13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
Received: 31 December 2017 Revised: 26 June 2018 Accepted: 27 June 2018
DOI: 10.1111/pce.13421

ORIGINAL ARTICLE

Reproductive success of soybean (Glycine max L. Merril)

cultivars and exotic lines under high daytime temperature
Maduraimuthu Djanaguiraman1,3 | William Schapaugh1 | Felix Fritschi2 |
2 1
Henry Nguyen | P.V. Vara Prasad

1
Department of Agronomy, Kansas State
University, Manhattan, Kansas
2
Division of Plant Sciences, University of
Missouri, Columbia, Missouri
3
Department of Crop Physiology, Tamil Nadu
Agricultural University, Coimbatore, India
Correspondence
P.V. Vara Prasad, Department of Agronomy,
Kansas State University, 2004 Throckmorton
Plant Science Center, Manhattan, KS 66506.
Email: vara@ksu.edu
Funding information
United States Agency for International Devel-
opment, Grant/Award Number: AID‐OAA‐L‐
14‐00006; United Soybean Board
Abstract
The objectives were to (a) quantify the effects of high daytime temperature (HDT)
from gametogenesis to full bloom on photosynthesis and pod set in soybean (Glycine
max L. Merril) genotypes and (b) assess the relationships among photosynthesis, car-
dinal temperatures for pollen germination, in vitro pollen germination percentage,
canopy reflectance, and pod‐set percentage. Three field experiments were conducted,
and Experiment I had HDT between gametogenesis and full bloom (36.5°C to 38.6°C)
compared with Experiments II and III (29.5°C to 31.6°C; optimum temperature). HDT
decreased photosynthesis (22%) and pod‐set percent (11%) compared with Experi-
ment III. Cultivars had higher photosynthesis and pod‐set percent than plant introduc-
tion (PI) lines. The cultivars (i.e., IA3023 and KS4694) and PI lines (i.e., PI393540 and
PI588026A) were HDT tolerant and susceptible, respectively. The decreased pod‐set
percentage in susceptible genotypes (PI lines) was associated with pollen characteris-
tics. Significant positive (r2 ≥ 0.67) association between photosynthesis, cardinal tem-
peratures for pollen germination (Topt and Tmax) with pod‐set percentage was
observed. However, a negative (r2 ≥ −0.43) association between photosynthesis
and pod set with canopy reflectance at visible spectrum was observed. In vitro pollen
germination and canopy reflectance at visible spectrum can be used as a high‐
throughput phenotypic tool for breeding HDT‐tolerant genotypes.

KEY W ORDS

cardinal temperature for pollen germination, high daytime temperature, high temperature, pod‐set
percentage, pollen morphology, soybean, spectral reflectance

1 | I N T RO D U CT I O N
Soybean (Glycine max L. Merril) is an important legume crop, which
contributes to one fourth of the global edible oil and two thirds of
the world's protein concentrate for livestock feeding (Agarwal, Billore,
Sharma, Dupare, & Srivastava, 2013). High daytime temperatures
(HDTs; >30°C) coinciding with reproductive stage can cause signifi-
cant damage to reproductive processes in soybean (Prasad,
Bheemanahalli, & Jagadish, 2017). The average daytime maximum
temperatures during the soybean reproductive stages often reach
>35°C (Choi, Ban, Seo, Lee, & Lee, 2016; Penalba, Bettolli, & Vargas,
2007; Schauberger et al., 2017), which is above the optimum temper-
ature (30°C) for soybean reproductive stages (Boote, Jones, &
Hoogenboom, 1998; Gibson & Mullen, 1996; Parent & Tardieu,
2012; Schauberger et al., 2017). For each 0.8°C increase above
26.7°C, the current mean temperature of the southern United States,
soybean yields are expected to decline by 2.4% (Hatfield et al., 2011).
Similar reductions in soybean yield by high temperature have been
reported for the United States (Lobell & Field, 2007; Mourtzinis
et al., 2015) and across the globe (Mohanty et al., 2015). The average

Plant Cell Environ. 2019;42:321–336. wileyonlinelibrary.com/journal/pce © 2018 John Wiley & Sons Ltd 321

322
reduction in soybean yield was 3.1%/°C rise above the present tem-
peratures (Lobell & Field, 2007; Schlenker & Roberts, 2009; Zhao
et al., 2017). Related with these maximum temperatures, prediction
models indicate that the average surface temperature could rise
between 2°C and 4°C by the end of the 21st century (IPCC, 2014),
with a frequent occurrence of extreme high temperatures (>40°C) in
soybean production areas (Urban, Ingwers, McGuire, & Teskey,
2017). These temperature extremes can affect the soybean yield sig-
nificantly (Schauberger et al., 2017). Understanding the response of
soybean reproductive processes to high temperature is critical for
improving stress tolerance.
In various crops, HDT coinciding with reproductive growth can
significantly decrease yield between 2.5% and 10% (Arshad et al.,
2017; Craufurd, Bojang, Wheeler, & Summerfield, 1998;
Djanaguiraman, Prasad, Boyle, & Schapaugh, 2013; Hatfield &
Prueger, 2015; Prasad et al., 2017; Prasad, Boote, Allen, & Thomas,
2002; Sita et al., 2017). However, the percent yield reduction varies
with species, duration and intensity of stress, and growth stage of
the crop (Prasad et al., 2017). In soybean, as daytime temperature
increased from 30°C to 35°C, the seed set decreased in male‐sterile,
female fertile plants regardless of nighttime temperature ranges of
18°C to 23°C (Wiebbecke, Graham, Cianzio, & Palmer, 2012). This is
in accordance with earlier observations that partially male‐sterile soy-
bean was converted to complete sterility when daytime temperatures
exceeded 35°C. This indicated that daytime temperatures during
flowering and pod set were important factors affecting reproductive
success in soybean (Caviness & Fagala, 1973). Controlled environment
experiments with limited genotypes have indicated that high daytime
or nighttime or combined daytime and nighttime temperatures during
flowering decreased pod‐set percentage and seed yield of soybean
(Gibson & Mullen, 1996; Djanaguiraman, Prasad, Boyle, & Schapaugh,
2011; Djanaguiraman, Prasad, Boyle, & Schapaugh, 2013;
Djanaguiraman, Prasad, & Schapaugh, 2013; Caviness & Fagala,
1973; Zhang, Zhu, Yu, & Zhong, 2016). However, little is known about
how soybean reproductive processes respond to HDT between game-
togenesis and flowering under natural field conditions in diverse soy-
bean genotypes.
Historical analyses of soybean cultivars released throughout the
past 90 years showed that a major driver of the increase in yields is
an increase in number of seeds per plant (seed‐set percentage) and
seed mass (individual seed weight; Ainsworth, Yendrek, Skoneczka, &
Long, 2012; Jin et al., 2010; Koester, Skoneczka, Cary, Diers, & Ains-
worth, 2014; Morrison, Voldeng, & Cober, 1999). Seed set primarily
depends on synchrony in the development of male and female repro-
ductive organs and coordinated signalling between the pollen and
ovule for normal fertilization and embryo development. In most
legumes, high temperature during reproductive growth causes
decreased male and female gamete viability (Kumar et al., 2013);
reduced fertilization (Porch & Jahn, 2001); and increased abortion of
flowers and pods (Djanaguiraman, Prasad, & Schapaugh, 2013), ulti-
mately resulting in decreased seed yield. In soybean, lower pollen ger-
mination under combined high daytime and nighttime temperature
was observed, and it was associated with reduced pollen viability,
thicker exine wall, disintegrated tapetum layer, decreased saturated
phospholipids, and increased phosphatidic acid levels in pollen grains
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DJANAGUIRAMAN ET AL.
(Djanaguiraman, Prasad, Boyle, et al., 2013; Djanaguiraman, Prasad,
& Schapaugh, 2013; Salem, Kakani, Koti, & Reddy, 2007).
In general, high‐temperature‐tolerant genotypes maintain a higher
level of pollen viability than sensitive genotypes under high tempera-
tures (Dane, Hunter, & Chambliss, 1991). Recent study showed that
soybean genotypes sensitive to high temperatures had longer
flowering duration and large reduction in number of flowers and pods
per plant compared with tolerant genotypes under high temperatures
(Jumrani, Bhatia, & Pandey, 2018). Similarly, high‐temperature‐sensi-
tive genotype had increased flower length and decreased number of
pollen grains per anther and pollen germination under high tempera-
tures than tolerant genotype (Salem et al., 2007). The use of pollen
viability as a phenotyping tool will provide valuable information about
the male gametophytic tolerance to high temperatures because pollen
traits are less complex than seed yield (Paupiere et al., 2017). Previous
research in rice (Oryza sativa L.), sorghum (Sorghum bicolor L. Moench),
pearl millet (Pennisetum glaucum L. R. Br.) peanut (Arachis hypogeae L.),
and cotton (Gossypium hirsutum L.) showed that the cardinal tempera-
tures for pollen germination vary among and within species (Coast,
Murdoch, Ellis, Hay, & Jagadish, 2016; Djanaguiraman et al., 2018;
Djanaguiraman, Prasad, Murugan, Perumal, & Reddy, 2014; Kakani
et al., 2005; Kakani, Prasad, Craufurd, & Wheeler, 2002) and that dif-
ferences in pollen cardinal temperatures among genotypes are related
to combined high daytime and nighttime temperatures tolerance or
susceptibility (Coast et al., 2016; Craufurd, Prasad, & Kakani, 2003;
Djanaguiraman et al., 2014; Djanaguiraman, Perumal, Jagadish, et al.,
2018). Under combined high daytime and nighttime temperature, a
significant, positive relationship between pollen viability or in vitro
pollen germination percentage and seed‐set percentage was observed
in rice (Shi et al., 2018); sorghum (Nguyen et al., 2013; Singh et al.,
2015); and peanut (Prasad, Craufurd, & Summerfield, 1999). However,
the effects of HDT on soybean pollen viability and its reproductive
success, namely, pod‐set percent, was not documented under field
conditions.
Large genetic diversity exists in legumes, but this diversity often is
not fully leveraged in active breeding programs (Considine, Siddique, &
Foyer, 2017). Current available genomic resources for soybean may be
employed to improve the resilience of soybean to abiotic stresses
(Considine et al., 2017; Li et al., 2017). To unravel the genetic basis
of abiotic stress tolerance, including HDT stress, genotypic informa-
tion needs to be associated with corresponding phenotypic data. Can-
opy spectral reflectance characteristics can be used for large‐scale
screening of genotypes for several traits (Babar, Ginkel, et al., 2006;
Babar, Reynolds, et al., 2006a, 2006b). Spectral reflectance can be
used to monitor plant pigments, canopy architecture, water status,
nitrogen, and phosphorus concentrations and detect abiotic and biotic
stresses (Babar, Ginkel, et al., 2006; Babar, Reynolds, et al., 2006a,
2006b; Penuelas & Filella, 1998; Chaerle & Van Der Straeten, 2000)
and has been used in soybean to identify genetic loci associated with
canopy pigmentation as well as the photochemical reflectance index
(Dhanapal et al., 2015; Dhanapal et al., 2016; Herritt, Dhanapal, &
Fritschi, 2016). However, the use of spectral reflectance to differenti-
ate soybean genotypes for their HDT stress tolerance during repro-
duction has not been explored. A better understanding of the
relationships between physiological and reproductive success of

