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Poor Field Emergence of Late-Maturing Peanut Cultivars (Arachis hypogaea L.) Derived from PI-203396


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1 POOR FIELD EMERGENCE OF LATE MATURING PEANUT CULTIVARS ( A rachis hypogaea L .) DERIVED FROM PI 203396 By BARRY R. MORTON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE R EQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Barry R. Morton

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3 ACKNOWLEDGMENTS This dissertation is th e 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 o f the Agronomy Department while study ing for a doctorate degree. Dr. Tillman chair and Dr. Kenneth J. Boote, co chair 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 experti se. Dr. Sus an Percival provided advice and access to her laboratory to assay antioxidants i n 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 i ndividuals 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.

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4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ............... 3 LIST OF TABLES ................................ ................................ ................................ ........................... 6 LIST OF FIGURES ................................ ................................ ................................ ......................... 8 ABSTRACT ................................ ................................ ................................ ................................ ... 10 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .................. 12 Cultivars ................................ ................................ ................................ ................................ .. 12 Poor Field Emergence ................................ ................................ ................................ ............. 12 Seed Handling and Storage ................................ ................................ ................................ ..... 14 Hypotheses ................................ ................................ ................................ .............................. 14 2 LITERATURE REVIEW ................................ ................................ ................................ ....... 16 Effect of Temperature and Relative Humidity on Germination ................................ ............. 16 Germination, Seed Vigor, and Field Emergence ................................ ................................ .... 16 Deterioratio n of Seed Quality ................................ ................................ ................................ 18 Water in Seeds ................................ ................................ ................................ ........................ 20 Seed Protection Mechanisms ................................ ................................ ................................ .. 22 Accelerated Aging ................................ ................................ ................................ .................. 23 Mode of Action of Aspergillus spp. ................................ ................................ ........................ 24 Effect of Aspergillus on Peanut Seed Germination ................................ ................................ 24 Hypotheses and Objectives ................................ ................................ ................................ ..... 26 3 MATERIALS AND METHODS ................................ ................................ ........................... 27 Cultivars and Storag e Locations ................................ ................................ ............................. 27 Germination Tests for Seed Viability ................................ ................................ ..................... 29 Seedling Emergence Field Tests ................................ ................................ ............................. 30 Storage Pathogen Assays ................................ ................................ ................................ ........ 31 Seed Vigor Tests ................................ ................................ ................................ ..................... 32 Electrolyte Conductivity Tests ................................ ................................ ............................... 33 Accelerated Ageing Tests ................................ ................................ ................................ ....... 34 Antioxidant Capacity Assay ................................ ................................ ................................ ... 35 Experimental Design and Data Analysis ................................ ................................ ................ 36

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5 4 RESULTS AND DISCUSSION ................................ ................................ ............................. 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 Locations ................................ ................................ ................................ ...................... 37 Field Emergence of Seeds from NFREC and FFSP Stored in Bags in Various Locations ................................ ................................ ................................ ...................... 39 Towel Germination and Field Emergence of Bulk St ored Seed from Production Year 2004 ................................ ................................ ................................ ..................... 40 Comparison of Treatment Effects on Bagged Seed Samples and Bulk Stored Seed Samples ................................ ................................ ................................ ........................ 42 Co rrelation of Towel Germination and Field Emergence ................................ ............... 43 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 ................................ ................................ ................................ .... 47 Measures of S eed Quality ................................ ................................ ................................ ....... 47 Comparative Vigor Index Tests ................................ ................................ ....................... 47 Electrolyte Conductivity and Comparative Vigor Tests ................................ ................. 49 Extended Accelerated Ageing Tests ................................ ................................ ................ 51 Antioxidant Capacity Assay ................................ ................................ ............................ 53 5 CONCLUSIONS ................................ ................................ ................................ .................... 83 Seed Deterioration and Poor Field Emergence ................................ ............................... 83 Scenario of Seed Quality Deterioration ................................ ................................ ........... 85 6 RECOMMENDATIONS ................................ ................................ ................................ ........ 88 Recommendations for Continued Research ................................ ................................ ............ 88 Recommendations for Improving Quality of See d Peanut ................................ ..................... 88 Improve Ventilation of Storage Facilities ................................ ................................ ....... 88 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

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6 LIST OF TABLES Table page 1 1 Performance of run ner market type peanut cultivars in two or three Flor ida locations over four years (2002 2005). ................................ ................................ ............................. 15 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 s eed (O), 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 ................................ ............... 56 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 locatio n 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 con ductivity 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 cro p 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. ................................ ............... 73 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 co nductivity of leachate, comparative vig or 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

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7 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 produ ced 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) 13 o C and 67% relative humidity, 2) 32 o C/100% relative humidity, and 3) 32 o C <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 sample. ................................ ................................ ................................ ........................... 81 4 19 Antio xidant 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 storag e environment/location and ye ar of crop production. ................................ ................................ ............................ 81

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8 LIST OF FIGURES Figure page 4 1 Effect of cultivar on moist towel germination and field e mergence 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. ................................ ................................ ........ 58 4 4 Effect of cultivars on moist towel germination and field emergence of bulk stored peanut produced in crop year 2004. ................................ ................................ ................... 59 4 5 E ffect 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 p eanut 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 cultiva r 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 10 0% 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 J anuary 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 Resear ch and Education Center (NFREC), Marianna, Florida for the periods October 15 to January 31, 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

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9 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 Compariso n 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. ................................ ............ 72 4 16 Comparative vigor index (CVI) versus electrolyte conductivity of leachate (LCH) from germinating peanut seed over all cultivars and storage environments ..................... 76 4 1 7 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 equivalents g 1 as af fected 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

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10 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 L ATE MATURING PEANUT CULTIVARS ( ARACHIS HYPOGAEA L.) DERIVED FROM PI 203396 By Barry R. Morton May 2007 Chair: Barry L. Tillman Cochair: Kenneth J. Boote Major: Agronomy Late maturing peanut cultivars DP 1, C 99R, Hull, and Florida MDR 98 ( Arachis hyp ogaea L.) have superior resistance to leafspot ( Cercosporidium personatum Berk & Curt.), white mold ( Sclerotium rolfsii Sacc.), and tomato spotted wilt virus The improved resistance s are primarily derived from PI 203396. The cultivars are high yieldin g. 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 germ ination 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 i s through commercial channels wit h large volumes being harvested stored in bulk bins and treated with fungicides The problem may be related to the commercial p ractice of storing seed peanuts in large piles with inadequate ventilation. F our 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 temperat ures and

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11 relative humidity had reduced field emergence. There was a genotype by st orage environment interaction. Field emergence was maintained when seed was stored at < 16 o C and < 70% relative humidity. Standard towel germination tests were not relia bl e indicators of field emergence. E lectrolyte 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 membran es wer e damaged during storage at elevated temperatures and r elative 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 te mperature 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.

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12 CHAPTER 1 INTRODUCTION Cultivars 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, 200 2 ), DP 1 (Gorbet, 2003), and Hull (Gorbet, 2003). These cultivars have superior resistance to late leafspot ( Cercosporidiu m personatum Berk & Curt.), white mold ( Sclerotium rolfsii Sacc.), and tomato spotted wilt virus ( genus Tospovirus ; family Bunyaviridae ) ( Table 1 1 ). The improved pathogen resistance s were derived primarily from lin e age to PI 203396 through a common par ent or grandparent UF81206. DP 1 has 50% genetic s 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 grow er 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 l onger and can compensate for unfavorable weather or pathogen damage. Poor F ield E mergence 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 an d, like Florida MDR 98 and DP 1; Southern Runner had g ermination problems (Gorbet et al. 1987) Unpublished studies found that Southern Runner

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13 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 bee n less frequent and less severe and seed is commercially produced Prasad et al. (2006) r eported 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 sensi tivity 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 NFRE C show that reduced field emergence of these cultivars se ldom occurs when the seed has been grown, harvested, and stored in research storage facilities (Tillman, 2004, Per. Comm.). The poor f ield emergence occurs when seed is produced in commercial channe ls with large volumes being harvested, stored in bulk in shell shelled, and treated with fungicides. The problem may be related to commercial storage o f 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 factor s 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 r elative humidity and temperature are

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14 the most important (McDonald, 2004). Relative humidity directly influences seed moisture. Increasing temperature increases the amount o f 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 S torage Seed p eanuts in the s outheast 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 separat e pods from stems. With forced heated air, pods and seeds a re dried in wagons to approximately 9.5% moisture content (Stadsklev 2004, Per. Comm.). The peanut pods are stored in large bulk bins at ambient atmospheric temperatures that may exceed 32 o C and relative humidity that may exceed 95%. Dimensions of the b ulk 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 s hows substantial fungal growth and the hulls may become dusty from spores. Hypotheses C 99R, DP 1, and Hull have important disease resistances for peanut production and PI 203396, the primary source of the se resistances appears in the pedigree of many l ines 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 hypothese s:

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15 High temperature or high relative humidity in commercial storage condition s may reduce field emergence There is an interaction of cultivar by storage environment on seed germination and field emergence. Reduced field emergence may be caused by storag e 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 run ner market type peanut cultivars in two or three Florida location s over four years (2002 2005). Entries are sorted by maturity and the four year average yield (in descending order) (Tillman, 2007) *E = early, M = medium, L = late; **High oleic oil chemistry. 3 YR = average of 2003, 2004 a nd 2005; 4 YR = average of 2002, 2003, 2004 and 2005. ***Tomato Spotted Wilt Virus ratings (1 10, 1 = no disease).

