**4. Research**

In order to quantify if phenotypic differences in peanut cultivar seed production could occur over different planting dates in the same year, multiple cultivars were planted over a 40-day planting window. After harvest and processing, evaluations of runner-type peanut seed germination and vigor were conducted from one location in 2008 (Dawson Georgia) and two locations in 2009 (Dawson Georgia and Headland Alabama). Initial data from this research was used to quantify TSWV effects on pod yield and quality [15], but seeds were also saved. Thus, the objectives of this research were to evaluate the same seed for different peanut cultivars grown using the same management practices each year to determine if there were differences in seed vigor when planted at different times in the same field. Multiple cultivars were evaluated for seed viability and vigor for two consecutive growing seasons using a thermal gradient device (**Figure 3**).

#### **4.1. Field trials**

Field experiments were conducted near Dawson Georgia in 2008 and 2009 and repeated near Headland Alabama in 2009. Soils were a Tifton, Fine-loamy, kaolinitic, thermic Plinthic Kandiudults at Dawson and a Dothan, Fine-loamy, kaolinitic, thermic plinthic kandiudults at Headland. Five peanut cultivars were planted and included Georgia Green, as the agronomic standard because of its tolerance to TSW. Other cultivars included Georgia-03 L [38], AT 3085RO and AT271516 [39] were considered to have greater field tolerance to TSW compared to Georgia Green. Cultivar Flavor Runner 458 was also included as a susceptible check [40]. For runnertype peanut seed production for eventual evaluation, seeds were planted at three different dates each year starting in April. The earliest planting date in each year and location was determined when the 10-cm soil temperature reached 18.3°C for 3 consecutive days after 10 April. Once the initial planting date was determined, planting was repeated in 20-day intervals two times for three in total (**Table 1**).

All plantings were on single rows on 0.9 m centers, with planting date at each location arranged as a randomized complete block design in a separate block [41]. All treatments were replicated four times in each planting date block. Agronomic management inputs and irrigation were

**Figure 3.** Thermal gradient table (left) and peanut seed after evaluation for germination (right).


a Rainfall, solar radiation, and GDD are reported from initial planting to digging. For Dawson, in 2008 digging dates were 2 Sept, 10 Sept, and 3 Oct, respectively; 2009 digging dates were 4 Sept, 23 Sept, and 8 Oct, respectively. For Headland, in 2009 digging dates were 4 Sept, 22 Sept, and 1 Oct.

b R, rainfall; SR, solar radiation; GDD, growing degree days.

**Table 1.** Monthly and total rainfall, solar radiation, and total growing degree days for various peanut planting dates up to time of digging from University of Georgia and Auburn University weather stations 1 km from experiments<sup>a</sup> .

applied according to University of Georgia [42] and Auburn University recommendations. All cultivars were considered to have similar maturity requirements, thus digging date was determined separately by location and planting date according to the hull scrape method [43] of Georgia Green in each respective planting date block. Border rows were planted to Georgia Green to use for maturity determination. Plots were 6.1 m by two rows in 2008 and 6.1 m by four rows in 2009. Peanut vines were threshed with a stationary harvester (Kingaroy Engineering Works, Kingaroy, Australia) after sufficient field drying, since all pods and seeds can be cleaned-out between plots to prevent mixtures and maintain cultivar purity with this machine. A uniform sample was obtained with divider, then this was graded and these seed were saved for germination and vigor testing.

#### **4.2. Seed screening**

seed germination and vigor were conducted from one location in 2008 (Dawson Georgia) and two locations in 2009 (Dawson Georgia and Headland Alabama). Initial data from this research was used to quantify TSWV effects on pod yield and quality [15], but seeds were also saved. Thus, the objectives of this research were to evaluate the same seed for different peanut cultivars grown using the same management practices each year to determine if there were differences in seed vigor when planted at different times in the same field. Multiple cultivars were evaluated for seed viability and vigor for two consecutive growing

Field experiments were conducted near Dawson Georgia in 2008 and 2009 and repeated near Headland Alabama in 2009. Soils were a Tifton, Fine-loamy, kaolinitic, thermic Plinthic Kandiudults at Dawson and a Dothan, Fine-loamy, kaolinitic, thermic plinthic kandiudults at Headland. Five peanut cultivars were planted and included Georgia Green, as the agronomic standard because of its tolerance to TSW. Other cultivars included Georgia-03 L [38], AT 3085RO and AT271516 [39] were considered to have greater field tolerance to TSW compared to Georgia Green. Cultivar Flavor Runner 458 was also included as a susceptible check [40]. For runnertype peanut seed production for eventual evaluation, seeds were planted at three different dates each year starting in April. The earliest planting date in each year and location was determined when the 10-cm soil temperature reached 18.3°C for 3 consecutive days after 10 April. Once the initial planting date was determined, planting was repeated in 20-day intervals two times for

All plantings were on single rows on 0.9 m centers, with planting date at each location arranged as a randomized complete block design in a separate block [41]. All treatments were replicated four times in each planting date block. Agronomic management inputs and irrigation were

**Figure 3.** Thermal gradient table (left) and peanut seed after evaluation for germination (right).

seasons using a thermal gradient device (**Figure 3**).