DJANAGUIRAMAN ET AL.
multiple soybean genotypes and high‐throughput phenotyping tools
may expedite breeding for HDT in soybean.
Most of the earlier studies have quantified the effects of high day-
time, high nighttime, and combined high daytime and nighttime tem-
peratures during full bloom on limited soybean genotypes on leaf
and reproductive physiology under controlled environments. How-
ever, quantification of effects of HDT on diverse genotypes from
gametogenesis to full bloom stages on soybean leaf physiology and
pollen functions was not attempted in either controlled environment
or field conditions. This study provides a mechanistic understanding
of the effects of HDT during gametogenesis and full bloom on soy-
bean reproductive success, its association with other physiological
traits, and a characterization of using high‐throughput phenotyping
to assess HDT response under field conditions. With this background,
we hypothesize that in soybean under HDT, the reproductive success
(pod‐set percent) will be associated with photosynthetic rate and
in vitro pollen germination percentage and cardinal temperatures for
pollen germination and canopy reflectance can be a proxy for identifi-
cation of soybean genotypes tolerant to HDT. The objectives were to
(a) quantify the effects of HDT from gametogenesis to full bloom on
photosynthesis and pod set in diverse soybean genotypes and (b)
assess the relationship among photosynthesis, cardinal temperatures
for pollen germination, in vitro pollen germination percentage, canopy
reflectance, and pod‐set percentage.
2 | MATERIALS AND METHODS
2.1 | Crop husbandry
Three field studies were conducted at the Kansas State University
research farm, Manhattan (39°8′N, 96°37′W, elevation of 322 m),
KS, during 2012 and 2013 to evaluate the response of soybean geno-
types to HDT stress. The experimental design was a randomized com-
plete block design with four replications. The soil type was coarse‐
silty, mixed, superactive, nonacid, mesic Typic Udifluvents. Twenty‐
two maturity groups III and IV soybean genotypes (12 cultivar and
10 plant introductions [PIs]) were used in this study. The cultivars rep-
resented genotypes that possessed excellent yield potential at the
time of their release in the United States between the 1960s. The
PIs were originated from Japan or China and were received into the
U.S. National Plant Germplasm System from 1975 to 1998. The PIs
were selected based on their high seed germinability when grown
under high temperature conditions (Smith, Mengistu, Nelson, & Paris,
2008). In both Experiments I and III, the replicated plots were planted
during the first week (third to fifth) of May 2012 and 2013 and har-
vested during the last week (28th to 30th) of October 2012 and
2013. Experiment II was planted on June (third to fifth) 2012 and har-
vested during first week (fifth to seventh) of November 2012. Each
experimental unit consisted of four rows (3.4 m long; spaced 76 cm
apart). Experiments I and III were grown under irrigated condition, by
providing flood irrigation as necessary to avoid water deficit condi-
tions. However, Experiment II was conducted under rainfed condi-
tions. Air temperature and relative humidity (RH) were measured
using WatchDog data loggers (1000 Series Micro Station, Spectrum
Technologies, Aurora, IL, USA). Incoming solar radiation was measured
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
323
using PAR sensors (LightScout Quantum Light Sensor, Spectrum Tech-
nologies, Aurora, IL, USA).
2.2 | Chlorophyll index, thylakoid membrane
damage, and photosynthetic rate
In Experiments I and III, the chlorophyll index, chlorophyll a fluores-
cence, and photosynthetic rate were recorded in 1‐week intervals
from beginning of flowering (R1) to full pod (R4; Fehr & Caviness,
1977), between 11:00 a.m. and 02:00 p.m. Three plants per replication
were randomly selected and tagged during vegetative development
for repeated measurements of above physiological traits. Physiological
traits were assessed on a fully expanded leaf (third leaf from the top of
the plant) on the tagged plants in each replication.
Thylakoid membrane stability was assessed by measuring chloro-
phyll a fluorescence using a pulse‐modulated fluorometer (OS 30p+,
OptiScience, Hudson, NH, USA). The minimum fluorescence ( F o) and
maximum fluorescence ( F m) were measured on 60‐min dark‐adapted
leaves. Thylakoid membrane damage was estimated as the ratio of
F o to F m (unitless; Krause & Weis, 1984; Ristic, Bukovnik, & Prasad,
2007). Chlorophyll index was measured using a self‐calibrating chloro-
phyll metre (Soil Plant Analytical Development, SPAD, Model 502,
Spectrum Technologies, Plainfield, IL, USA) and expressed as SPAD
units. Leaf photosynthetic rate (μmol m−2 s−1) were measured with
an infrared gas analyser (CI‐340 portable photosynthesis system,
CID Inc., Camas, WA, USA). The leaf was allowed to habituate for
5 min before measurements began; and measurements were taken
with the leaves oriented perpendicular to the sun with the PPFD
reaching the leaf surface ≥1,600 μmol m−2 s−1.
2.3 | Canopy spectral reflectance
In Experiments I and III, the canopy spectral reflectance was recorded in
1‐week intervals from beginning of flowering (R1) to full pod (R4; Fehr
& Caviness, 1977), between 11:00 a.m. and 02:00 p.m. Canopy reflec-
tance was measured using an ASD LocationSpec 3 spectroradiometer
(Analytical Spectral Devices, Boulder, CO, USA). Solar radiation
reflecting from the soybean canopy was captured between 350 and
1,050 nm with the sampling intervals of 1.4 nm in the electromagnetic
spectrum using fibre optics with a 25° field of view. Moving averages
were calculated automatically to achieve 1‐nm‐width continuous
bands. A Spectralon (Labsphere, Inc., North Sutton, NH, USA) reflec-
tance panel was used to calibrate the spectroradiometer with a dark
current and white reference for every 22 plots or when needed (this
ranged from every plot to 22‐plot intervals, depending on conditions)
to create reflected light percentages. The sensor was mounted on an
adjustable monopod pole and held vertically, approximately 1 m above
the canopy, to achieve approximately a 50‐cm diameter collection area.
Two measurements were taken per plot on the second and third rows,
excluding the first metre of each plot to eliminate border effect. Each
measurement was the average of 10 scans, which was calculated auto-
matically. Spectral data were collected on nearly cloud‐free days within
2 hr of solar noon. The spectral data were collected on the same days
on which other physiological traits were recorded.