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16 CHAPTER 2 LITERATURE REVIEW Effect of Temperature and Relative H umidity on G ermination Ideal conditions for storage of peanut seed are 10 o C and 65% relative humidity (Ketring, 1992). The resulting seed moisture content is approximate ly 6%. Maintaining these conditions is difficult when peanuts are stored in warehouses without climate control Navarro et al (1989) studied the interact ion of temperature and mo isture concentration on the g ermination of peanut seeds. Germination of peanuts stored in shell at 15 o C and 79 83 % relative humidity (RH) remained above 80% for 150+ days. In the same peanut cultivars stored at 15 o C and 85 89 % RH germination decreased dramatically to 30% in 80 days. If the storage temperature increased to 20 o C and RH was at 79 83 %, germination steadily decreased to 80% at 80 days and, if stored at 20 o C and 85 89 % RH, germination dropped to 20% in 80 days. Nava rro 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 se ed tolerated temperatures of 44 o C 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. H ypocotyl radicle length was more adversely affected than germination. G ermination Seed Vigor, and Field E mergence Germination tests are required for the commercial sale of peanut seed and are intended to be d emergence. Germination is variously defined. For the producer, it is the appearance of the seedling at the soil surface. For t he peanut seed tester, it is the initial protrusion of the radicle from the seed and germination is reported as the percent o f normal seedlings at the conclusion of the test (Association of Official Seed Analysts,

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17 1970 ). Germination is triphasic (Black and Bewley, 1994) In phase I when water is imbibed rapidly, leakage of ions and soluble sugars o ccur s until the organelle me mbranes are repaired. In phase II, the lag phase, organelle membrane s 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 elongat ion and cell division commence. The producer accessing field emergence obs erves the conclusion of phase III ; whereas, t he seed tester observes the commencement of phase III. The Seed Vigor Testing Handbook ( Association of Official Seed Analysts, 2002) states for rapid, uniform emergence and development of normal seedlings under a w V i gor testing quantifies the vitality of the seed and places a premium upon uniform emergence. Ketring (199 3) developed a Vigor Index for peanut tha t takes into account germination and r ate of seedling growth. Peanut seed exposed to short periods of eleva ted temperature had reduced se edling 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 growin g 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 conc luded that Vigor Index is a better indicator of potential field emergence and final plant stand than germination tests.

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18 Deterioration of Seed Quality Seed deterioration is the result of changes within the seed that decrease the ability of the seed to sur vive. 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 cot yledons. 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 198 8). 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 w ater present in the atmosphere may be attracted by the same matric forces as soil water resulting in higher water content in the radicle c ompared to the storage reserves ( McDonald, 1998 ) The higher moisture content could selectively accelerate seed deterioration in the axis He concluded that s tudies 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 ac ids, 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

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19 susceptibility of fatty acids t o peroxidation increases exponentially with increasing unsaturation. Antioxidants, such as 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 (Wi lson and McDonald, 1986). Me mbrane 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 de compose into volatile aldehydes such as m alondialdehyde (MDA). The volatile aldehydes produce a wide array of cytotoxic effects including reaction with sulfhydryl groups ca using inactivation of proteins Harman et al. (1982) demonstrated an association between volatile aldehyde production durin g early germination and low soybean seed vigor. Har man 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 storag e 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 compet ence (Simon, 1974). The changes in me mbrane 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 g ermination tests by Internat ional Seed Testing Association Rules (ISTA) were not a sensitive assay for detecting the de gr ee of

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20 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 w hole 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 addit ion, using MDA as an indicator of hydroperoxides and lipid pero xidation in peanut they determined that MDA increase was most pronounced in the axis. Perez and Arguello (1995) concluded that biochemical changes which take p lace in the membranes of peanut seed as they age may be detected best in the embryonic axes, e ither through changes in the leakage of electrolytes or in MDA content and that the embryonic axi s may be the active center in relation to vigor. Water in S eeds Although lipid peroxidation occurs in al l cells, in fully imbibed cells water acts as a buf fer 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 serv e as a buffer against autoxidation, but is insufficient to activate lipoxygenase mediated free radica l production (McDonald, 2004). Below 6% moisture lipid autoxidation may be the pri me factor in seed deterioration as water is unavailable to buffer the f ree 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 temper ature increase s 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 incre ases anabolic enzyme activity and cellular repair of the damage created by free radicals Seed vigor

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21 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 c entral 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 versu s 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 envir onment, 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 str ucture becomes incr easingly fluid allowing reactions to occur more rapidly. Thus temperature above Tg may have a disproportionate effect on the stability of biological materials. A dry seed is metabolically incompetent and chemical aberrations go unrepair ed. Over time, damage from a suite of degrading reactions w ill accumulate. A change occurs from strong viability to a weaker seed to a non lines. The m odel demonstrates that different reactions are involved in seed deterioration, and that if the kinetics of these reactions

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22 are differentially controlled by temperature and water concentration the relative importance of each reaction in the overall loss o f 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 soybea n at 1 and 8 day s of storage at temperatures of 45 o and 3 5 o C, respectively and for wheat at 4 and 11 days at 4 5 o and 3 5 o C, respectively. They concluded that soybean seeds have a higher water activity and are more sensitive to changes in water status compared to wheat se eds. Seed Protection M echanisms 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 (McD onald, 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 (toco pherol), 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 mole cules (Bewley, 1986). Soybean s eed 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 damag e resulting from free radical attack during seed storage (McDonald, 2004). Talcott et al. (2005b) found that antioxidant capacity varied with genoty pe and oleic acid concentration and Talcott et al. (2005a) in analysis of oxidative stability of polypheno lics reported that, in dry roasted peanut, rates of lipid oxidation are directly proportional to the degree of fatty acid unsaturation. Hashim et al. (1993) suggested

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23 that natural antioxidants should always be considered as a parameter when predicting pe anut seed stability. Some seed degradation is inevitable (McDonald, 2004). In addition to the protective action of antioxidants, r epair enzymes and repair pathways exist specifically to fix damage caused by free radicals During imbibition t he pathways can repair DNA by removing damaged bases and oxidative lesions and correct misincorporation of nucle otides during DNA replication. If the damage is not severe, the cellular mechanisms are quickly repaired and the seed germinates into a vigorous seedling. Accelerated A ging Accelerated aging (AA ) tests expose seeds for short time periods to elevated temperature and rela tive 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) p rovides standards for using AA t o test peanut seed vigor. Ac celerated 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 tend ed to have less soluble protein and reduced activi ty of peroxide scavenging enzymes G ermination decreased and the number of weak seedlings increased co mpared to the control. These e ffects are consistent with increased lipid peroxidation in the axes and cotyledons The AA test is easy to conduct and re latively fast. Howeve r, researchers debate whether AA produces the same biochemical events as occur in natural aging (McDonald, 1999) Liklatchev 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.

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24 Mode of A ction of Aspergillus 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 protein s, fatty acids, and sterols ( Moss, 1994). Syn thesis 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 secondary met abolites. E ffect of Aspergillus on Peanut Seed G ermination 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 of Aspergillus and Penicil lium 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. halophil us Sartory & Mey is active at 7 0 to 73% relative humidity; A. flavus Link is active at 85 95 percent relative humidity. Damage to the pod predispose s the seed to invasion. Invasion ca n occur at seed moisture concentration levels as low as 13.2 percent. Seed water concentration determines the species of Aspergillus and a s mall 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 (a w ). The development of storage fungi depends on the water activity of the seed rather th an i ts relative moisture concentration Water activity differs for species and assuming that lipid is non miscible the

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25 critical moisture concentration should be computed on the non l ipid portion of the seed. For example, in so ybean ( Glycine max L. ) which has lipid concentration of 18%, fungi will grow at a moisture concentration 1.5 percent age points lower than in ce real grains, whi ch have lipid concentration of < 5%. Seed may be affected internally without showing any external evidence. Infected seed c an 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. (2001) studied the effect of Aspergillus ru ber Thom & Church on soybean seed stored at 2 5 o C a nd 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 decli ne differed with water activity (a w ) and was slower in the samples of lower a w and increased as a w increased. Concurrently free fatty acids (FFA) increased significantly. At a w of 0.66 (11.3% moisture) emergence decreased to zero by 140 day s while free fatty ac ids increased approximately 0.5 units The p roportion 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 stora ge 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 resultin g 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

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26 mitochondria and inhibit tissue respiration ( B assir and Bababunmi 1972 ). Ratnavathi and Sashidhar (2000) looked at the e ffect of aflatoxin on enzyme activity within germinating seed of sorghum ( Sorghum bicolor L. ) Although sorghum is not a high oil content seed, increasing aflatoxin r educed the act ivity of lipase. amylase and 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 increas e 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 p ercent germination as result of Aspergillus infection of seed. Hypotheses and Objectives P oor field emergence of late maturing, disease resistant peanut cultivars occurs when seeds are produced in commercial channels with large volumes being harvested, sto red 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 l iterature, the m aterials 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 lea chate of germinating peanuts.