**4.1. Field trials**

108 Advances in Seed Biology

three in total (**Table 1**).

After threshing, peanut pods were dried with forced 30–40°C warm air to 7% moisture. All samples of pods were then hand-cleaned over a screen table. For each cultivar and planting date sample, grades were determined according to Federal State Inspection Service procedures [44] for runner-type peanut. Pods were mechanically shelled on a small scale unit and seeds were sized according to diameter via round holed screens with selection based on what passed through a size 8.3 mm (Screen No. 21) but retained over 7.1 mm (Screen 18). Further screening was then conducted and sound mature seeds retained over a 7.1 by 19.1 mm slotted screen from each plot (four plots for each cultivar and planting date combination each year) were then evaluated for seed size based on Georgia Federal-State Inspection Service regulations [44]. Seeds were then stored at 16–18°C at approximately 30% humidity for up to 7 months prior to testing. Seed response to temperature and time for all seed to germinate were then evaluated on a thermal gradient table [31, 37].

#### **4.3. Thermal gradient testing**

The thermal gradient table was constructed from solid aluminum block measuring 2.4 m long by 0.9 m wide by 7.6 cm thick with a mass of 470 kg (**Figure 3**). On each end of the aluminum block, a 1.0 cm hole was drilled across the side section to allow fluid to be pumped into the table. On each end of the table, ethylene glycol plus water (1:10 mixture) at 14 or 35°C were pumped at 3.8 L per min to generate the thermogradient. Approximately 1.0°C increments occurred every 10 cm along the length of the thermogradient with a constant temperature across the width. This produced 24 increments across the length to obtain different temperatures, with nine increments across the width at each temperature. Thermocouples made from duplex insulated wire (PR-T-24 wire, Omega Engineering, Inc. Stamford, CT) were mounted to the underside of the table from the hot to cold ends. These were inserted vertically into a hole on the bottom of the table. Holes measuring 8 mm wide by 7 cm deep were drilled to allow the thermocouple to be placed within 5 mm of the upper table surface, at 10 cm intervals along the length of the table. This created a continuous temperature gradient ranging from 14 to 35°C along the length of the table. Temperatures were monitored continuously for each thermocouple and recorded at 30 minute intervals with a Graphtec midi data logger (MicroDAQ,com Ltd., Contoocook, NH).Temperature data for each thermocouple was recorded daily.

#### **4.4. Seed testing**

Peanut seeds for the appropriate plot of each cultivar by planting date were evenly distributed on germination paper (SDB 86 mm, Anchor Paper Co., St. Paul, MN), which was placed in a 100 by 15 mm sterile plastic Petri dish (Fisher Scientific Education, Hanover Park, IL). Twenty seeds were placed in each Petri dish followed by 10 ml of distilled water. A single Petri dish was then placed at each 1.0°C increment every 10 cm along the length of the table for a total of 24 dishes per plot (**Figure 3**). Beginning within 68–72 hours after seeding, peanut seed germination was counted when the radicle extended more than 5 mm beyond the seed, and then the seed was removed from the dish. Peanut seed with radicles longer than 2 mm from the seed coat are considered germinated [45] but 5 mm was chosen as it has been used in previous research [46]. Distilled water in 5 ml increments was added as needed to maintain adequate moisture in each Petri dish, and varied by temperature increment. Tests were run for 7 days with counts taken daily. All counts were taken in less than one hour each day at approximately the same time, depending upon when an experiment was started on day zero. Counts were conducted from the cold end working toward the warm end. Seeds availability were limited each year, so individual field plots were considered replications with 24 Petri dishes for each replication (n = 480 seed per field replication, n = 1920 seed per cultivar by planting date each year). Germination data was converted to a percentage by day, and cumulative germination was determined for each Petri dish over the duration of that assay. Temperature data was recorded by the data loggers for each experiment. Data included temperature maximum and minimum (±0.5°C for each thermocouple) by individual Petri dish. Maximum and minimum temperatures were the highest and lowest measures, respectively taken during one germination experiment for a specific Petri dish.