324
2.4 | Cardinal temperatures for pollen germination
Cardinal temperatures for pollen germination was studied in Experi-
ments I and II. To determine the cardinal temperatures for pollen
germination, flowers (corolla slightly opened) from tagged plants of
different genotypes were collected at the time of anthesis
(07:00 a.m. to 09:00 a.m.). Collected flowers were air‐dried for
1 hr in the laboratory (~25°C), and pollen grains from each flower
were dusted on germination medium by brushing anthers with a
nylon hairbrush. The germination medium was prepared by dissolv-
ing 15 g of sucrose, 0.03 g of calcium nitrate, and 0.01 g of boric
acid in 100 ml of deionized water (Salem et al., 2007). To this liquid,
0.5 g of agar was added and slowly heated on a hot plate. After the
agar was completely dissolved, 3 ml of the germinating medium was
poured on clean glass slides and allowed to cool for about 15 min to
let the agar solidify. Each glass slide layered with germinating
medium was kept in an empty petri dish lined with moistened filter
paper to provide a humid atmosphere and incubated at different
temperatures ranging from 10°C to 45°C at 5°C increments for
30 min in an incubator (Echotherm, Torrey Pines Scientific, Inc.,
Carlsbad, CA, USA). A total of 100 to 200 grains were counted on
each slide. The percentage of pollen germination was estimated by
counting the total number of pollen grains and number of germi-
nated pollen grains in a random microscopic field on each micro-
scope slide using a compound light microscope (Nikon Instruments
Inc., Melville, NY, USA). Pollen was considered germinated if the
length of the pollen tube was greater than the diameter of the pol-
len grain (Prasad, Boote, & Allen Jr, 2006). The germination percent-
ages were used to determine pollen temperature response curves
and model cardinal temperatures (Tmin [minimum temperature below
which pollen grains did not germinate], Topt [temperature where
maximum germination occurred], and Tmax [temperature above which
pollen grains did not germinate]).
2.4.1 | In vitro pollen germination at 30°C and 38°C
In vitro pollen germination percentages at 30°C and 38°C were mea-
sured on all the genotypes in Experiments I and III as previously
explained.
2.5 | Pollen morphology
Pollen morphology was studied in Experiments I and II. To determine
pollen morphological characteristics, the pollen grains were collected
from at least four randomly tagged plants of different genotypes as
explained above. The pollen grains were dusted on double stick
carbon tape affixed to a carbon stub, dipped immediately in super‐
cooled ethanol for 1–3 s, and stored at −80°C until further analysis.
The carbon stub was placed in a vacuum desiccator for ~2 min to
remove excess moisture from the stub at the time of analysis.
Pollen grains were viewed under a scanning electron microscope
(SEM; Nova NanoSEM 430, FEI, Hillsboro, OR, USA) using a low vac-
uum detector. At least, 25 pollen grains per genotype per replication
was analysed for morphological features. The SEM was operated in a
vacuum, 5 kV, with a spot size of 3 and a pressure of 72.5 Pa.
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DJANAGUIRAMAN ET AL.
Images were taken at 3,000×; 12,000×; and 24,000× magnifications
(Djanaguiraman, Boyle, Welti, Jagadish, & Prasad, 2018).
2.6 | Pod‐set percentage
Pod‐set percentage was documented in all the three experiments. To
follow the fate of individual flower buds, 25 floral buds (corolla not
opened) were randomly tagged with twine in each replication on five
different plants (five flowers per plant). The flowers were tagged on
the top third and fourth main‐stem node from each plant. These floral
buds opened 3–4 days after tagging. The ability of floral buds to set
pods (pod set) was estimated after 30 days. The pod‐set percentage
was determined based on the number of pods that formed on tagged
flower buds. Pods greater than 2 cm long having seeds were consid-
ered to be fertile pods.
2.7 | Statistical analyses
The data were analysed using SAS 9.4 (SAS Institute, 2008). The
PROC GLM procedure of SAS was used for analysis of variance for
cardinal temperatures for pollen germination, in vitro pollen germina-
tion, and pod‐set percentage. However, PROC MIXED procedure
was used for repeated measure analysis. Because the main objective
was to examine the overall response of the genotypes, the physiolog-
ical data from the different measurement dates were averaged for
each plot. Data on physiological traits from Experiment II were not
included in the analysis because it had combined drought and high
temperature effects from postflowering to maturity stages. A separate
PROC GLM analysis was carried out to assess the responses of culti-
vars and PI lines to various growth temperatures (experiments) by
nesting different cultivars and PI lines within group. Standard errors
are shown as an estimate of variability, and the means of various var-
iables are separated for significance by a least significant difference
test using a probability level of 0.05. The response of pollen germina-
tion to temperature was analysed from three randomly selected,
tagged plants from each replication at three different times. Cardinal
temperatures for pollen germination were estimated following a pro-
cedure similar to that presented by Djanaguiraman et al. (2014).
Briefly, to determine cardinal temperatures (Tmin, Topt, and Tmax) for
different genotypes, different regression equations (linear, broken
stick, and quadratic) were compared for variation, determined by cal-
culating the coefficient of determination (r2) and root mean square
deviation for observed and fitted values. A quadratic equation best
described the responses of pollen germination to temperature for
most of the genotypes. The response surface regression procedure
in SAS was used to estimate parameters in the quadratic equation,
as it uses least squares to fit quadratic response surface regression
models.
Spectral reflectance data (350 to 2,500 nm) were initially trimmed
to 400 to 1,050 nm to eliminate the noise from atmospheric absorp-
tion regions focused around the water absorption wavebands in the
infrared and the atmospheric scatter of blue light in the ultraviolet
portion of the spectrum. The data were combined to form 10‐nm
means to reduce the dataset size and eliminate some of the collinear-
ity associated with hyperspectral reflectance wavebands in proximity

DJANAGUIRAMAN ET AL.
(De Jong, Pebesma, & Lacaze, 2003). Combined bands contain less
variation from sample to sample than single‐band measurements
(Lin, Yang, & Kuo, 2012), and due to the high correlation with
wavebands in proximity, minimal information is thought to be lost
due to waveband combination. The PROC REG procedure of SAS
was used to characterize the relationship among the physiological
traits, in vitro pollen germination, cardinal temperatures for pollen ger-
mination, spectral reflectance, and pod‐set percentage. The classifica-
tion of soybean genotypes for HDT tolerance was performed using
principal component analysis (PCA) as described by Kakani et al.
(2005) using the data (photosynthetic rate, pollen germination at
30°C and 38°C, pod‐set percentage, and cardinal temperatures for
pollen germination) from Experiment I. Eigenvectors generated by
PCA were used to identify soybean genotypes tolerant to HDT.
3 | RESULTS
3.1 | Environmental conditions
The average daytime maximum temperature during 7 days before the
start of flowering (R1), namely, gametogenesis in Experiments I, II, and
III, was 36.5°C, 29.5°C, and 31.6°C, respectively. The corresponding
average nighttime minimum temperature was 21.8°C, 17.0°C, and
20.0°C, respectively (Figure S1). Similarly, the average daytime maxi-
mum temperature from start of flowering (R1) to full bloom (R2)
flowering stage was 38.6°C, 30.4°C, and 30.0°C, and the correspond-
ing nighttime minimum temperature was 22.5°C, 17.0°C, and 19.4°C
during Experiments I, II, and III, respectively (Figure S1). From 2010
to 2017, average daytime maximum and nighttime minimum tempera-
tures from gametogenesis to start of flowering (R1) of irrigated soy-
bean grown in Shawnee county representing the study area were
32.8°C and 20.7°C, respectively (Figure S1). The average daytime
maximum and nighttime minimum temperatures from start of
flowering (R1) to full bloom (R2) were 33.6°C and 21.6°C, respectively
(Figure S1). The average day and nighttime relative humidities during
gametogenesis and flowering stages (R1 to R2) of Experiment I was
50% and 47%, respectively. Similarly, for Experiments II and III, the rel-
ative humidities were 58% and 60%, and 61% and 73%, respectively
(Figure S1). In‐season total precipitation was 272 and 244 mm in
Experiments I and II, respectively. However, in‐season total precipita-
tion was 39.5 mm in Experiment III. Experiments I and III were main-
tained under irrigated conditions by supplemental flood irrigation.
Experiment II was rainfed, and it received 98 and 107 mm of precipi-
tation during vegetative and flowering stages (R1 to R2), respectively.
Seddigh and Jolliff (1984) observed no significant differences in seed
number at nighttime temperatures ranging from 10°C to 24°C. Simi-
larly, Djanaguiraman, Prasad, Boyle, and Schapaugh (2013) have
reported that no significant difference between 20°C and 23°C for
pod set and seed yield of soybean. A rise in temperature will lead to
a decrease in RH of the ambient air and an increase in vapour pressure
deficit (VPD). Comparing the experiments (I with II or III), an increase
of 7°C and 5.5°C during daytime and nighttime has decreased the day-
time and nighttime RH by ~10% and 25%, respectively (Figure S1). In
both experiments (I and III), the plants are maintained under irrigated
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
325
condition; therefore, the observed differences might be primarily asso-
ciated with HDT, but with little VPD effects, because high tempera-
ture effect will be confounded with VPD effect. This was validated
from canopy temperature and stomatal conductance values of cultivar
and PI lines (Figure S3). From these observations, it is clear that Exper-
iment I experienced HDT during gametogenesis and flowering stages
(R1 to R2) compared with Experiments II and III, which experienced
more optimum temperatures (Figure S2).
3.2 | Effect of HDT, comparison between PI lines
and cultivars, and genetic variability for
photosynthesis‐related traits
Irrespective of genotypes, HDT (Experiment I) significantly (P ≤ 0.001)
decreased the chlorophyll index (6%) and photosynthetic rate (22%)
and increased the thylakoid membrane damage (8%) over optimum
temperature (Experiment III; Table 1). Across the experiments, the cul-
tivars had increased chlorophyll index (7%) and photosynthetic rate
(43%) than PI lines (Table 2). However, the thylakoid membrane dam-
age was higher in PI lines (19%) than the cultivars. There was a signif-
icant (P ≤ 0.001) difference among the genotypes for chlorophyll
index, photosynthetic rate, and thylakoid membrane damage (Figure
S3). In Experiment I, across the genotypes, the chlorophyll index and
photosynthetic rate ranged between 32.7 and 42.0 SPAD units and
11.4 and 23.4 μmol m−2 s−1, respectively, and the thylakoid membrane
damage ( F o/ F m ratio) ranged from 0.270 to 0.340 (Figure S3). In
Experiment III, chlorophyll index, photosynthetic rate, and thylakoid
membrane damage ranged from 36.7 to 42.0, 12.4 to 30.7, and
0.233 to 0.376, respectively (Figure S3). Overall, HDT decreased chlo-
rophyll index and photosynthetic rate and increased thylakoid mem-
brane damage compared with optimum temperature. The cultivars
had higher photosynthetic rate than PI lines through less thylakoid
membrane damage. There was a wide variability among the genotypes
for photosynthesis‐related traits.
3.3 | Effect of HDT, comparison between PI lines
and cultivars, and genetic variability for pod‐set‐
related traits
In both the experiments, cultivars had higher cardinal temperatures for
pollen germination (Topt and Tmax) than PI lines (Table 2). The Topt and
Tmax varied significantly (P ≤ 0.001) among genotypes in Experiments I
and II (Tables 3 and 4). Across all genotypes, in Experiment I, the
means Topt and Tmax were 28.5°C and 47.2°C, respectively (Table 3).
Similarly, in Experiment II, the means Topt and Tmax were 27.7°C and
47.3°C, respectively (Table 4). Among the genotypes, Topt in Experi-
ments I and II ranged from 25.5 to 31.6, and between 23.7°C and
31.3°C, respectively (Tables 3 and 4). The Tmax was ranged from 43.7
to 50.5, and between 41.0°C and 50.6°C, in Experiments I and II,
respectively (Tables 3 and 4).
Across the genotypes, significant (P ≤ 0.001) decrease in pollen
germination percentage at 30°C (9%) and 38°C (7%) was observed
under HDT (Table 1). The percent decrease in in vitro pollen germina-
tion by HDT was smaller among the cultivars (7%) than the PI lines
(20%) at 38°C (Table 2). Across the experiments, the in vitro pollen
 13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
326 DJANAGUIRAMAN ET AL.