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27 CHAPTER 3 MATERIALS AND METHOD S 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 m aturing 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 yie lds and good grades (Table 1 1). However, field emergence has been unreliable after storage in commercial bulk peanut bins. AP 3 wa s chosen as the control, mainly because AP 3 has no lineage of PI 20 3396 and has good field emergence after storage in commer cial 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 ob tained 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 o C) 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 cult ivar 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 fro m each seed origin in each year were placed into storage treatment locatio ns as follows: NFREC temperature and relative humidity cont rolled storage facility (UNICOOL ), Approximately 1.5 meters into the b ase of the bulk in shell peanut pile within the FFSP commercial peanut storage barn ( STACKS ),

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28 In bags on pal lets in the FFSP w arehouse (WHSE ), In the center of bulk peanuts being stored in wagons housed in open sided sheds at FFSP (WAGON ). A HOBO Pro Series temperature/relative humidity recorder manufactured by Onset Computer Corp., Bourne, Mass. was placed adjacent to the stor ed samples at each treatment location. U NICOOL location provided uniform air temperatures of 12 15 o C and relative humidity at 60 7 0 % throughout October to December. In the STACKS piles varied in heig ht to 15 meters and had a volume of 7 ,000m 3 Ventilati on 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 min ute 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 50 m from the FFSP storage facilities. In December, FFSP began shelling peanuts When a bin became empty, t he treatments were ended; the peanut bags were removed from the ir storage places and placed into controlled atmospheric conditions at 1 2 13 o C and 66 6 8 % 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 STA CKS and WAGON, a second set of samples designat ed Bulk Stored Seed was collected at the end of the storage period from pods deep

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29 within the bulk pile s of STACKS and the center of WAGO N. 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 t he bulk peanuts of the WAGON. In the analysis of towel germination and field emer gence 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 gr ade 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 dum ped into an elevator, lifted to the top of the elevator leg, dropped to the sh eller 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 th e Association of Official Seed A nalysts (2004). S eeds were treated with Vitavax PC for control of fungi. Active ingredients in Vitavax by we ight are Captan 45.0%, p entochloronitro benzene 15% an d 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, germin ation tests were conducted in a Model I 35LVL germination chamber manufactured by Percival Mfg. Co., Boone, Iowa. Temperature was set at 25 o C and seedlings cm radicle were counted 10 days after imbibition. Each year towel germination test s were run just prior to sowing field emergence tests In April 2005 t he 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 field origins, NFREC and FFSP,

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30 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, Flori da 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 E1 Tallahassee, Florida, 32301. Seedling Emerg ence Field Tests Four replicat ions 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 fine sand in field research plots located at the University of Florida Gainesville, Florida. Soil temperatures exceeded 15.5 o C 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 pl ots 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, so wn seed in April came from both the bagged s eed (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 13 o C 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 J an, 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 five d ay intervals.

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31 For the 2005 production year seed, a seedling emergence field test was sown May 5, 2006. T hree additional cultivars McCloud, Florida 07, and York, were added to the previously selected four cultivars Seed origin was FFSP only; since pri or 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 field emergence test. Storage Pathogen Assays Pathogen assays were conducted using Difc o Sabouraud Maltose Agar mixed thoroughly a t 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 dissolve d by boiling the water for one minute. The solute was autoclaved at 121 o C 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 32 o C for 84 hours and then pathogen s were identified. The assay consisted of 20 seeds for each of the four cultivars, AP 3, C 99R, DP 1, and Hull, from t hree 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 froze n at harvest 2) seed stored in UNICOOL and 3) seed stored in the STACKS

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32 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 Analy sts (2002) and a etring (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. V ig or tests in this research were condu cted to test cultiv ar effect and the interaction between cultivar and storage location. The results of the vigor tests are reported as for the specific purpose of comparing seedling growt h within a specif ied 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 germin ation test procedure for peanut (Association of Of ficial 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. N umerical values were assigned to th e 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 t he class. Percent germination was computed by counting all seeds with protruding radicles For the Seed V igor Te st s #1 and #2, t he seed source was from production year 2005. For Vigor Test #1, t he treatments were four cultivars, AP 3, C 99R, DP 1, and Hull, from each of four storage locations. Two replica tions of 25 seeds were held for 44 hours at 40 o C and 100% relative humidity in an accelerated ageing chamber. Two replications of 25 seeds were placed into the controlled storage at 13 o C and 67% relative humidity for 44 hours. All four replica tions of 25 seeds were germinated at 30/20 o for 7 days in the University of Florida Seed Laboratory

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33 germination chamber In Vigor Test #2 t he treatments were seven cultivars, AP 3, C 99R, DP 1, Hull, McClou d, Florida 07, and York, each stored in three locations, UNICOOL, STACKS and WHSE Four replica tions of 25 seeds were germinated at 25 o C for 7 days in the University of Florida Seed L aboratory germination chamber. Electrolyte Conductivity Test s The pro cedure 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 in to 200 mL de ionized water in beakers in a cont rolled temperature chamber at 30/20 o C for 24 hours. After 24 hours, 10 mL of leachate per treatment were withdrawn from the leachate beakers to test for electrolyte conductivity. T he leachate was agitated by swirling gently and electrol yte conductivity was determined using a Fisher Scientific Digital Conductivity Meter ( Fisher Scientific Pittsburgh, PA ) Data were expressed as mhos and were divided by the seed g weight to obtain mhos g 1 Unless otherwise stated, electrolyte conduct ivity 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 acceleratin g 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 mois t towels and incubated at 30/20 o C for an additional 5 days. In the leachate conductivity test s eac h year the s eed source was from excess seed stored at temperature < 0 o C 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

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34 Accelerated Ageing Tests Accelerated a geing 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 s eed for this test ca me from seed of production year 2005 stored in WHSE until March 2006 and then stored in UNICOOL at 12 13 o C 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 13 o C and 67% relativ e humidity, ( 2) accelerated ageing chamber at 32 o C and 100% RH (EAA5wks) and ( 3) the University germination chamber at 32 o C and r elative humidity < 10 % (HT/LRH) All seeds were treated with Vitavax For the EAA5wks treatment seeds were placed in a single layer on a copper sc reen above a water surface in small plastic germination box es sealed with lids (Association of Seed Analys ts, 200 2). The boxes were placed in an accelerated ageing c hamber 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 desiccan ts 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 30 C for 24 hours. The electrolyte conductivity of the resulting leachates wa s recorded. A fter 24 hours of imbibition the samples of 20 seeds were wrapped in moist towels and returned to the germination chamber at 30 o C for 96 hours for determination of comparative seed vigor and percent germination. The lots of 200 seeds selecte d for the seedling field emergence test were divided into four replicati ons of 50

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35 seeds each and sown in to the field plots at the University of Florida beginning September 20, 2006. Soil temperatures exceeded 22 o C at the sow ing depth of 5 cm. T he number of vigorous plants was counted at 8 DAP and 12 DAP A plant was considered vigorous if t he trifoliate leaves were open horizontal, and the plant was green and healthy The 8 DAP counts indicate d relative vigor of the treatment sample and the 12 DAP coun ts provide d final percen t field emergence. Antioxidant Capacity Assay Antioxidant capacity of see ds 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 see d 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 13 o C until January 31 and then stored at temperature < 0 o C. S eed from 2005 harvest and 2006 came from samples stored at temperature < 0 o C since harvest. Seed from STACKS was stored at temperature < 0 o C upon removal from STACKS. There were three replications. For this assay, the testa was removed and the seeds wer e trimmed to approximately uniform weights of 0.400 g. Peanuts were ground individually with mortar and pedestal and soaked in 5 mL of methanol for 15 minutes. The mixture was centrifuged at 2500 rpm for 10 minutes at room temperature. A sample of 150L of methanol ext ract was drawn from each sample and placed into a small test tube containing 850L of DPPH at 0.25mM. DPPH is 90% 1 Diphenyl 2 picryl hydrazyl (C 18 H 12 N 5 O 6 ). 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 6 h ydroxy 2,5,7,8 tetra m ethych romane 2 carboxylic acid 6 h ydroxy 2,5,7,8 tetra methyl

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36 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 5M, 10 M, 25M, 50M, 75M, and 100M of Trolox. A blank standard consisted of zero Trolox made from 100L of methanol and 900L DPPH. The resulti ng total number of samples was three replicates x four cultivars x four storage treatments with each sample assay ed three times. Samples were pipetted into 96 well Corning Costar plates and assayed for antioxidant capacity using a Spectra Max 340 PC manu factured 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 Desig ns (RBD). Analyses of Variance (ANOVA) was accomplished by the procedures in SAS System generated using the general linear model (PROC GLM) Pearson correlation coe fficients were generated using the correlation procedures (PROC CORR). Regression analyses were conducted using PROC REG. Unless stated otherwise, d ifferences reported were significant at alpha of less than or equal to 0.05.

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37 CHAPTER 4 RESULTS AND DI SCUSSION 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 199 9 2002 Poor field emergence and unacceptable stands occurred (Tilllman, 2004 Per. Comm.). The 2002 and 2003 NFREC field emergence trials included limited entries of seeds produced by Florida Foundation Seed Prod ucers (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 G ermination 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 ger mination 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 field emergence tests were designed to identify ( 1) the process and fa ctors in commercial seed production that affected seed vigor and field emergence, ( 2) the reliability of germination tests in predicting field emergence, and ( 3) year to yea r variation in field emergence. At the conclusion of the storage season s mean towe l germination for crop production year 2004 was 93.6% and was superior to th e mean towel germination of 87.1 % for production year 2005 (P <0.0001 ) ( Tables 4 1 and 4 2 ). M ean towel germination for seed from FFSP origin was 94.4% and was superior to the m ean germination of 92.8% of the NFREC origin (P =0.0321 ) ( Tables 4 1 and 4 3 ). The l ower 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

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38 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 condi tions during seed production. The main effects of cultivar and storage location fo r 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 i n 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 ( F igure 4 1 ) In 2005, towel germination of AP3 and C 99R was similar and greater than that of H ull; towel germination of DP 1 was inter mediate. Combined across both years, towel germination of C 99R was 92.6%, which was greater than both DP 1 and Hull T owel germination of AP 3 was intermediate. Storage environment/location a ffected towel germina tion (2004, P=0.0493; 2005, P=0.0498; c ombined, 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 t he 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 germi nation tests showed interaction of cultivars with storage locations for crop productio n year 2004 (P=0.0021), but not for crop production year 2005 (P =0.6526) ( Table 4 1 ).

