The pile temperature difference could alternatively be related to crop maturity or seed moisture
at harvest going into storage. However, the environment within the STACKS in 2004 consisted
of elevated temperatures and high relative humidity, an environment which would allow
interaction of seed moisture and temperature and increase the incidence of lipid peroxidation.
Data from the seed vigor and leachate conductivity tests of 2004 suggest that increased
peroxidation of lipids occurred and damaged cellular membranes, especially in the seeds of Hull
and DP-1. The data from 2005 seed vigor and leachate conductivity tests suggest that with the
cooler temperatures in the pile in 2005, peroxidation may have been less than in 2004.
Seed deterioration is an individual seed event (McDonald, 2004). As peanuts are removed
from bin storage with a front-end loader, the face of the pile keeps sliding down so that peanuts
close to the pile surface and perhaps of higher quality become mixed with peanuts of perhaps
lower quality from the center of the pile. As a result, the peanuts bagged for sale to the grower
are a composite of peanut quality and may not emerge as a uniform and vigorous stand.
Antioxidants have the ability to scavenge free radicals and suppress autoxidation
(McDonald, et al., 1988). In the analysis of antioxidant capacity for the four principal cultivars,
antioxidant capacity at harvest varied by cultivar, oleic acid content, and year. This is in
agreement with Amaral et al. (2005) and Talcott et al. (2005b). For example, the antioxidant
capacity of AP-3 at harvest, measured in Clequivalents g- was 68.4 in 2004, 75.0 in 2005, and
87.6 in 2006. The antioxidant capacity of Hull, a high oleic peanut, at harvest measured in
Clequivalents g- was 76.8 in 2004 and 71.2 in 2005. During subsequent storage in stacks of seed
produced in 2004, antioxidant capacity of AP-3 decreased from 68.4 to 32.9, C-99R from 50.0 to
33.1, DP-1 from 38.3 to 36.2, and Hull from 76.8 to 71.9. In AP-3 the lost antioxidant capacity
may have been consumed in suppressing autoxidation, thus protecting the seed from free radical
attack. For C-99R, DP-1, and Hull, antioxidant capacity reduction was substantially less and
suppression of autoxidation may have been insufficient to protect the seed from free radical
attack, thus resulting in the cellular membrane damage and poor seed vigor, as evidenced in
increased leachate conductivity, reduced seed vigor, and poor field emergence of C-99R, DP-1,
and Hull. At this time there is no explanation for the failure of DP-1 and Hull antioxidant
capacity to suppress autoxidation nor conclusive evidence that for AP-3 antioxidant capacity was
instrumental in protecting its seed from autoxidation.
Recommendations for Continued Research
AP-3, C-99R, DP-1, and Hull can be ranked by percentage of lineage to PI 203396, which
relates to degree of intolerance to elevated temperature and relative humidity during storage.
Perhaps decreased antioxidant capacity is linked to PI 203396. The literature reports that in
many species antioxidant capacity depends upon year of production and genotype (Amaral et al.,
2005) and that it may be possible in a breeding program, to select for elevated a-tocopherols by
selecting for altered response to temperature (Britz and Kremer, 2002). Based on the data from
this research, storage quality of peanut may be improved by including antioxidant capacity as a
standard in cultivar evaluation. Additional research is necessary to refine the evaluation of
antioxidant capacity and verify the relationship of antioxidant capacity to seed vigor and field
Recommendations for Improving Quality of Seed Peanut
Field emergence of peanut seedlings is affected by many factors, including seed maturity,
seed size within the cultivar, seed damage during harvesting and processing, loss of viability
during seed storage, and soil tilth, temperature, and moisture at planting time (McDonald 2004).
The following suggestions may improve seed quality and field emergence:
Improve Ventilation of Storage Facilities
Peanut seed quality may be improved by developing a low capital investment, low
operating cost, and fully automated forced air ventilation system for reducing temperature and
relative humidity within the peanut pile during the October-February storage period. Butts et al.
(2006) published research comparing four possible ventilation methods for peanut barns. Peanut
Company of Australia (PCA) found that lowering the percent moisture in seed with high oil
content helped to maintain field emergence (PCA, 2006. Per. Comm.). Alternatively, effective
storage may require refrigeration/air conditioning capacity, especially for these storage-sensitive
Replace Towel Germination Tests with a Test that Measures Seed Vigor
Develop a method of evaluating vigor of peanut seed at the time of bagging the seed for
sale to the grower. One method is an electrolyte conductivity test of seed leachate derived by
soaking a sample of peanuts in water for 18 hours. The test can be completed within 24 hours,
takes very little space, requires an inexpensive conductivity meter, and can be accomplished
accurately with minimal training of technicians. A second method is to use seed vigor tests.
These tests require 5 days, more space, germination chambers, and the data can reflect the bias of
the technicians measuring the linear length of hypocotyl-radicles.
SEED VIGOR DIFFERENCES IN GERMINATING PEANUT SEED
An example of seed vigor differences as evident in variation of hypocotyl/radicle length of seeds
germinating in a moist towel test.
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Barry Morton earned a Bachelor of Arts in English literature at Brown University and a
Master of Science in agronomy from Pennsylvania State University. His master's thesis was a
"Study of Pollination and Pollen Tube Growth in Buckwheat (Fagopyrum sagittatum Gilib.)".
His dissertation "Poor Field Emergence of Late-Maturing Peanut Cultivars (Arachis hypogea
L.) Derived from PI 203 396", completes his doctoral program toward a Doctor of Philosophy in
agronomy from the University of Florida in May 2007. Mr. Morton' s experience is a unique
combination of farm owner/operator of a cash crops and swine farrowing operation, agricultural
lender, agricultural consultant, and teacher at the secondary and university level. As a
commercial farm owner/producer, he raised corn, alfalfa, small grains, and potatoes in
Southeastern Pennsylvania. The swine farrowing operation, with a total of three employees and
an on-farm feed mill, produced 9000 pigs/year. As an agricultural loan officer, Mr. Morton
analyzed credit, appraised collateral, made cash flow proj sections, and tracked loan progress of
farm operating loans and mortgages for a regional bank in Pennsylvania. As an agricultural
consultant, Mr. Morton negotiated bank financing and provided crop/livestock planning, cash
flow projections, and on-farm management. Clients included a 2500 acre Manor Farm located in
Maryland producing comn, soybeans, and hay using sewage sludge to supplement fertilizer. Mr.
Morton taught biology at York College of Pennsylvania and secondary level English classes at
Desert Pride Academy located at the border of New Mexico and Mexico. He was instrumental
in the initial phases of establishment of a program for returning students and co-authored
guidelines and curriculum for the program.