TABLE 1 Effect of temperature regime (high daytime temperature [Experiment I] and optimum temperature [Experiment III]) on chlorophyll
index (SPAD units); thylakoid membrane damage ( F o/ F m ratio, unitless); photosynthetic rate (μmol m−2 s−1); in vitro pollen germination at 30°C
and 38°C (%) and pod‐set percentage in diverse soybean genotypes

Temperature regime
Traits High daytime temperature (Experiment I) Optimum temperature (Experiment III)
Chlorophyll index (SPAD units) 37.21 ± 0.311b 39.63 ± 0.297a
a
Thylakoid membrane damage ( F o/ F m ratio, unitless) 0.301 ± 0.002 0.280 ± 0.003b
−2 −1 b
Photosynthetic rate (μmol m s ) 17.42 ± 0.307 22.40 ± 0.446a
b
In vitro pollen germination at 30°C (%) 64.09 ± 1.467 70.54 ± 1.544a
In vitro pollen germination at 38°C (%) 50.93 ± 1.722b 54.56 ± 1.794a
b
Pod‐set percentage 41.73 ± 1.728 46.60 ± 1.668a

Note. SPAD: Soil Plant Analytical Development. Mean values are shown with standard error. Means followed by same letter(s) were not significantly dif-
ferent (P ≤ 0.05) as determined by the least significance difference test.

TABLE 2 Effect of temperature regime (high daytime temperature [Experiment I] and optimum temperature [Experiment III]) on chlorophyll
index (SPAD units); thylakoid membrane damage ( F o/ F m ratio, unitless); photosynthetic rate (μmol m−2 s−1); cardinal temperatures (°C) for pollen
germination (Tmin, Topt, and Tmax); in vitro pollen germination at 30°C and 38°C (%); and pod‐set percentage in diverse soybean genotypes
Temperature regime
High daytime temperature (Experiment I) Optimum temperature (Experiment III)
Traits Cultivar PI lines Cultivar PI lines
a b A
Chlorophyll index (SPAD units) 39.30 ± 0.301 34.70 ± 0.482 40.04 ± 0.311 39.14 ± 0.322B
b a B
Thylakoid membrane damage ( F o/ F m ratio, unitless) 0.29 ± 0.003 0.31 ± 0.004 0.24 ± 0.003 0.32 ± 0.005A
Photosynthetic rate (μmol m−2 s−1) 21.27 ± 0.250a 12.81 ± 0.187b 28.27 ± 0.235A 15.36 ± 0.347B
a b A
Cardinal temperature for pollen germination (°C)—Tmin* 10.02 ± 0.064 9.59 ± 0.150 7.50 ± 0.014 8.35 ± 0.022B
a b A
Cardinal temperature for pollen germination (°C)—Topt* 29.50 ± 0.113 27.39 ± 0.127 29.11 ± 0.011 25.98 ± 0.014B
a b A
Cardinal temperature for pollen germination (°C)—Tmax* 49.13 ± 0.123 45.54 ± 0.120 49.48 ± 0.025 43.35 ± 0.027B
In vitro pollen germination at 30°C (%) 75.17 ± 0.753a 50.81 ± 1.031b 82.01 ± 1.013A 56.80 ± 1.069B
a b A
In vitro pollen germination at 38°C (%) 64.33 ± 0.942 34.87 ± 0.949 68.94 ± 0.740 37.31 ± 0.868B
a b A
Pod‐set percentage 55.48 ± 0.869 25.25 ± 0.731 59.29 ± 0.913 31.38 ± 1.152B

Note. PI: plant introduction; SPAD: Soil Plant Analytical Development. Mean values are shown with standard error. The cultivar and PI lines were compared
within each temperature regime. Means followed by same letter(s) were not significantly different (P ≤ 0.05) as determined by the least significance dif-
ference test.
*Data from Experiments I and II, others from Experiments I and III.

germination was consistently lower at 38°C compared with 30°C in
most of the genotypes (Figure 1a,b). The in vitro pollen germination
at 30°C and 38°C during Experiment I ranged between 43% and
79% and 25–70%, respectively (Figure 1a,b). Similarly, in Experi-
ment III, the in vitro pollen germination ranged between 45% and
90% and 27–75%, respectively.
When averaged across all genotypes, the pod‐set percentage was
significantly (P ≤ 0.01) lower (10%) in Experiment I than in Experiment
III (Table 1). In both the experiments, the pod‐set percentages of PI
lines were lower than those of the cultivars (Table 2). Significant geno-
typic variability (18% to 67%) was observed for pod‐set percentage in
all experiments (Figures 2 and 3). Overall, HDT decreased in vitro pol-
len germination and pod‐set percent. Cultivars had higher Topt, Tmax,
in vitro pollen germination, and pod‐set percent than PI lines. Among
the genotypes, there was a wide genetic variability for pod‐set‐related
traits.
germination at 30°C (15%) and 38°C (15%), and photosynthetic rate
(15%) among the soybean genotypes. The second principal component
(PC2; 6.8%) have captured the variability for cardinal temperature
(Topt) for pollen germination (39%; Figure 4). Based on the eigenvec-
tors and factor loading values, the cultivars IA3023, Williams,
KS4694, Macon, and Pella86 were grouped in quadrant I (+PC1 and
+PC2). The genotypes in this quadrant had the highest pod‐set per-
cent, pollen germination percentage at 30°C and 38°C, photosynthetic
rate under HDT stress, and Topt value. Conversely, the PI lines,
PI588026A, PI603719A, PI393540, PI423932, PI603723, and
PI587982A were grouped in quadrant IV (−PC1 and −PC2), and these
PI lines had the lowest pod‐set percent, pollen germination percentage
at 30°C and 38°C, photosynthetic rate under HDT stress, and Topt
value. In general, cultivars are grouped under tolerant, and PI lines
under susceptible, category.

3.5 | Pollen morphology

3.4 | Principal component analyses
Under HDT (Experiment I) and optimum temperature (Experiment II), all
The PCA has indicated that the first principal component (PC1; 86%) the pollen grains of all cultivars appeared globular in shape with clear
captured the variability for pod‐set percentage (16%), pollen columella head; however, the pollen grains of most of the PI lines were
 13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DJANAGUIRAMAN ET AL. 327

TABLE 3 Quadratic equation constants (y = ax2 + bx + c) and cardinal temperatures for pollen germination of soybean cultivar and PI lines in
response to temperature (the data are from Experiment I, irrigated condition)

Equation constant Cardinal temperature

Genotype a b c r2 RMSE Tmin Topt Tmax
Calland −0.280 16.17 −148.24 89.97 13.85 11.4 28.9 46.4
Clark63 −0.211 12.15 −82.67 94.01 8.30 7.9 28.7 49.8
Harper −0.225 12.73 −104.57 88.44 11.92 10.0 28.1 45.5
IA3023 −0.187 11.29 −86.49 90.00 9.35 9.0 31.6 50.5
KS4694 −0.235 14.37 −126.80 90.48 12.70 10.7 30.5 50.1
LD00‐3309 −0.243 14.66 −128.99 90.30 13.47 10.7 29.5 49.7
Macon −0.235 14.19 −126.76 86.38 14.41 10.9 29.8 49.5
Pella86 −0.199 11.91 −98.77 80.88 15.89 9.9 29.7 49.8
Pioneer9411 −0.235 14.09 −120.24 92.97 10.84 10.3 29.5 49.6
Stressland −0.207 12.19 −96.02 90.06 11.23 9.4 28.9 49.6
Union −0.198 11.62 −94.11 83.64 15.14 9.7 28.7 48.9
Williams −0.216 13.06 −112.66 92.53 10.07 10.4 30.2 50.2
PI393540 −0.219 11.77 −89.08 85.07 12.26 9.1 26.8 44.5
PI423932 −0.157 8.16 −56.27 88.88 7.87 8.3 26.1 43.7
PI423941 −0.130 7.37 −60.88 70.90 12.29 10.0 28.0 46.8
PI587982A −0.249 13.98 −118.46 77.67 17.84 10.4 27.7 45.7
PI588026A −0.265 13.47 −82.50 98.41 4.54 7.1 25.5 43.7
PI603670 −0.219 12.69 −120.78 95.32 4.80 12.0 28.3 45.8
PI603719A −0.152 8.07 −59.60 82.00 9.49 8.9 26.5 44.3
PI603723 −0.202 10.87 −80.08 76.60 14.12 8.8 26.9 45.0
PI603746 −0.187 10.58 −83.82 91.44 8.36 9.5 28.1 47.0
PI603751A −0.157 8.91 −80.97 80.24 10.36 11.4 27.9 45.4
Mean −0.212 12.20 −100.12 87.01 11.49 9.9 28.5 47.2
LSD ‐ ‐ ‐ ‐ ‐ 0.25*** 0.63*** 0.20***

Note. LSD: least significant difference; PI: plant introduction. The data are based on incubator temperature and not medium temperature. “‐” means data not
analysed statistically.
***Significant at P ≤ 0.001.