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39 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 t he t owel 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 superio r to other storage locations. Germination of c ultivar s 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 find ing and the fact that in the towel germination tests, seed of FFSP origin was not inferior to seed of NFREC origin, and the fact that poor seed emergence occurred frequently with peanut produced and stored by FFS P 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 f ield 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

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40 99R was similar and greater than that of DP 1 and Hull. Combined across both years, field emergence of AP 3 and C 99R was about 89%, which was greater than both DP 1 and Hull. Storage location affected field emergence (2004, P=0.0208; 2005, P=0.0957; combined, P=0.0063) ( Table 4 1 ). From the 2004 crop field 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 field emergence of seeds stored in the STACKS was less than those stored in the WHSE and emergen ce of seeds stored in UNICOOL and WAGON was intermediate. In the combined analysis, field emergence of seeds stored in the STACKS was l ess than all other locations (P=0.0711). In summary f ield emergence tests confirmed that seed origin was not a signifi cant factor in poor field emergence; that the seed quality of crop production year 2004 was superior to the seed quali ty of crop production year 2005; and that cultivars AP 3 and C 9 9R had superior field emergence, compared to DP 1 and Hull. In contras t t o towel germination tests, fi eld emergence of seed st ored in STACKS had lower field emergence than all other storage locations. Towel Germi nation and Field Emergence o f 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, t he bulk stored seed samples were collected from the same STACKS and WAGONS e ither 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 interac tion of cultivar and storage environment and provide a comparison to the towel germination and field emergence of the shallower buried bagged peanuts.

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41 The main effects of cultivar and storage location for towel ge rmination tests and field emerg ence 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 a nd October (towel, P=0.0002; April fiel d emergence P <0.0001 ; October field emergence P <0.0001 ) ( Table 4 4 ). In towel germination and the April field emergence c ultivars 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 Octobe r field emergence AP 3 was superior to C 99R, DP 1, and Hull; C 99R was superior to DP 1 and Hull. Storage locati on also affected towel germination and April a nd Oc tober field emergence (towel, P =0.0309; Apr il field emergence P <0.0001 ; October field emergence P <0.0001 ) ( Table 4 4 ). T owel 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 c ultivar and storage location is presented in Figur es 4 6 and 4 7 The P values for interaction of culti var and storage loca tion for towel germinat ion, 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 STACK S, 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 culti var 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 e mergence

PAGE 42

42 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 whi le stored in STACKS decreased. The implication is that STACKS storage may reduce vigor for all cultivars and, therefore, in conditions of stress, reduce se edling populations in the field. Comparison of Treatment Effects on Bagged Seed Samples and Bulk St ored Seed Samples In comparing the April field emergence of bagged seed and the April field emergence of bulk stored seed from production year 2004, the re 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 environmen t/location was ve ry 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 degrad ing 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 ger mination tests indicated acceptable seed

PAGE 43

43 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 13 o C and 66 68 % relative humidity. The additional stress was uniform and minimal f or all samples and, yet, in October field emergence the number of emerged seedli ngs was dramatically lower ( Table 4 6 ). The greatly reduced field emergenc e 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 se ed deterioration graph ( Figure 4 8 ) Correlation of Towel Germination and Field Emergence Towel germination tests were completed one we ek preceding sowing peanut seed in April. A comparison of towel germination data the germination tests conducted by Tallahassee Seed T esting, LLC, and the field emergence in April of bulk stored seed is presented in Table 4 7 G ermination in the Tallah a ssee Seed Tests and the germination in the towel germination tests of this research are similar. F ield 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. T owel germination was not correlated with Ap ril 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 c oncept that

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44 vigor de creases 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 T owel 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 fail ures 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 hamp ered 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 o f these studies support the conclusions that : Seed production origin is not a significant factor in eventual field emergence ( Tables 4 1 and 4 3 ). There may b e an effect of season upon seed vigor and field emergence ( Table 4 1 ). Across years the field eme rgence 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 < 16 o C and relative humidity < 7 0% ( Figures 4 2, 4 6 and 4 7 ). Field emergence of seed may decrease w hen 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 AP 3. Towel g erminat ion tests are not a reliable measure of field emergence for peanut ( Table s 4 7 and 4 8 )

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45 S torage Environment Characteristics as Possible Factors in Declining Seed Vigor External atmospheric temperature and relative humidity data at 2m height for 2004 and 2005 were recorde d at the Florida Aut omated Weather Network (FAWN) adjacent to the st orage 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 .8 o C in 2004 and 15.7 o C in 2005. Although there is n o meaningful difference in the mean daily temperatures of the external air for ventilation, t he temperatures within the center of STACKS October 11 to January 24 for the 2004 crop year averaged 22.8 o C compared to 15.8 o C for the 2005 crop a difference of 7.0 o C ( 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 13.4 o C which was 8.6 o C cooler than the average of 22.0 o C for the same period in 2004. During that time interval, temperature in STACKS in 2005 decreased 5 o C and temperature in STACKS in 2004 increased 3 o C. However, the 11 day period of higher temperatures in 2004 by itself doe s not account for more than a part of the higher temperatures throughout the storage period in 2004. A lthough external air temperature may change rapidly, sometimes as much as 10 o C within 24 hours, the temperatu res at the center of the STACKS changed slow ly, usually less than 2 o C per day. The temperature and relative humidity for the DP 1 in WAGON storage fluctuated with the external atmos pheric 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 unde r sheds suggests that the temperature in STACKS in 2004 is unexpectedly higher, possibly associated with heating in STACKS by peanut respiration resulting fr om insufficient drying or a climate event in 2004 that may have affected maturity of the seed prior to drying.

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46 T emperatures in UNICOOL varied between 10 and 15 o C in 2004 and fluctuated from 7 to 21 o C in 2005 (Figure 4 1 2 ) The UNICOOL 2005 seed was stor ed in an inadequately insulated building and temperatures could not be held constant. Mean temperature in UNICOOL was 12.3 o C in 2004 and 14.4 o C in 2005. Compared to STACKS, i n 2004 temperature in the center of STACKS exceeded that in UNICOOL by 5 15 o C un til the end of December ( Figure 4 13 ). By contrast, in 2005 the mean temperature of 15.8 o C in the center of STACKS exceeded the UNICOOL mean temperature of 14.4 o C by only 1.4 o C 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 se ed deterioration and that seed moisture and temperature interact. Relative humidity in all storage conditions varied throughout the storage period ( Figure s 4 11, 4 14 and 4 15 ). In the UNICOOL the relativ e 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 sto rage 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 vigor. The temperature and relative humidity data support the conclusions: In 2004, temperatures in STACKS averaged 7.0 o C higher than temperatures in STACKS in 2005 and 10.5 o C higher than temperatures in UNICOOL in 2004 ( Figure s 4 10 and 4 13 ). Temperatures at the center of the STACKS during the first months of storage may range f rom 20 o C to >30 o C ( Figure 4 1 3 ).

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47 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 prese nt 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 inter acted to reduce the seed vigor during the sto rage of the 2004 seed E ffect of Storage Pathogens Five storage fungi were isolated; Aspergillus f lavus 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. w ere the most numerous fungi isolated and were found on 5.8% of the seed assayed Seed stored i n the stacks had more incidence s of fungi than seed at ha rvest or seed stored in UNICOOL The highes t incidence of storage fungi was Penicillium spp. present in seed stored in the stacks during the winter of 200 5. The very l ow incidence of storage fungi and the random distribution of fungal contamination support the conclusions: Storage fungi were not an important factor contributing to poor field emergence in 2 004 and 2005. Storage environmental conditions did not cause excessive growth of Aspergillus spp. Measures of Seed Quality Comparative Vigor Index Tests Seed vigor is reported here radicle s of germinatin g seeds were measured and placed into classes with numerical values. T he CVI for a seed sample equals the sum of the products of number of seedlings per class times the value

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48 as signed to the class. A photograph presented in the appendix demonstrates the variation in rate o f growth of germinating peanuts. There are 11 seedlings with excellent vigor (v alue 3 ), 10 seedli ngs with medium vigor (value 2 ), 3 see dlings with low vigor (value 1 ), and one seed counted as zero vigor (value 0). The CVI computes to 5 6 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 subjected to accelerated ageing ( AA) at 40 o C 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 location s, UNICOOL, STACKS, and WHSE. Cultivar had a si gnificant e ffect 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 corr elates with the April and Octobe r field emergence ( Table 4 8 ). P values for correlation of Vigor Te st #1 with field emergence were 0.108 4 for April and 0.0003 for October In Vigor Test #2, P values for correlation were 0.0027 for A pril 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 stan ds. This is in agreement with the conclusions of Ketring (1993). L ocation 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 reflect s the lo w towel germination tests of seed stored in UNICOOL ( Figure 4 2 ). In contrast, field

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49 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 we re intermediate in vigor ( Table 4 11 ). It was expected that peanut seed subjected to accelerated ageing temperatures of 40 o C 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 1 1 ). In a physical examination of the accelerated aged seed it was obvious that the seeds had absorbed water. The unintended consequence of this acc elerated ageing was seed priming The increased moisture concentration of the seed enabled the seed to commence germination earlier than seeds in the c ontrol 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 lo cation had no effect, then Vigor Test #2 may reflect genotype differences in rate of germination of t he 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 Test s An electrolyte conductivity test measures the electrical conductivity of the leachate of ger minating seed. A higher electrolyte conductivity value indicates greater leakage of electrolytes through the cellular membranes and dec reased 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 vig or along with the means from April a nd Octobe r field emergence are presented in Table 4 13 Seed st ored in STACKS had higher electrolyte conductivity than seed stored in