#### **4.5. Data analysis**

passed through a size 8.3 mm (Screen No. 21) but retained over 7.1 mm (Screen 18). Further screening was then conducted and sound mature seeds retained over a 7.1 by 19.1 mm slotted screen from each plot (four plots for each cultivar and planting date combination each year) were then evaluated for seed size based on Georgia Federal-State Inspection Service regulations [44]. Seeds were then stored at 16–18°C at approximately 30% humidity for up to 7 months prior to testing. Seed response to temperature and time for all seed to germinate

The thermal gradient table was constructed from solid aluminum block measuring 2.4 m long by 0.9 m wide by 7.6 cm thick with a mass of 470 kg (**Figure 3**). On each end of the aluminum block, a 1.0 cm hole was drilled across the side section to allow fluid to be pumped into the table. On each end of the table, ethylene glycol plus water (1:10 mixture) at 14 or 35°C were pumped at 3.8 L per min to generate the thermogradient. Approximately 1.0°C increments occurred every 10 cm along the length of the thermogradient with a constant temperature across the width. This produced 24 increments across the length to obtain different temperatures, with nine increments across the width at each temperature. Thermocouples made from duplex insulated wire (PR-T-24 wire, Omega Engineering, Inc. Stamford, CT) were mounted to the underside of the table from the hot to cold ends. These were inserted vertically into a hole on the bottom of the table. Holes measuring 8 mm wide by 7 cm deep were drilled to allow the thermocouple to be placed within 5 mm of the upper table surface, at 10 cm intervals along the length of the table. This created a continuous temperature gradient ranging from 14 to 35°C along the length of the table. Temperatures were monitored continuously for each thermocouple and recorded at 30 minute intervals with a Graphtec midi data logger (MicroDAQ,com Ltd., Contoocook, NH).Temperature data for each ther-

Peanut seeds for the appropriate plot of each cultivar by planting date were evenly distributed on germination paper (SDB 86 mm, Anchor Paper Co., St. Paul, MN), which was placed in a 100 by 15 mm sterile plastic Petri dish (Fisher Scientific Education, Hanover Park, IL). Twenty seeds were placed in each Petri dish followed by 10 ml of distilled water. A single Petri dish was then placed at each 1.0°C increment every 10 cm along the length of the table for a total of 24 dishes per plot (**Figure 3**). Beginning within 68–72 hours after seeding, peanut seed germination was counted when the radicle extended more than 5 mm beyond the seed, and then the seed was removed from the dish. Peanut seed with radicles longer than 2 mm from the seed coat are considered germinated [45] but 5 mm was chosen as it has been used in previous research [46]. Distilled water in 5 ml increments was added as needed to maintain adequate moisture in each Petri dish, and varied by temperature increment. Tests were run for 7 days with counts taken daily. All counts were taken in less than one hour each day at approximately the same time, depending upon when an experiment was started

were then evaluated on a thermal gradient table [31, 37].

**4.3. Thermal gradient testing**

110 Advances in Seed Biology

mocouple was recorded daily.

**4.4. Seed testing**

Maximum and minimum temperatures were then used to determine the thermal time [30, 31] or growing degree day (GDD) accumulation for the following equation.

Maximum and minimum temperatures were then used to determine the thermal time [30, 31] for growing degree day (GDD) accumulation for the following equation.

$$t\_u = \sum\_{i=1}^{k} \left[ \frac{T i\_{\text{max}} + T i\_{\text{min}}}{2} - T\_b \right] \tag{1}$$

where *t*n is the sum of GDD for *n* days, T*i* max and T*i* min are the daily maximum and minimum temperature (°C) of Day *i* [47], and *T*<sup>b</sup> is the base temperature for peanut, in this model *T*<sup>b</sup> was set at 15°C [48].

For all measurements, analysis of variance (ANOVA) was applied to the data combined across cultivar, planting date, experiment replication in time, and year to test for the differences among group means of variables and interactions. Years were regarded as random factors while cultivars (seed lot cultivar within a year) and seed germination thermal times were considered fixed effects. Interactions between cultivar and these factors were used as error terms.

Nonlinear regression using the logistics growth curve with three parameters was used to model data [49]. The equation *<sup>Y</sup>* <sup>=</sup> \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ *<sup>a</sup>* <sup>1</sup> <sup>+</sup> [((*<sup>a</sup>* <sup>−</sup> *<sup>b</sup>*1)/*b*1)<sup>∗</sup> *<sup>e</sup>*(−*b*2*x*)

$$Y = \frac{a}{1 + \left[ ((a - b1)/b1)^\* e^{(\cdot + b2a)} \right]} \tag{2}$$

with the parameters *a* being the height of the horizontal asymptote at a very large *X*, *b*1 the expected value of *Y* at time *X* = 0, *b*2 is the measure of growth rate, and *Y* is the predicted seed germination. One indicator of seed vigor is the number of GDD required to reach the 80% germination rate (Germ80). Germ80 was then determined by solving the logistic growth curve equation using the parameter estimates for each seed lot cultivar setting *Y* = 80%. Data for cultivar by planting date equations were subjected to ANOVA using the general linear models procedures with mean separation using 95% asymptotic confidence intervals. The 95% confidence limits of three parameters in the equations were used to compare the significant differences for Eq. (2). Nonlinear regressions were graphed using SigmaPlot 13.0 (SigmaPlot 13.0. SPSS Inc. 233 S. Wacker Dr., Chicago, Illinois).