collapsed, flattened with no clear columella head under HDT (Figures
S4–S7). Among the cultivars, the pollen grains of Clark63, Harper,
LD00‐3309, Calland, and Union had rough surface under HDT com-
pared with optimum temperature; however, the pollen grains of most
of the PI lines had deep pits except PI603746, PI603751A, and
PI423941 under HDT (Figures S4–S7). The morphology of cultivar/PI
lines representing four quadrants (I to IV) was shown in Figure 5. The
pollen grains of the genotypes in quadrant I (+PC1 and +PC2) had
smooth exine ornamentation, globular pollen grains with clear colu-
mella head with mucilage secretion on the surface under HDT and opti-
mum temperature (Figure 5a–d). In contrast, the pollen grains of
genotypes in Quadrant IV (−PC1 and −PC2) had abnormal exine orna-
mentation, collapsed, and flattened with no clear columella head under
HDT compared with optimum temperature (Figure 5m–p). The pollen
grains of genotypes in Quadrant II (+PC1 and −PC2) and Quadrant III
(−PC1 and +PC2) was globular with clear columella head under HDT,
but with abnormal exine ornamentation (Figure 5e–l).
3.6 | Relationships among spectral reflectance,
physiological traits, and pod‐set percentage
There were significant (P ≤ 0.001) positive relationships between
chlorophyll index (r2 = 0.37, Figure 2a); photosynthetic rate (r2 = 0.76,
Figure 2c); Topt (r2 = 0.52, Figure 3a); Tmax (r2 = 0.73, Figure 3b); in vitro
pollen germination at 30°C (r2 = 0.82, Figure 1a); and 38°C (r2 = 0.84,
Figure 1b) with pod‐set percentage. However, a significant (P ≤ 0.001)
negative relationship between thylakoid membrane damage and pod‐
set percentage was observed (r2 = 0.44, Figure 2b).
Strong correlations were observed between photosynthetic rate,
thylakoid membrane damage, and pod‐set percentage and selected
spectral reflectance wavebands of Experiments I and III (Figure 6a,
b). However, the chlorophyll index showed a strong correlation with
spectral reflectance in Experiment I. The association between green
(505–595 nm) and red (605–695) regions of the spectrum with pho-
tosynthetic rate, chlorophyll index, and pod‐set percentage was sig-
nificant (P ≤ 0.001) and negative (r ≥ −0.42) in Experiment I.
However, thylakoid membrane damage had a significant (P ≤ 0.001)
positive (r ≥ 0.42) association in these spectral ranges (Figure 6a).
However, in Experiment III, significant (P ≤ 0.001) negative
(r ≥ −0.33) correlations between the visible spectrum with photosyn-
thetic rate and pod‐set percentage and significant (P ≤ 0.001) posi-
tive (r ≥ 0.49) correlations with infrared regions of the spectrum
were observed (Figure 6b). However, the thylakoid membrane dam-
age was positively (r ≥ 0.33, P ≤ 0.001) and negatively (r ≥ −0.49,
P ≤ 0.001) correlated with visible and infrared regions of the spec-
trum, respectively.
 13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
328 DJANAGUIRAMAN ET AL.

TABLE 4 Quadratic equation constants (y = ax2 + bx + c) and cardinal temperatures for pollen germination of soybean cultivar and PI lines in
response to temperature (the data are from Experiment II, dryland condition)

Equation constant Cardinal temperature

Genotype a b c r2 RMSE Tmin Topt Tmax
Calland −0.143 8.42 −69.62 90.59 7.13 10.0 29.4 48.9
Clark63 −0.154 9.09 −75.22 91.47 7.28 10.0 29.4 48.9
Harper −0.116 6.05 −25.62 93.87 4.34 4.6 26.1 47.6
IA3023 −0.151 9.26 −79.25 82.96 11.65 10.3 30.6 50.6
KS4694 −0.094 5.72 −34.54 90.77 4.89 6.8 30.4 50.2
LD00–3309 −0.109 5.83 −30.60 83.56 7.01 5.9 26.7 47.5
Macon −0.111 6.92 −48.53 94.51 4.72 8.0 31.3 50.5
Pella86 −0.074 4.56 −27.46 90.36 4.24 6.7 31.0 50.5
Pioneer9411 −0.150 8.76 −65.15 92.76 6.32 8.7 29.2 49.6
Stressland −0.100 5.46 −23.97 95.72 2.95 4.8 27.4 49.9
Union −0.150 8.54 −58.32 92.48 6.26 7.9 28.5 49.1
Williams −0.105 6.16 −34.59 91.74 4.77 6.3 29.3 50.4
PI393540 −0.106 5.74 −36.18 83.02 6.03 7.3 26.5 46.8
PI423932 −0.172 9.14 −85.37 89.89 5.21 12.1 27.0 41.1
PI423941 −0.113 5.67 −40.35 89.28 4.42 8.5 25.8 41.8
PI587982A −0.147 7.25 −45.78 88.13 6.06 7.4 25.2 42.0
PI588026A −0.119 6.42 −47.52 89.14 6.04 8.9 26.9 44.9
PI603670 −0.128 6.01 −25.41 92.96 4.14 4.6 23.7 42.3
PI603719A −0.086 4.43 −27.37 91.66 2.86 7.1 25.9 44.4
PI603723 −0.084 4.64 −35.74 91.73 3.33 9.2 27.4 46.6
PI603746 −0.124 6.74 −67.78 90.69 3.48 13.3 27.3 41.0
PI603751A −0.109 5.21 −23.99 86.81 5.02 5.1 24.1 42.6
Mean −0.120 6.64 −45.83 90.19 5.37 7.9 27.7 47.3
LSD ‐ ‐ ‐ ‐ ‐ 0.59*** 0.36*** 0.85***

Note. LSD: least significant difference; PI: plant introduction. The data are based on incubator temperature and not medium temperature. “‐” means data not
analysed statistically.
***Significant at P ≤ 0.001.

FIGURE 1 Scatterplot illustrating relationship between (a) pollen germination at 30°C, fitted line y = −25.1 + 1.02×; r2 = 0.82 (P < 0.001), and (b)
pollen germination at 38°C, fitted line y = −3.2 + 0.89×; r2 = 0.84 (P < 0.001) with pod‐set percentage in diverse soybean genotype as determined
by linear regression. represents Experiment I dataset, and represents Experiment III dataset. PG; pollen germination. The closed circles
represent cultivars, and open circles represent plant introduction lines. The standard error of mean for both dependent and independent variables
is shown

4 | DISCUSSION percentage) in diverse soybean genotypes; (b) cultivars had higher

photosynthetic rate and reproductive success than PI lines; (c) there
The study showed that (a) HDT from gametogenesis to full bloom was a significant positive (r2 ≥ 0.67) association between photosyn-
decreased photosynthetic rate and reproductive success (pod‐set thesis, Topt, Tmax, and pollen germination with pod‐set percentage; (d)

DJANAGUIRAMAN ET AL.
FIGURE 2 Scatterplot illustrating relationship between (a)
chlorophyll index (Soil Plant Analytical Development [SPAD] units),
fitted line y = −300.0–3.74×; r2 = 0.37 (P < 0.001); (b) thylakoid
membrane damage ( F o/ F m ratio, unitless), fitted line y = 132.9–
307.5×; r2 = 0.44 (P < 0.001); and (c) photosynthetic rate (μmol m−2 s
−1
), fitted line y = 2.2–0.22×; r2 = 0.76 (P < 0.001) with pod‐set
percentage in diverse soybean genotype as determined by linear
regression. represents Experiment I dataset, and represents
Experiment III dataset. The closed circles represent cultivars, and open
circles represent plant introduction lines. The standard error of mean
for both dependent and independent variables is shown
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
329
FIGURE 3 Scatterplot illustrating relationship between (a) Topt for
pollen germination, fitted line y = −119.7 + 5.9×; r2 = 0.52
(P < 0.001), and (b) Tmax for pollen germination, fitted line
y = −159.7 + 4.38×; r2 = 0.73 (P < 0.001) with pod‐set percentage in
diverse soybean genotype as determined by linear regression.
represents Experiment I dataset, and represents Experiment II
dataset. The closed circles represent cultivars, and open circles
represent PI lines. The standard error of mean for both dependent and
independent variables is shown
in vitro pollen germination at 38°C and canopy reflectance at visible
spectrum can be used as a high‐throughput phenotypic tool for iden-
tification of HDT‐tolerant genotypes; and (e) PCA showed that
IA3023, Williams, KS4694, Macon, and Pella86 and PI588026A,
PI603719A, PI393540, PI423942, PI603723, and PI587982A were
HDT tolerant and susceptible, respectively.
High temperature is a major environmental stress that limits the
yield of soybean. Modelling data have shown that for each 0.8°C
increase above 26.7°C, the soybean yields are expected to decline
by 2.4% (Hatfield et al., 2011). In this study, during Experiment I, the
mean daytime temperature from gametogenesis to flowering stages
was 37.5°C, which is ~8°C and 7°C increase over Experiments II and
III, respectively (Figure S1). HDT decreased photosynthetic rate and
chlorophyll index and increased thylakoid membrane damage com-
pared with optimum temperature (Table 1). Earlier studies in soybean