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50 UNICOOL, WHSE or WAGON. The mean electrolyte conductivity by storage locat ion was 1.39 for STACKS, 0.88 for WAGON, 0.63 for UNICO OL, and 0.59 for WHSE. In the subsequent seed vigor test, the comparative me an 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. I n th e April field emergence the corresponding means for fi eld emergence were 69.6% for STACKS, 91% for UNICOOL, 88.5% for WHSE, and 77.7% for WAGON. In th e 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 Coeffici ent 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 f ield emergence was 0 .8150 (P = 0.0002 ) and with October f ield emergence was 0.5022 (P =0.05 64 ) ( Table 4 1 4 ). Comparative vigor index (CVI) correlated with the April field emergence. D uring 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 tes t 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 th e slow er rate of hypocotyl/radicle elongation. Figure 4 17 shows the negative regression of April field emergence as electrolyte conductivity increases. The elect rolyte cond uctivity 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 correlation s of electrolyte

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51 conductivity, seed vigor test, and April field emergence confirm that both the electrol yte conductivity test and the seed vigor tests are reliabl e 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 UNICO OL at 13 o C and 67% relativ e humidity, ( 2) the accelerated ageing chamber (EAA5wks) at 32 o C and 100% RH, and ( 3) the laboratory germination chamber (HT/LRH) at 32 o C and relative humidity < 10 %. Seeds were evaluated for electrolyte cond uctivity, seedling vi gor, towel germination, and field emergence at 8 DAP and 12 DAP. Seed m oisture changed with seed treatment ( Table 4 15 ). Seed moisture in UNICOOL remained constant. Seed moisture in the EAA5wks increased from an average of 5.6% to a final moisture avera ge 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 Yor k 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 4 17 ). Seedling field emergence at 8 DAP and 12 DAP of UNICOOL and HT/LRH treatments was superior to EAA5wks (P <0.0001 ) ( Tables 4 16 and 4 17 ). The EAA5wks treatment of 32 o C and 100% relative humidity resulted in high average seed moisture of 38.2%, low electrolyte conductivi t y, high vigor index, and high towel germination, but low seedling emergence in the

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52 field ( Table 4 17). The EAA test was designed as an attempt to duplicate possible post harvest storage conditions in bulk bin s. The temperature was 2 3 o C above the expect ed 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 EAA5wks treatment had a head start and the hypocotyl radicle elongated faster than seeds from the other treatment s At the time of sowing, the EAA5wks treatments appeared fully imbibed with radicles protruding. The extent of priming varied by cultivar and within cultivar The seed was fr agile and field emergence of EAA5wks was reduced possibly by damage to the rad icle 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 EAA5wks treatment 12 DAP was low compar ed to the treatments of UNICOOL and HT/LRH ( Figure 4 18 ). Seed priming was a n unintended consequence in the EAA5wks treatment. Both the vigor test and t he field emergence results are not representative here and should be discounted, because vigor index w as inflated by priming effect of EAA5wks but was reduced by HT/LRH treatment, while field emergence was reduced by EAA5wks as expected and not reduced by warm, dry treatment (HT/LRH). Table 4 17 summarizes the m ean values of the EAA tests sorted by both t reatment and cultivar. As mhos g 1 increased, vigor index, towel germination, and field emergence decreased. This pattern is uniform throughout the table, except for the field emergence of EAA5 wks, which was confounded by the unintended priming of EAA5w ks seed samples. The

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53 pattern is in agreement with Table 4 8 which shows the strong negative correlation of electrolyte conductivity of leachate with seed vigor and field emergence. The data from electrolyte conductivity tests, seed vigor tests, and fie ld trials support the conclusions: The cultivars were affected differentially by temperature and relative humidity in the bulk storage bins ( Figures 4 4, 4 1 6 and 4 17 ). Electrolyte conductivity is negatively correlated to seed vigor and field emergence ( Table 4 8 ). Electrolyte conductivity tests and seed vigor tests correlate with field emergence and are reliable indicators of seed quality ( Table 4 8 ). In the accele rated ageing tests the elevated temperature and relative humid ity primed the accelerat ed aged seed, resulting in good towel germination but in poor field emergence ( Table 4 17 ). 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 produc tion year after storage for 4 months in STACKS. Peanut seed from production years 2005 and 2006 after harvest were placed in storage at temperature < 0 o C. Peanuts fro m production year 2004 at harvest were stored in UNICOOL at 12 13 o C until January 31 and then stored at temperature < 0 o C. Seed from the STACKS of 2004 production seed year was placed in < 0 o C 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 w as similar ( Table 4 19 ). A ntioxidant capacity at harvest for all years was greater than antioxidant capacity of seed stored in STACKS in 2004 for four months ( Table 4 20 ).

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54 Listed by year of sampling, the mean equivalents/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 equivalents/g peanut was 43.5. The variation in antioxidant capacity by year may reflect different growing conditions during the crop year, or, fo r harvest 2004, the lower antioxidant capacity o f 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 st orage 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 antioxidant s 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 a ctivity 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 conductivit y, 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 cul tivar 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 prog rams.

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55 Table 4 1. ANOVA for towel germination and April field emergence of peanut seed as affected by year (Y), cultivar (C), origin of seed (O), and storage environment/location (L) for crop production years 2004 and 2005 Table 4 2. Means of towel germination and field emergence tests of peanut seed from crop production years 2004 and 2005

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56 Table 4 3. Means of cultivar and seed origin in towel germination tests and April field emergence test of pe anut seed produced in crop year 2004 Figure 4 1. E ffe ct of cultivar o n moist towel germination and field emergence at the end of winter storage of seed peanut from crop production years 2004 and 200 5. Within a grouping, means with the same letter are not significantly different.

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57 Figure 4 2. E ffect of environment/sto rage location o n moist towel germination and field emergence of seed peanut from crop production years 20 04 and 2005. Within a grouping, means with the same letter are not significantly different.

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58 Figure 4 3. Moist towel germination of bagged seed produced in crop year 2004 as affected by cultivar and storage environment/locat ion. 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. Source df Towel Germination April Field Emergence Oct Field 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

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59 Figure 4 4. Effect of cultivars on moist towel germination and fiel d emergence of bulk sto red peanut produced in crop year 2004. Within a grouping, means with the same letter are not significantly different.

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60 Figure 4 5. Effect of storage environment/location on moist towel germination and field emerg ence of bul k 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 locatio n 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.

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61 Figure 4 6. F ield emergence in April 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.

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62 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.

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63 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 t he AOSA 50(1):136.

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64 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. Table 4 7. Compa rison 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.

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65 Table 4 8. Correlation of tow el germination, field emergence, comparative vigor index and leachate conductivity of peanut as affected by cultivar and storage location for production years 2004 an d 2005.

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66 Figure 4 9. M ean daily air tem perature at 2 meters above ground level as measured at the Florida Automated Weather Network (FAWN) substat ion, Marianna, Florida, September 16 to January 24 fo r crop years 2004 and 2005.

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67 Figure 4 10. Mean daily temperature within the bulk pile of peanut cultivar AP 3 stored in a traditional storage bin at the Florida Foundatio n Seed Producers (FFS P) for October 15 to January 24 fo r crop years 2004 and 2005.

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68 Figure 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 Octob er 15 2004 to January 31, 2005

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69 Figure 4 1 2 Mean t emperature of the seed storage room located at the University of Florida Research and Education Center (NFREC), Marianna, Florida for the period s October 15 to January 31, 2004 and 2005.

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70 Figure 4 13 Comparison of mean daily temperature in the seed storage room located at the University of Florida Research and Education Center (NFREC) and the mean daily temperature within the bulk pile of pean ut cultivar AP 3 stored in a traditional storage bin at the Florida Foundation Seed Producers (FFSP) for the per iod October 17 2004 to January 24, 2005.

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71 Figure 4 14 Comparison of relative humidit y (RH) in the seed storage r oom at the University of Florida Research and Education Center (NFREC) for October 15 to January 31 for crop years 2004 and 2005.

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72 Figure 4 15. Comparison of relative humidity (RH) within the bulk pile of peanut cultivar AP 3 s tored in a traditional storage bin at the Florida Foundation Seed Producers (FFSP) for October 15 to January 31 for crop years 2004 and 2005.

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73 Table 4 9. Incidence of fungi per 20 seeds per storage location in bulk stored peanut for crop production in 20 04 and 2005. Table 4 10. ANOVA for vigor tests #1 and #2 of peanut germination as affected by cultivar and storage environment/location for crop produ ction in 2005.

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74 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. 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.

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75 Table 4 13. Electrolyte conductivity of leachate, comparative vigor index (CVI), April fi eld emergence and October field emergence as affected by cultivar and seed storage environment/location of seed peanuts produced in crop year 2004. Table 4 14. Pearson c orrelation 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.

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76 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 cro p production year 2004 Seed with a LCH > 0.1 were considered to be non viable. Linear with LCH <0.1 CVI = 179.7 227.0*LCH R 2 = 0.7974, P value = 0.0005 3 rd order polynominal CVI = 366.9 935.2*LCH + 776.7*LCH 2 209.5*LCH 3 R 2 = 0.8855, P value <0.0001

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77 Figure 4 17 April fiel d emergence versus electrolyte conductivity of leachate (LCH) from germinating peanut seed over all cultivars and storage environments for seed from crop production year 2004.