330
FIGURE 4 Classification of 22 soybean genotypes for high daytime temperature stress tolerance based on the factor scores of first (PC1) and
second (PC2) principal components. represents cultivars, and represents plant introduction (PI) lines
have indicated that irrespective of timing (day, night, and day and
night), high temperature stress has decreased photosynthetic rate to
the same level (~8% decrease over optimum temperature;
Djanaguiraman, Prasad, Boyle, & Schapaugh, 2013; Gibson & Mullen,
1996). Similar to combined high daytime and nighttime temperature,
HDT may have caused structural and functional changes in the thyla-
koid membrane, leading to loss of chlorophyll pigments, because it is
bound to chloroplastic membranes (Djanaguiraman, Perumal,
Ciampitti, Gupta, & Prasad, 2018; Narayanan, Prasad, Fritz, Boyle, &
Gill, 2015; Ristic et al., 2007; Tewari & Tripathy, 1999). In soybean,
the number of seeds per plant is linearly correlated with photosyn-
thetic rate under optimum growth conditions (Egli, 1988; Egli & Yu,
1991; Jiang & Egli, 1993) and mild drought stress conditions (Liu,
Jensen, & Andersen, 2004). Similarly, a positive relationship between
photosynthetic rate and pod‐set percentage was observed under HDT
(Figure 2c). In soybean, vegetative growth lasts until the middle or late
stages of reproductive growth (pod development stage), causing a
strong competition between developing vegetative and reproductive
organs for photosynthate (Brun & Betts, 1984). In soybean, shading
treatments from flowering to pod development significantly decreased
the number of pods per plant (Kurosaki & Yumoto, 2003); however,
increased light intensities by supplemental lighting have increased
the number of pods per plant, suggesting that inadequate supply of
photosynthates to the developing reproductive organ is the major rea-
son for pod abortion (Egli & Yu, 1991; Abdin et al., 1998). Confirming
these findings, in the present experiments, the genotypes having
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DJANAGUIRAMAN ET AL.
higher photosynthetic rates also had higher pod‐set percentages
(Figure 2c). Several studies have reported the natural variation in leaf
photosynthetic rate in soybean (Jin et al., 2010; Morrison et al.,
1999; Sakoda, Tanaka, Long, & Shiraiwa, 2016). The linear increase
in photosynthetic rate against year of release was reported, and the
newer cultivars had 23% greater photosynthetic rate than older culti-
vars and local genotypes (Koester et al., 2014; Sakoda et al., 2016).
This explains the increased photosynthetic rate and pod‐set percent-
age of cultivars over the PI lines.
Pod‐set percent (number of pods formed from flowers) depends
on the functionality of male and female gametes. The environmental
conditions from gametogenesis to anthesis stage can influence the
performance of gametes (Prasad et al., 2017). Similar to combined high
daytime and nighttime temperatures or high daytime or nighttime
temperatures during full bloom (Djanaguiraman, Prasad, Boyle, &
Schapaugh, 2013), HDT from gametogenesis to full bloom has
deceased the pod‐set percentage in soybean compared with optimum
temperature (Table 1). The decrease in pod‐set percentage was attrib-
uted to a decrease in pollen viability, because a positive linear relation-
ship between pollen germination percentage and pod‐set percentage
was observed (Figure 1). Similar to this observation, Prasad et al.
(1999) in peanut; Aloni, Peet, Pharr, and Karni (2001) in bell pep-
per (Capsicum annuum L.); Djanaguiraman et al. (2014); Nguyen et al.
(2013); and Singh et al. (2015, 2016) in sorghum have established a
high positive correlation between in vitro pollen germination and
fruit/seed set under combined high daytime and nighttime
 13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DJANAGUIRAMAN ET AL. 331

FIGURE 5 Scanning electron microscopic images of pollen grains of representative soybean genotypes (Williams [Quadrant I, +PC1 and + PC2]);
Stressland (Quadrant II, +PC1 and −PC2); PI603746 (Quadrant III, −PC1 and + PC2); and PI393540 (Quadrant IV, −PC1 and −PC2) of Experiments I
and II. † indicates columella head; * indicates the exine ornamentation. The first and third column images were recorded at 3,000×, and the second
and fourth column images were recorded at 12,000×

temperatures. The genotypes having lower pod‐set percentages under
HDT had shrivelled pollen grains with abnormal exine ornamentation,
and few columellae heads (Figure 5). The exine originates from the
tapetum, and premature degeneration of the tapetal layer under high
temperature stress might have affected the translocation of carbohy-
drates to developing pollen grains leading to abnormal exine ornamen-
tation and loss of pollen viability (Djanaguiraman, Prasad, &
Schapaugh, 2013; Djanaguiraman, Boyle, Welti, et al., 2018). Studies
in sorghum have shown that apart from tapetum degeneration, high
temperatures also induce accumulation of reactive oxygen species in
pollen grains leading to loss of pollen viability (Djanaguiraman et al.,
2014; Djanaguiraman, Boyle, Welti, et al., 2018; Prasad &
Djanaguiraman, 2011). However, in pearl millet, the pistil is found to
be more sensitive to high temperature than pollen grains
(Djanaguiraman, Perumal, Ciampitti, et al., 2018). Such information is
not available in soybean; thus, understanding the effects of high tem-
peratures on pistil fertility and associated pod‐set percentage will also
help in developing high‐temperature‐tolerant genotypes.
Results from in vitro studies showed that soybean genotypes var-
ied in response to temperature for cardinal temperatures for pollen
germination (Tmin, Topt, and Tmax; Table 3 and 4; Figure 3). Overall,
between Experiments I and II, the cardinal temperatures for pollen
germination (Topt and Tmax) varied little (<1°C; Tables 3 and 4), and
the mean Topt and Tmax value of cultivars was higher than PI lines in
both experiments I and III (Table 2). The high Topt and Tmax value of cul-
tivars is associated with high pod‐set percentage (Figure 3). The differ-
ences in Topt and Tmax reflected cultivar susceptibility or tolerance to
high temperature because the cultivars that had a higher Topt also
had higher Tmax values or vice versa as observed in rice (Coast et al.,
2016) and peanut (Craufurd et al., 2003). However, in sorghum (Singh

332
et al., 2016) and cotton (Kakani et al., 2005), high‐temperature toler-
ance is not associated with cardinal temperatures for pollen germina-
tion. This study provides evidence that in soybean, cardinal
temperature for pollen germination (Topt and Tmax) is associated with
pod‐set percentage (Figure 3). The observed Tmax value for soybean
was higher than or equal to most of the field crops (rice, 42.2°C, Coast
et al., 2016; sorghum, 43.2°C, Singh et al., 2016; cotton, 46.2°C,
Kakani et al., 2005; peanut, 46.3°C, Kakani et al., 2002; soybean,
47.2°C, Salem et al., 2007; and pearl millet, 50°C, Djanaguiraman,
Perumal, Jagadish, et al., 2018). The cardinal temperatures are based
on incubator temperatures (the actual tissue or medium temperatures
could be about 1°C to 2°C cooler because of air circulation and loca-
tion of the sensor that controls incubator temperature), and the qua-
dratic equation used to calculate the Tmax does not account for the
frequently observed decline of developmental rate at supraoptimal
temperatures (Yin, Kropff, McLaren, & Visperas, 1995). Rodriguez‐
Garay and Barrow (1988) bred high‐temperature‐tolerant cotton
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DJANAGUIRAMAN ET AL.
FIGURE 6 Correlation coefficients (r) for the
relationship between spectral reflectance in
400 to 1,050 nm and photosynthetic rate
(μmol m−2 s−1); thylakoid membrane damage
( F o/ F m ratio, unitless); chlorophyll index (Soil
Plant Analytical Development units); and pod‐
set percentage. (a) Experiment I and (b)
Experiment III. Wavebands are 10‐nm
intervals, and reflectance is the percentage of
a white reference panel. Dashed lines
represent significance at the P < 0.05 level
genotypes by crossing female plants with pollen grains exposed to
35°C for 15 min, suggesting that pollen could be used to screen cotton
cultivars for high‐temperature tolerance. Our earlier studies have
showed that percent pollen germination at 38°C was ~50% of the pol-
len germination percent at optimum temperature (Djanaguiraman,
Prasad, Boyle, & Schapaugh, 2013; Djanaguiraman, Prasad, &
Schapaugh, 2013). Therefore, the ability of pollen grain to germinate
and grow well at temperature of 38°C was assessed, and the result
indicated a strong positive association between in vitro pollen germi-
nation at 38°C and pod‐set percent (Figure 1a,b). Based on the pollen
germination, it can be concluded that cardinal temperatures for pollen
germination (Tmin and Topt) and pollen germination at 38°C could be
used as a high‐throughput phenotypic tool to identify soybean geno-
types tolerance to high temperatures.
Spectral reflectance of a plant is closely associated with the struc-
tural and physiological characteristics of the canopy (Lobos & Han-
cock, 2015). The results present in Figure 6 reveals that it is possible

DJANAGUIRAMAN ET AL.
to capture the effect of HDT on photosynthetic properties of soybean
genotypes in terms of their spectral signature in two regions (visible,
VIS; 400–700 nm) and near‐infrared (NIR, 700–1300 nm). There was
a subtle difference in the shape of the canopy reflectance curves
between cultivars and PI lines in the above regions under HDT (Figure
S8). All the cultivars had low reflectance in the green and red regions
and high reflectance in the red‐edge and near‐infrared regions com-
pared with the PI lines (Figure S8). Leaf pigments, including chlorophyll
a, b, carotenoids, anthocyanins, and other accessory pigments are
associated with light harvesting reactions of photosynthesis and are
abundant in healthy plants causing less canopy reflectance (Asner,
1998; Babar, Ginkel, van Klatt, Prasad, & Reynolds, 2006; Chappelle,
Kim, & McMurtrey III, 1992; Yao, Zhu, Tian, Feng, & Cao, 2010). The
higher reflectance in the visible spectrum in the PI lines indicated
unhealthy and loss of the chlorophyll pigments under HDT. This was
validated from chlorophyll index values of cultivar and PI lines
(Figure 2a). Apart from this, low reflectance in NIR regions of the cul-
tivars might be due to a higher ratio of spongy mesophyll to palisade
mesophyll cells (Ollinger, 2011). Increased spongy to palisade meso-
phyll cells helps in rapid diffusion of CO2 to the chloroplasts causing
higher photosynthetic rate. This is consistent with the finding of
Slaton, Hunt Jr., and Smith (2001), who found a strong correlation
between NIR leaf reflectance and the photosynthetic rate. This was
confirmed in this study by a significant negative correlation between
visible spectrum reflectance and chlorophyll index and photosynthetic
rate (Figure 6). Generally, thylakoid membrane damage is negatively
related to the SPAD value under high temperature conditions (Ristic
et al., 2007), and the leaves having higher chlorophyll indices will scat-
ter more light leading to higher near‐infrared reflectance (Peng et al.,
2017), as evidenced by reflectance of the cultivars. The decrease in
the reflectance in the near infrared regions may be attributed to a col-
lapse in the leaf cellular structure caused by the high temperature
(Feng et al., 2014; Knipling, 1970), which might have occurred more
in the PI lines compared with the cultivars. Based on these data, it is
apparent that spectral reflectance at visible spectrum could be used
as a high‐throughput phenotypic tool to identify soybean genotypes
tolerance to high temperatures.
5 | C O N CL U S I O N S
Our results indicate that under field conditions, an increase in daytime
temperature from gametogenesis to full bloom decreased photosyn-
thetic rate and pod‐set percentage in diverse soybean geno-
types through thylakoid membrane damage and loss of pollen
viability, respectively. Pollen morphological characteristics under
HDT are associated with pollen viability. A strong (r2 ≥ 0.67) positive
association between pod‐set percentage and photosynthetic rate, car-
dinal temperature for pollen germination (Topt and Tmax), and pollen
germination was observed. Cardinal temperatures for pollen germina-
tion (Topt and Tmax), pollen germination at 38°C, and spectral reflec-
tance at visible spectrum can be a high‐throughput phenotypic tool
for identification of HDT‐tolerant soybean lines. PCA showed that
IA3023, Williams, KS4694, Macon, and Pella86 and PI588026A,
PI603719A, PI393540, PI423942, PI603723, and PI587982A were
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
333
HDT tolerant and susceptible, respectively. The presence of extensive
genotypic variation for photosynthetic rate, pollen germination, and
pod set supports the view that plant breeding can potentially mitigate
some of the adverse effects of HDT. However, further research is
required to understand the sensitivity of female gametophyte to
HDT, response of pollination and fertilization process to HDT, and
whether the differences in HDT tolerance among the genotypes are
associated with differences in sensitivity to HDT per se or diurnal tem-
perature difference or combined temperature and VPD effects.
ACKNOWLEDGMENTS
We thank the United Soybean Board for financial support. Feed the
Future Innovation Lab for Collaborative Research on Sustainable
Intensification (Grant No. AID‐OAA‐L‐14‐00006) 593 funded by
United States Agency for International Development is acknowledged.
We also thank Dr. Dan Boyle, Division of Biology, Kansas State Uni-
versity, for the help in pollen morphology analysis. We thank Tamil
Nadu Agricultural University, India, for permitting Dr. M.
Djanaguiraman to conduct postdoctoral research at Kansas State Uni-
versity. This publication has a Contribution No. 18‐255‐J from the
Kansas Agricultural Experiment Station.
ORCID
Henry Nguyen http://orcid.org/0000-0002-7597-1800
P.V. Vara Prasad http://orcid.org/0000-0001-6632-3361
RE FE RE NC ES
Abdin, O. M., Zhou, X., Coulman, B. E., Cloutier, D., Faris, M. A., & Smith, D.
L. (1998). Effect of sucrose supplementation by stem injection on the
development of soybean plants. Journal of Experimental Botany, 49,
2013–2018.
Agarwal, D. K., Billore, S. D., Sharma, A. N., Dupare, B. U., & Srivastava, S.
K. (2013). Soybean: Introduction, improvement, and utilization in India‐
Problems and prospects. Agricultural Research, 2, 293–300.
Ainsworth, E. A., Yendrek, C. R., Skoneczka, J. A., & Long, S. P. (2012).
Accelerating yield potential in soybean: Potential targets for biotechno-
logical improvement. Plant, Cell & Environment, 35, 38–52.
Aloni, B., Peet, M., Pharr, M., & Karni, L. (2001). The effect of high temper-
ature and high atmospheric CO2 on carbohydrate changes in bell
pepper (Capsicum annuum) pollen in relation to its germination.
Physiologia Plantarum, 112, 505–512.
Arshad, M. S., Farooq, M., Asch, F., Jagadish, S. V. K., Prasad, P. V. V., &
Siddique, K. H. M. (2017). Thermal stress impacts reproductive devel-
opment and grain yield in rice. Plant Physiology and Biochemistry, 115,
57–72.
Asner, G. P. (1998). Biophysical and biochemical sources of variability in
canopy reflectance. Remote Sensing of Environment, 64, 234–253.
Babar, M., Ginkel, M., van Klatt, A., Prasad, B. & Reynolds, M. (2006) The
potential of using spectral reflectance indices to estimate yield in
wheat grown under reduced irrigation Euphytica 150, 155‐172.
Babar, M. A., Reynolds, M. P., van Ginkel, M., Klatt, A. R., Raun, W. R., &
Stone, M. L. (2006a). Spectral reflectance indices as a potential indirect
selection criteria for wheat yield under irrigation. Crop Science, 46,
578–588.
Babar, M. A., Reynolds, M. P., van Ginkel, M., Klatt, A. R., Raun, W. R., &
Stone, M. L. (2006b). Spectral reflectance to estimate genetic variation
for in‐season biomass, leaf chlorophyll and canopy temperature in
wheat. Crop Science, 46, 1046–1057.
Boote, K. J., Jones, J. W., & Hoogenboom, G. (1998). Simulation of crop
growth: CROPGRO Model. In R. M. Peart, & R. B. Curry (Eds.),

334
Agricultural systems modeling and simulation. (pp. 651–692). New York,
USA: Marcel Dekker, Inc.
Brun, W. A., & Betts, K. J. (1984). Source/sink relations of abscising and
non‐abscising soybean flowers. Plant Physiology, 75, 187–191.
Caviness, C. E., & Fagala, B. L. (1973). Influence of temperature on partially
male‐sterile soybean strain. Crop Science, 13, 503–504.
Chaerle, L., & Van Der Straeten, D. (2000). Imaging techniques and the
early detection of plant stress. Trends in Plant Science, 5, 495–501.
Chappelle, E. W., Kim, M. S., & McMurtrey, J. E. III (1992). Ratio analysis of
reflectance spectra (RARS): An algorithm for the remote estimation of
the concentrations of chlorophyll a, chlorophyll b, and carotenoids in
soybean leaves. Remote Sensing of Environment, 39, 239–247.
Choi, D. H., Ban, H. Y., Seo, B. S., Lee, K. J., & Lee, B. W. (2016). Phenology
and seed yield performance of determinate soybean cultivars grown at
elevated temperatures in a temperate region. PLoS One, 11, e0165977.
Coast, O., Murdoch, A. J., Ellis, R. H., Hay, F. R., & Jagadish, K. S. V. (2016).
Resilience of rice (Oryza spp.) pollen germination and tube growth to
temperature stress. Plant, Cell & Environment, 39, 26–37.
Considine, M. J., Siddique, K. H. M., & Foyer, C. H. (2017). Nature's pulse
power: Legumes, food security and climate change. Journal of Experi-
mental Botany, 68, 1815–1818.
Craufurd, P. Q., Bojang, M., Wheeler, T. R., & Summerfield, R. J. (1998).
Heat tolerance in cowpea: Effect of timing and duration of heat stress.
Annals of Applied Biology, 133, 257–267.
Craufurd, P. Q., Prasad, P. V. V., & Kakani, V. G. (2003). Heat tolerance in
groundnut. Field Crops Research, 80, 63–77.
Dane, F., Hunter, A. G., & Chambliss, O. L. (1991). Fruit set, pollen fertility,
and combining ability of selected tomato genotypes under high tem-
perature field conditions. Journal of the American Society for
Horticultural Science, 116, 906–991.
De Jong, S. M., Pebesma, E. J., & Lacaze, B. (2003). Above‐ground biomass
assessment of Mediterranean forests using airborne imaging spectrom-
etry: The DAIS Peyne experiment. International Journal of Remote
Sensing, 24, 1505–1520.
Dhanapal, A. P., Ray, J. D., Singh, S., Hoyos‐Villegas, V., Smith, J. R., Purcell,
L. C., … Fritschi, F. B. (2015). Association mapping of total leaf caroten-
oids in diverse soybean [Glycine max (L.) Merr.] genotypes based on leaf
extracts and canopy spectral reflectance. PLoS One, 10, e137213.
Dhanapal, A. P., Ray, J. D., Singh, S. K., Hoyos‐Villegas, V., Smith, J. R., Pur-
cell, L. C., & Fritschi, F. B. (2016). Genome‐wide association mapping of
soybean chlorophyll traits based on canopy spectral reflectance and
leaf extracts. BMC Plant Biology, 16, 174.
Djanaguiraman, M., Boyle, D. L., Welti, R., Jagadish, S. V. K., & Prasad, P. V.
V. (2018). Decreased photosynthetic rate under high temperature in
wheat is due to lipid desaturation, oxidation, acylation, and damage
of organelles. BMC Plant Biology, 18, 55.
Djanaguiraman, M., Perumal, R., Ciampitti, I. A., Gupta, S. K., & Prasad, P. V.
V. (2018). Quantifying pearl millet response to high temperature stress:
Thresholds, sensitive stages, genetic variability and relative sensitivity
of pollen and pistil. Plant, Cell & Environment, 41, 993–1007.
Djanaguiraman, M., Perumal, R., Jagadish, S. V. K., Ciampitti, I. A., Welti, R.,
& Prasad, P. V. V. (2018). Sensitivity of sorghum pollen and pistil to
high‐temperature stress. Plant, Cell & Environment, 41, 1065–1082.
Djanaguiraman, M., Prasad, P. V. V., Boyle, D. L., & Schapaugh, W. T.
(2011). High‐temperature stress and soybean leaves: Leaf anatomy
and photosynthesis. Crop Science, 51, 2125–2131.
Djanaguiraman, M., Prasad, P. V. V., Boyle, D. L., & Schapaugh, W. T.
(2013). Soybean pollen anatomy, viability and pod set under high tem-
perature stress. Journal of Agronomy and Crop Science, 199, 171–177.
Djanaguiraman, M., Prasad, P. V. V., Murugan, M., Perumal, M., & Reddy, U.
K. (2014). Physiological differences among sorghum (Sorghum bicolor L.
Moench) genotypes under high temperature stress. Environmental and
Experimental Botany, 100, 43–54.
Djanaguiraman, M., Prasad, P. V. V., & Schapaugh, W. T. (2013). High day‐
or night‐time temperature alters leaf assimilation, reproductive
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
DJANAGUIRAMAN ET AL.
success, and phosphatidic acid of pollen grain in soybean (Glycine max
(L.) Merr.). Crop Science, 53, 1594–1604.
Egli, D. B. (1988). Alterations in plant growth and dry matter distribution in
soybean. Agronomy Journal, 80, 86–90.
Egli, D. B., & Yu, Z. W. (1991). Crop growth rate and seeds per unit area in
soybean. Crop Science, 31, 439–442.
Fehr, W. R., & Caviness, C. E. (1977). Stages of soybean development. Coop-
erative Extension Service: Agriculture and Home Economics
Experiment Station Iowa State University, Ames, Iowa.
Feng, B., Liu, P., Li, G., Dong, S. T., Wang, F. H., Kong, L. A., & Zhan, J. W.
(2014). Effect of heat stress on the photosynthetic characteristics in
flag leaves at the grain‐filling stage of different heat‐resistant winter
wheat varieties. Journal of Agronomy and Crop Science, 200, 143–155.
Gibson, L. R., & Mullen, R. E. (1996). Influence of day and night tempera-
ture on soybean seed yield. Crop Science, 36, 98–104.
Hatfield, J. L., Boote, K. J., Kimball, B. A., Ziska, L. H., Izaurralde, R. C., Ort,
D., … Wolfe, D. W. (2011). Climate impacts on agriculture: Implications
for crop production. Agronomy Journal, 103, 351–370.
Hatfield, J. L., & Prueger, J. H. (2015). Temperature extremes: Effect on
plant growth and development. Weather and Climate Extremes, 10,
4–10.
Herritt, M., Dhanapal, A. P., & Fritschi, F. B. (2016). Identification of geno-
mic loci associated with the photochemical reflectance index by
genome‐wide association analysis in soybean. Plant Genome, 9, 1–12.
IPCC (2014). Climate change 2014: Synthesis report. Contribution of
working groups I, II and III to the fifth assessment report of the inter-
governmental panel on climate change. In Core writing team CW, R.
K. Pachauri, & L. A. Meyer (Eds.). (p. 151). Geneva, Switzerland: IPCC.
Jiang, H., & Egli, D. B. (1993). Shade induced changes in flower and pod
number and flower and fruit abscission in soybean. Agronomy Journal,
85, 221–225.
Jin, J., Liu, X., Wang, G., Mi, L., Shen, Z., Chen, X., & Herbert, S. J. (2010).
Agronomic and physiological contributions to the yield improvement
of soybean cultivars released from 1950 to 2006 in Northeast China.
Field Crops Research, 115, 116–123.
Jumrani, K., Bhatia, V. S., & Pandey, G. P. (2018). Screening soybean geno-
types for high temperature tolerance by in vitro pollen germination,
pollen tube length, reproductive efficiency and seed yield. Indian Jour-
nal of Plant Physiology, 23, 77–90.
Kakani, V. G., Prasad, P. V. V., Craufurd, P. Q., & Wheeler, T. R. (2002).
Response of in vitro pollen germination and pollen tube growth of
groundnut (Arachis hypogaea L.) genotypes to temperature. Plant, Cell
& Environment, 25, 1651–1661.
Kakani, V. G., Reddy, K. R., Koti, S., Wallace, T. P., Prasad, P. V. V., Reddy, V.
R., & Zhao, D. (2005). Differences in in vitro pollen germination and
pollen tube growth of cotton cultivars in response to high temperature.
Annals of Botany, 96, 59–67.
Knipling, E. B. (1970). Physical and physiological basis for the reflectance of
visible and near infrared radiation from vegetation. Remote Sensing of
Environment, 1, 155–159.
Koester, R. P., Skoneczka, J. A., Cary, T. R., Diers, B. W., & Ainsworth, E. A.
(2014). Historical gains in soybean (Glycine max Merr.) seed yield are
driven by linear increases in light interception, energy conversion, and
partitioning efficiencies. Journal of Experimental Botany, 65,
3311–3321.
Krause, G. H., & Weis, E. (1984). Chlorophyll fluorescence as a tool in plant
physiology. II. Interpretation of fluorescence signals. Photosynthesis
Research, 5, 139–157.
Kumar, S., Thakur, P., Kaushal, N., Malik, J. A., Gaur, P., & Nayyar, H. (2013).
Effect of varying high temperatures during reproductive growth on
reproductive function, oxidative stress and seed yield in chickpea
genotypes differing in heat sensitivity. Archives of Agronomy and Soil
Science, 59, 823–843.