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78 Table 4 15. Changes in seed moisture in 5 week accelerated ageing test of peanut cultivars stored in 3 environments: 1) 13 o C and 67% relative humidity (UNICOOL) 2) 32 o C and 1 00% relative hu midity (EAA5wk), and 3) 32 o C and <10% relative humidity (HT/LRH). Seed source was crop year 2005 seed that was stored in WHSE location Table 4 16. ANOVA for field emergence of peanut seed as affected by 3 treatments: 1) 13 o C a nd 67% relative humidity, 2) 32 o C/100% relative humidity and 3) 32 o C <10% relative humidity in 5 week accelerated ageing test Seed source was crop year 2005 seed that was stored in WHSE location

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79 Table 4 17. E lectrolyte conduc tivity of germinating peanut lea chate, 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) 13 o C and 67% relative humidity (UNICOOL ), 2) 32 o C and 100% relative humidity (EAA5wk) and 3) 32 o C and <10% relative humidity (HT/LRH). Within a grouping means with the same letter are not significantly different.

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80 Figure 4 18 F ield em ergence at 12 DAP of peanut cultivar s from crop production year 2005 as affected by treatment in an extended accelerated ageing test (EAA). The treatments o f five week duration were: 1) seed stored in a University of Florida seed storage room (UNICOOL) at 13 o C and 67% relative humidity, 2) seed placed in an accelerated ageing chamber (EAA5wks) at 32 o C and 100% relative humidity, and 3) seed placed in a University of Florida germination chamber (HT/LRH) at 32 o C and relative humidity < 10%. Within a groupin g, means with the same letter are not significantly different.

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81 Table 4 18. ANOVA for antioxidant capacity of peanut seed as affected by cultivar and date/year of sample. 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 significantly different. Table 4 20. Antioxidant capacity of peanut seed as affected by seed storag e environment/location and year of crop production. In 2006 seed of Hull was not produced. Means with the same letter are not significantly different.

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82 Figure 4 19 Antioxidant capacity of seed pe anut measured in equivalents g 1 as affected by year of production, cultivar, and sample date. Sample dates were harvest 2006 (HAR 06), harvest 2005 (HAR 05), harvest 2004 (HAR 04), and March 15, 2005 of peanut s stored in stacks (STACK S ) from crop produc tion year 2004. Within a grouping, means with the same letter are not significantly different.

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83 CHAPTER 5 CONCLUSIONS Seed Deterioration and Poor Field Emergence Late maturing peanut cultiv ars with genetics related to PI 203396 frequently have poor fi eld 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 p eanut production. This research looked at effects of storage environment on three of these cultivar s compared to the AP 3 check cultivar as revealed by seed vigor and field emergence. Analysis of data from the 20 04 and 2005 crop storage treatments show: The field of origin in crop 2004 year was not a factor in field emergence The seed storage envir onment/location was a factor affecting leachate conductivity, seedling vigor, and field emergence in 2004 and 2005. The bulk storage environm ent differed in 2004 and 2005. Temperatures within the peanut stack piles from October 11 to January 24 in 2004 averaged 7.0 o C higher than temperatures within the stack piles for the same period in 2005. Temperature within the peanut stack piles from October 11 to Janua ry 24 for crop production year 2004 averaged 10.5 o C higher than temperatures within the University climate controlled storage room for the same pe riod There was a cultivar by seed storage env ironment (location) interaction affecting seedling vigor and fie ld emergence. Cultivars stored in the bulk bin location had reduced seed vigor and reduced field emergence. 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. S eed vigor and field emergence were maintained when seeds were stored at < 16 o C and < 70% relative humidity. Storage fungi were not an important contributing cause to poor field emergence in 2004 and 2005.

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84 Standard towel germination tests were not reliable indicators of seed vigor or field emergence. Electrolyte conductivity tests and seed vigor tests were highly correlated wi th seed quality as indicated by field emergence. At harvest the antioxidant capacity of peanut seed varied by cultivar and year of p roduction. The Seed Vigor Testing Handbook which determine the potential for rapid, uniform emergence and development of normal n 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 deterio rate. 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 see d qual ity, 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 bi n (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 sam e samples was stored for an additional 5.5 months at < 16 o C 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 wa s 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 c ool storage,

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85 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 Figure 4 8 Sce nario of Seed Quality D eterioration The data in conjunction with the literature review, supports the following possible scenario of seed quality d eterioration: 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 concentrate d 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 autoxida tion 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 se vere, 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 d ifference in field emergence of seed from production years 2004 and 2005 may be related to the 7.0 o C higher temperature within the peanut piles (STACKS) during the common period of October 11 to January 24, 2005 compared to the 2005 production year The c ause for

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86 this temperature dif feren ce has not been determined, but, is speculated to be associate d with heating inside the pile; outside ambient temperatures during this period were similar over years The pile temperature difference could alternatively be related to cr op maturity or seed moisture at harvest going into storage However, the environment within the STACKS in 2004 consisted of elevated temperature s and high relative humidity, an environment which would allow interaction of seed moisture and t emperature and increase the incidence of lipid peroxidation. Data from the seed vigor and leachate conductivity te sts of 2004 suggest that increased peroxidation o f lipids occurred and damaged cellular membranes, especially in the seed s of Hull and DP 1. The data from 2005 seed vigor and leachate conductivity tests suggest that with the cooler temperatures in t he pile in 2005, peroxid ation may have been less than in 2004 Seed deterioration is an individual seed event (McDonald, 2004). As peanuts are r emoved 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, t he 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 a ntioxidant capacity for the four principal cultivars, antioxidant capaci ty at harvest varied by cultivar oleic acid content, and y ear. This is in agreement with Amaral et al. (2005) and Talcott et al. (2005 b ) For example, the antioxidant capacity of AP 3 at harvest, measured in equivalents g 1 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 equivalents g 1 was 76.8 in 2004 and 71.2 in 2005. During subsequent storage in s tacks of seed produced in 2004, antioxidant capacity of AP 3 decreased from 68.4 to 32.9, C 99R from 50.0 to

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87 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 i n 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 c apacity to suppress autoxidation nor conclusive evidence that for AP 3 antioxidant capacity was instrumental in protecting its seed from autoxidation.

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88 CHAPTER 6 RECOMMENDATIONS Recommendations for Continued Research AP 3, C 99R, DP 1, and Hul l 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 antio xidant capacity is linked to PI 203396. The literature reports that in many species antioxidant capacity d epends upon year of production and genotype (Amaral et al ., 2005) tocopherols by selecting for altered r esponse to temperature (Britz and Kremer 20 02). 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 antio xidant capacity to seed vigor and field emergence. 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 dama ge 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 P eanut 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 s torage 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

PAGE 89

89 content helped to maintain fie ld emergenc e ( PCA, 2006. Per. Comm.). Alternatively e ffective storage may require refrigeration/air conditioning capacity especially for these storage sensitive cultivars. Replace Towel Germination Tests with a Test that Measures Seed Vigor Develop a method of ev aluating vigor of peanut seed at the time of bagging the seed for sale t o the grower. One method is an electrolyte conductivity test of seed leachate derived by soaking a sample of peanuts in wat er 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 min imal training of technicians. A second method is to use seed vigor tests. These tests require 5 days, more space, germination chambers, and t he data can reflect the bias of the technicians measuring the linear length of hypocotyl radicles.

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90 APPENDIX SEED VIGOR DIFFERENC ES IN GERMINATING PE ANUT SEED An example of seed vigor differences as ev ident in variation of hypocotyl /radicle length of seeds germinating in a moist towel test.

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91 LIST OF REFERENCES Abdul Baki, A.A. and J.E. Baker (1973) Are changes in cellular organelles or membranes related to vigor loss in seeds? Seed Sci. and Technol. 1 : 89 125 Amaral, J.S., S.C. Cunha, M.R.Alves, J .A. Pereira, R.M. Seabra, and B.P.P. Oliveira, (2004) Triacylglycerol composition of walnut ( Juglans regia L.) cultivars: characterizaton by HPLC ELSD and chemometrics, J. Ag and Food Chem.52(26):7964 7969. Association of Official Seed Analysts, (1970) Tet razolium Testing Handbook for Agricultural Seeds AOSA Handbook Contr No. 29. Association of Official Seed Analysts, (2002) Seed Vigor Testing Handbook AOSA Handbook Contr No. 32. Association of Official Seed Analysts, (2004) Rules for Testing Seeds Bass ir, O. and E.A. Bababunmi, (1972) Liver function and histology in various species of animal treated with n on lethal doses of aflatoxin B1, J.Pathol. 1:85 90. Bewley J.D., (1986) Membrane changes in seeds as related to germination and the perturbations res ulting from deterioration in storage. Physiology of seed deterioration : proceedings of a symposium / sponsored by Divisions C 2 and C 4 of the Crop Science Society of America in Las Vegas, NV, 28 Nov. 1984 ; editors, M.B. McDonald, Jr. and C.J. Nelson. (p p. 27 45). Madison, Wis. Black, M. and J.D. Bewley (1994) Seeds: Physiology of Development and Germination, Plenum Press, New York. Britz, S.J., and D.F. K r emer, (2002) Warm temperatures or drought during seed maturation increase free alpha tocopherol in seeds of soybean (Glycine max [L.]Merr.), J.Ag and Food Chem.. Butts, C.L., J.W. Dorner, S.L. Brown, and F.H. Arthur, (2006) Aerating farmer stock peanut storage in the Southeastern U.S. Transactions of the ASAE. 49(2):457 465. Christensen, C.M., (1957) De terior ation of stored grains by f ungi, Bot. Rev. 23:108 134. Copeland, L.O. and M.B. McDonald, (1985) Principles of Seed Science and Technology Burgess Publishing Co.,Minneapolis, Minn. Dey, G., R.K. Mukherjee, and S. Bal, (1999) Influence of harvest and post harvest conditions on the physiology germina tion of peanut kernels, Peanut Sci. 26(2):64 68. Dhingra, O.D., E.S.G. Mizubuti, I.T. Napolea o and G. Jham, (2001) Free fatty acid a ccumulation and quality loss of stored soybean seeds invaded by Aspergill us ruber Seed Sci. and Tech. 29(1):193 203.

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92 Egli, D.B. and D.M. TeKrony (1995) Soybean seed germination vigor and field emergence, Seed Sci. Technol. 23:595 609. Glavind, J., (1963) Antioxidants in animal ti ssue, Acta Chemica Scandinavica. 17:1635 1640 Gorbet, D.W., (2003). Hull A new multiple disease resi stant high oleic peanut variety, Marianna NFREC Research Report 03 6 University of Florida North Florida Research and Education Center, Marianna, FL Gorbet, D.W., (2003) DP 1 A new late maturity multiple disease resistant peanut variety. Mari anna NFREC Research Report 03 7, University of Florida North Florida Research and Education Center, Marianna, FL Gorbet, D.W. (2006) Personal communication, University of Florida North Florida Research and Education Center, Marianna, FL Gorbet, D.W. and F.M. Shokes, (2003) Registrati on of 'C 99R' peanut, Crop Sci. 42(6):2207. Gorbet, D.W. and F.M. Shokes, (2002) Registra tion of 'Florida MDR 98' peanut Crop Sci. 42(6):2207 2208. Gorbet, D.W., A.J. Norden, F. (4) :817 Harman, G.E. and L.R. Mattick, (1976) Association of lipid oxid ation with seed aging and death, Nature 260:323 324. Harman, G.E., B.L. Nedrow, B.E. Clark, and L.R. Mattick, (1982) Association of volatile aldehyde production during germination with poor soybean and pea seed quality soilborne fungi an d bacteria, vigor and viability, Crop Sci. 22(4):712 716. Harrington, J.F., (197 2) Seed storage and longevity i n Seed Biology 3: 145 240, T.T. Kozlowski, (ed.), Academic Press, New York. Hashim, I.B., P.E. Koehler, R.R. Eitenmiller and C.K. Kvien, (1993) Fatty acid composition and tocopherol content of drought stressed F lorunner peanuts, Peanut Sci. 20(1):21 24. Jeng, T.L. and J.M. Sung, (1994) Hydration effect on lipid peroxidation and peroxide scavenging enzymes activity of artificially aged pe anut seed, Seed Sci. and Technol. 22:531 539. Ketring, D.L., (1992) Physiology of oil seeds. X. Seed quality of peanut genoty pes as affected by ambient storage temperature, Peanut Sci. 19(2):72 77. Ketring, D.L., (1993) Physiology of oil seeds. XI. Laboratory asses sment of peanut seed vitality, i n PSWCL Special Report 93 001, Plant Sci ence Research Laboratory Stillw ater, OK.

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93 K rishnan, P., S Nagarajan, and A.V. Moharir, (2004) Thermodynamic characterization of seed deterioration during storage unde r accelerated ageing conditions, Biosystems Engineering 89(4):425 433. Lacey, J. (1994) Aspergilli in Feeds and Seeds in The Genus Aspergillus (1994) K.A. Powell, A. Renwick, and J.F. Pe berdy (eds.), Plenum Press, New York and London. Liklatchev, B.S., G.V. Zelensky, G.Y. Kiashko, and Z.N. Schevchenko (1984) Modeling of seed ageing, Seed Sci. and Technol. 12:385 393. McDonald, M.B., (2004) Orthodox se ed deterioration and its repair, i n Handbook of Seed Physiology, eds. R.L. Benech Arno ld and R.J. Sanchez, pp.273 296 Food Products Press and Haworth Reference Press, New York. McDonald, M.B., (1999) Seed deterioration: physiology, rep air and assessment, Seed Sci & Technol. 27:177 237. McDonald, M.B., (1998) Seed quality assessment, Seed Sci. Res. 8(2):265 275. McDonald, M.B. and L.O. Copeland (1989) Seed Science and Technology Laboratory Manual published by Iowa State University Pre ss. McDonald, M.B., C.W. Vertucci, and E.E. Roos, (1988) Soybean seed imbibition:water absorption of seed parts, Crop Sci. 28:993 998 McDonald, M.B., J. Sullivan, and M.J. Lauer, (1994) The pathway of water uptake in maize ( Zea mays L.) seeds, Seed Sci. a nd Tech nol 22:79 90. Mead, J.F., (1976) Free radical mechanisms of lipid damage and conseq uences for cellular membranes i n Free Radicals in Biology 1: 51 68, ed.W.A. Pryor, Academic Press, New York. Moreau F., (1978) The electron transport system of oute r membranes of plant mitochondria, Developments Plant Biology pp.77 84. Moss, M.O., (1994) Biosynthesis of Aspergillus toxins non aflatoxins in The Genus Aspergillus (1994) K.A. Powell, A.Renwick, and J.F. Peberdy (eds.): Plenum Press, New York and Lond on. Navarro S., E. Donahaye, R. Kleinerman, and H. Haham, (1989) The influence of temperature and moisture content on the germi nation of peanut seeds, Peanut S ci. 16(1):6 9. Neergaard, P. (1977) Seed Pathology Published by Wiley, New York. Peanut Compan y of Australia, (2006) Personal communication, 133 Haly Street, Kingaroy, Queesland, 4610, Australia

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94 Perez, M.A. and J.A. Arguello, (1995) Deterioration in peanut ( Arachis hypogaea L. c v. Florman) seeds under na tural and accelerated aging, Seed Sci. and Te chnol. 23:439 445. Prasad, P.V.V., K.J. Boote, J.M.G.Thomas, L.H. Allen Jr., and D.W. Gorbet, (2006) Influence of soil temperature on seedling emergence and early growth of peanut cultivars in field conditions, J. Agron. & Crop Sci. 192:168 177. Ra tnavath i, C.V. and R.B. S ashidhar, (2000) Changes in enzyme activities and aflatoxin elaboration in sorghum genotypes following Aspergillus parasiticu s infestation, J. S ci. Food and A g. 80 (12):1713 1721. SAS Institute, System 9.1 SAS User's Guide: Statistics, S AS Institute, Cary, NC. Simon, E. (1974) Phospholipids and plant membrane permeability, New Phytology 73:377 420. Singh, R., A. Singh, S. Vadhera, and I.S. Bhatia (1974) Effect of aflatoxins on the changes in fats and carbohydrates during germination and on the symbiotic fixation of nitrogen in Stadsklev, T., (2004) Pers onal communication, Florida Foundation Seed Producers, P.O. Box 309, Greenwood, FL, 32443. Talcott, S.T., C.E. Duncan, D. Del Pozo Insfran, and D.W. Gorbet, (2005 a ) Polyphenolic and anti oxidant changes during storage of normal, mid, and high oleic acid peanuts, Food Chem 89:77 84. Talcott, S.T., S. Passeretti, C.E. Duncan, and D.W. Gorbet, (2005b) Polyphenolic content and sensory properties of normal and high oleic acid peanuts, Food Chem 90:379 388. TeKrony, D.M., (2004) Accelerated aging test: principles and procedures, Seed Technology/a Symposium: Seed Vigor 27(1):135 146. Tillman, B.L., (2004) Pers onal communication, University of Florida North Florida Research and Education Center, Ma rianna, FL. Tillman, B.L., (2007) Unpublished data, University of Florida North Florida Research and Education Center, Marianna, FL. Tillman, B.L. and D.W. Gorbet, (2004) Unpublished data, University of Florida North Florida Research and Education Center, Marianna, FL. Trawatha, S.E., D.M. TeKrony, and D.F. Hildebrand, (1995) Relationship of soybean seed quality to fatty acid and C6 aldehyde levels during storage, Crop Sci. 35:1415 1422. Tuite, J., (1969) Plant Pathological Methods, Fungi and Bacteria p. 45, Burgess Publishing Co.,Minneapolis, Minn.

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95 v an den Hondel, C.A.M.J.J., P.J. Punt and R.F.M. (1994 ) Production of extr a cellular proteins by the filamentous fungus Aspergillus i n The Genus Aspergillus (1994) K.A. Powell, A. Renwick, and J.F. Peberdy (e ds.) :Plenum Press, New York and London. Walters, C., (1998) Understanding the mechanisms and kinetics of seed ageing, Seed Sci. Res. 8(2):223 244. Wilson, D.O.Jr, and M.B. McDonald, Jr (1986) The lipid peroxidat ion model of seed ageing, Seed S ci and Te chnol. 14(2):269 300.

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96 BIOGRAPHICAL SKETCH Barry Morton earned a Bachelor of Arts in English l iterature at Brown University and a Master of Science in a gronomy from Pennsylvania State University His hesis was a Study of Pollination and Pol len Tube Growth in Buckwheat ( Fagopyrum sagittatum Gilib.) His dissertation Maturing Peanut Cultivars ( Arachis hypogaea L.) Derived from PI completes his doctoral program toward a Doctor of Ph il osophy in a gronomy f rom 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 leve l. As a commercial farm owner/producer, he raised corn, alfalfa, small grains, and potatoes in Southeastern Pennsylvania. The sw ine farrowing operation with a total of three employees and an on farm feed mill, produced 9000 pigs/year As an agricultura l loan officer, Mr. Morton a nalyzed credit, appraised collateral, made cash flow projections, and tracked loan progress of farm operating loans and mortgages for a regional bank in Pennsylvania. As an agricultural consultant, Mr. Morton ne gotiated bank fi nancing and provided crop/livestock planning, cash flow projections, and on farm management Clients included a 2500 acre Manor Farm located in Maryland producing corn, soybeans, and hay using sewage slud ge to supplement fertilizer Mr. Morton taught bio logy 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 i nstrumental in the initia l phases of establishment of a program for returning students and c o authored guidelines an d curriculum for the program


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Title: Poor Field Emergence of Late-Maturing Peanut Cultivars (Arachis hypogaea L.) Derived from PI-203396
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Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
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Title: Poor Field Emergence of Late-Maturing Peanut Cultivars (Arachis hypogaea L.) Derived from PI-203396
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
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POOR FIELD EMERGENCE OF LATE-MATURING PEANUT CULTIVARS (Arachis
hypogaea L.) DERIVED FROM PI-203396


















By

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

2007

































02007 Barry R. Morton









ACKNOWLEDGMENTS

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


page

ACKNOWLEDGMENT S .............. ...............3.....


LI ST OF T ABLE S .........._.... ...............6..._.........


LIST OF FIGURES .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 10...


CHAPTER


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


Table page

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


Fiare page

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

By

Barry R. Morton

May 2007

Chair: Barry L. Tillman
Cochair: Kenneth J. Boote
Major: Agronomy

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.









CHAPTER 1
INTRODUCTION

Cultivars

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.

Hypotheses

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
field emergence.

* There is an interaction of cultivar by storage environment on 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.


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).









CHAPTER 2
LITERATURE REVIEW

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

biological materials.

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

seed stability.

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

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

secondary metabolites.

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.









CHAPTER 3
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

follows:

* 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
(WAGON) .

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

emergence test.

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

emergence.

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.









CHAPTER 4
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

production.

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

peanuts.









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

field.

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

conclusions that:

* Seed production origin is not a significant factor in eventual field emergence (Tables 4-1
and 4-3).

* 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
AP-3 .

* 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

vigor.

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

conclusions:

* Storage fungi were not an important factor contributing to poor field emergence in 2004
and 2005.

* 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

Ketring (1993).

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

4-17).

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

conclusions:

* 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
4-8).

* 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
(Table 4-17).

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
P-values
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
Towel Towel


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.














100

95A
B ^r AB pA
AE A nR B.
90--BAA

BI~8 HCBI HI H





70 -- *- *
MM I M


60 --*'. .



50
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


WAGON
P"WHSE
STACK
UNICOOL


AP-3


Hull


C-99R


DP-1


Figure 4-3. Moist towel germination of bagged seed produced in crop year 2004 as affected by
cultivar and storage environment/location.













S100 A
o B A
90 --BA
A
80 --
S70
60 AP-3
FC ii C-99R
50 -
0 40 -t~ -rrrre Orrr~ Drrr P-1
30 -~ 5 Hull


20


Towel Germination April Field October Field
Emergence Emergence


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.













ABA A
il-


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


100
90
80
70
60
50
40
30
20
10
-


B


SUNICOOL
O STACKS
O WAGON


Towel
Germination


April Field


October Field













100


60
Hull
UICOOL~
WAGONw
STACKS


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


-~~ 3"1*20
C-99R .

DP-1

Hull
UNICOOL
WAGON
STACKS



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.






















U
PI


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
Test Test
April Field 0.0532
Emergence 0.6763
64
October Field -0.13819 0.25924
Emergence 0.4507 0.1519
32 32
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
















25 .~z











II

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













35



21







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.











100

90








80


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





















25









15 to Jaur 31 204nd205





















53


30 t
10/1 103 111 112 212/61912
UNCO 204---AP3SAK


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














80

75







5 0 na*



40
10/1 103 114 1/8 1212261912
NCO 04---UIOL20

Fiur 414 Cmprionofreatvehuidty(R ) n hesed toag romatth Uivrstyo
Flrd eerchadEuainCne NRC frOtbr1 oJnay3 o
crop~I yer 200 an 05











90
85
80

75 .
S70
65
S60

S55
50
45
40
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.















Harvest
Harvest
Harvest
Harvest
Unicool
Unicool
Unicool
Unicool
Stacks
Stacks
Stacks
Stacks
Harvest
Harvest
Harvest
Harvest
Unicool
Unicool
Unicool
Unicool
Stacks
Stacks
Stacks
Stacks


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.
Penicilliu


Treatment m A. flavus A. niger Rhizopu


Year Cultivar
2004 AP-3
2004 C-99R
2004 DP-1
2004 Hull
2004 AP-3
2004 C-99R
2004 DP-1
2004 Hull
2004 AP-3
2004 C-99R
2004 DP-1
2004 Hull
2005 AP-3
2005 C-99R
2005 DP-1
2005 Hull
2005 AP-3
2005 C-99R
2005 DP-1
2005 Hull
2005 AP-3
2005 C-99R
2005 DP-1
2005 Hull
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.
Duncan
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
Duncan
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.
Duncan
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

Duncan
Location Means Grouping N
STACKS 58.6 A 28
WHSE 57.9 A 28
UNICOOL 56.8 A 28


















94.0
95.5
94.5
80.0
91.0
81.5
76.0
46.5
74.5
69.6
88.5
90.5
86.0
89.0
88.5
97.3
86.7
60.0
66.7
77.7


89.0
74.5
78.5
71.0
78.3
69.0
57.0
25.5
36.5
47.0
71.0
62.0
24.0
37.0
48.5
87.5
38.5
24.0
28.0
44.5


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
Conductivity
(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.


October
Field
Emergence


April Field
Emergence


Location
UNICOOL
UNICOOL
UNICOOL
UNICOOL

STACKS
STACKS
STACKS
STACKS

WHSE
WHSE
WHSE
WHSE

WAGON
WAGON
WAGON
WAGON


Cultivar
AP-3
C-99R
DP-1
Hull
Means
AP-3
C-99R
DP-1
Hull
Means
AP-3
C-99R
DP-1
Hull
Means
AP-3
C-99R
DP-1
Hull
Means


Clmhos g-1
0.607
0.663
0.458
0.791
0.630
1.159
1.218
1.531
1.638
1.390
0.546
0.522
0.554
0.733
0.590
0.520
0.614
1.159
1.261
0.880


CVI
25.0
41.0
68.0
4.0
34.5
5.0
0.0
1.0
0.0
1.5
40.0
73.0
68.0
7.0
47.0
72.0
35.0
4.0
0.0
27.8












3rd Order polynominal
80 CVI= 366.9 -935.2*LCH + 776.7*LCH2 209.5*LCH3
70 R2 = 0.8855, P-value <0.0001

60
0 50 Linear with LCH <0.1
CVI= 179.7 -227.0*LCH
S40 R2= 0.7974, P-value = 0.0005

S30

20

10


-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-
viable.












120
CVI= -30.036*(LCH) +107.89
R2 = 0.6852, P-value = 0.0002
100


8 80 *








40



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


31.23
35.04
34.89
36.59
30.23


0.5
0.4
0.4
3.7
-0.6
0.8


0.06
0.02
-0.01
1.19
-0.25
0.17


4.0-6.2
4.9-6.5
4.6-7.1
4.3-6.3
4.1-6.0


5.2
5.8


C-99R
DP-1
Hull
FL-07
York


UNICOOL
UNICOOL
UNICOOL
UNICOOL
UNICOOL
Average

EAA
EAA
EAA
EAA
EAA
EAA
Average

HT/LRH
HT/LRH
HT/LRH
HT/LRH
HT/LRH
HT/LRH
Average


31.29
35.07
34.88
37.78
29.98


33.06 33.23


AP-3
C-99R
DP-1
Hull
FL-07
York



AP-3
C-99R
DP-1
Hull
FL-07
York


32.28 43.54 11.26


5.5 4.6-6.5


42.7


34.3
36.8
37.0
40. 1
38.2


1.2
1.0
1.8
1.8
0.4
2.1
1.4


37.2


28.5
30.7
31.7
34.9
32.6


-4.3
-4.2
-4.0
-4.3
-4.9
-3.1
-4.2


extensive fungal growth
35.61 45.05
34.10 43.81
34.42 44.66


9.44
9.71
10.24
9.54
10.04


-1.43
-1.41
-1.43
-1.71
-1.89
-0.93
-1.47


4.9-6.5
4.6-7.1
4.3-6.3
4.1-6.0



4.6-6.5
4.0-6.2
4.9-6.5
4.6-7.1
4.3-6.3
4.1-6.0


29.04
33.09


32.42
32.84
34.43
38.66
38.15
29.14
34.27


38.58
43.13


30.99
31.44
33.00
36.95
36.26
28.21
32.81










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

Cultivar
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












100


90
AA A .A
80 -- *
A *

o 60 -

50 -

40 --

20 -

10 -


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
significantly different.











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
significantly different.
Duncan
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.
Duncan
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














90


A AA
70

S60 --B
E B BB


oB B
S30 -

20

10

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.









CHAPTER 5
CONCLUSIONS

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
emergence.

* 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
and 2005.










* Standard towel germination tests were not reliable indicators of seed vigor or field
emergence.

* 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
product on.

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.









CHAPTER 6
RECOMMENDATIONS

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

emergence.

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

cultivars.

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.









APPENDIX
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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BIOGRAPHICAL SKETCH

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.