DJANAGUIRAMAN ET AL.
Kurosaki, H., & Yumoto, S. (2003). Effects of low temperature and shading
during flowering on the yield components in soybeans. Plant Production
Science, 6, 17–23.
Li, M. W., Xin, D., Gao, Y., Li, K. P., Fan, K., Munoz, N. B., … Lam, H. M.
(2017). Using genomic information to improve soybean adaptability
to climate change. Journal of Experimental Botany, 68, 1823–1834.
Lin, W. S., Yang, C. M., & Kuo, B. J. (2012). Classifying cultivars of rice
(Oryza sativa L.) based on corrected canopy reflectance spectra data
using the orthogonal projections of latent structures (O‐PLS) method.
Chemometrics and Intelligent Laboratory Systems, 115, 25–36.
Liu, F., Jensen, C. R., & Andersen, M. N. (2004). Pod set related to photo-
synthetic rate and endogenous ABA in soybeans subjected to
different water regimes and exogenous ABA and BA at early reproduc-
tive stages. Annals of Botany, 94, 405–411.
Lobell, D. B., & Field, C. B. (2007). Global scale climate‐crop yield relation-
ships and the impact of recent warming. Environmental Research Letters,
2, 1–7.
Lobos, G. A., & Hancock, J. F. (2015). Breeding blueberries for a changing
global environment: A review. Frontiers in Plant Science, 6, 782.
Mohanty, M., Sinha, N. K., Lenka, S., Hati, K. M., Somasundaram, J., Saha,
R., … Rao, A. S. (2015). Climate change impacts on rainfed soybean
yield of central India: Management strategies through simulation
modelling. In A. Singh, J. Dagar, A. Arunachalam, & K. Shelat (Eds.), Cli-
mate change modelling, planning and policy for agriculture. New Delhi:
Springer.
Morrison, M. J., Voldeng, H. D., & Cober, E. R. (1999). Physiological
changes from 58 years of genetic improvement of short‐season soy-
bean cultivars in Canada. Agronomy Journal, 91, 685–689.
Mourtzinis, S., Specht, J. E., Lindsey, L. E., Wiebold, W. J., Ross, J., Nafziger,
E. D., & Conley, S. P. (2015). Climate‐induced reduction in US‐wide
soybean yields underpinned by region‐ and in‐season‐specific
responses. Nature Plants, 1, 1–4.
Narayanan, S., Prasad, P. V. V., Fritz, A. K., Boyle, D. L., & Gill, B. S. (2015).
Impact of high night‐time and high daytime temperature stress on win-
ter wheat. Journal of Agronomy and Crop Science, 201, 206–218.
Nguyen, C. T., Singh, V., van Oosterom, E. J., Chapman, S. C., Jordan, D. R.,
& Hammer, G. L. (2013). Genetic variability in high temperature effects
on seed‐set in sorghum. Functional Plant Biology, 40, 439–448.
Ollinger, S. V. (2011). Sources of variability in canopy reflectance and the
convergent properties of plants. New Phytologist, 189, 375–394.
Parent, B., & Tardieu, F. (2012). Temperature responses of developmental
processes have not been affected by breeding in different ecological
areas for 17 crop species. New Phytologist, 94, 760–774.
Paupiere, M. J., van Haperen, P., Rieu, I., Visser, R. G. F., Tikunov, Y. M., &
Bovy, A. G. (2017). Screening for pollen tolerance to high temperatures
in tomato. Euphytica, 213, 130.
Penalba, O. C., Bettolli, M. L., & Vargas, W. M. (2007). The impact of cli-
mate variability on soybean yields in Argentina. Multivariate
regression. Meteorological Applications, 14, 3–14.
Peng, Y., Zeng, A., Zhu, T., Fang, S., Gong, Y., Tao, Y., … Liu, K. (2017). Using
remotely sensed spectral reflectance to indicate leaf photosynthetic
efficiency derived from active fluorescence measurements. Journal of
Applied Remote Sensing, 11, 026034. https://doi.org/10.1117/1.
JRS.11.026034
Penuelas, J., & Filella, I. (1998). Visible and near‐infrared reflectance tech-
niques for diagnosing plant physiological status. Trends in Plant Science,
3, 151–156.
Porch, T. G., & Jahn, M. (2001). Effects of high temperature stress on
microsporogenesis in heat sensitive and heat tolerant genotypes of
Phaseolus vulgaris. Plant, Cell & Environment, 24, 723–731.
Prasad, P. V. V., Bheemanahalli, R., & Jagadish, S. V. K. (2017). Field crops
and the fear of heat stress—Opportunities, challenges and future direc-
tions. Field Crops Research, 200, 114–121.
Prasad, P. V. V., Boote, K. J., & Allen, L. H. Jr. (2006). Adverse high temper-
ature effects on pollen viability, seed‐set, seed yield and harvest index
13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
335
of grain‐sorghum (Sorghum bicolor L. Moench) are more severe at ele-
vated carbon dioxide due to higher tissue temperatures. Agricultural
and Forest Meteorology, 139, 237–251.
Prasad, P. V. V., Boote, K. J., Allen, L. H., & Thomas, J. M. G. (2002).
Effects of elevated temperature and carbon dioxide on seed‐set
and yield of kidney bean (Phaseolus vulgaris L.). Global Change Biol-
ogy, 8, 710–721.
Prasad, P. V. V., Craufurd, P. Q., & Summerfield, R. J. (1999). Fruit number
in relation to pollen production and viability in groundnut exposed to
short episodes of heat stress. Annals of Botany, 84, 381–386.
Prasad, P. V. V., & Djanaguiraman, M. (2011). High night temperature
decreases leaf photosynthesis and pollen function in grain sorghum.
Functional Plant Biology, 38, 993–1003.
Ristic, Z., Bukovnik, U., & Prasad, P. V. V. (2007). Correlation between heat
stability of thylakoid membranes and loss of chlorophyll in winter
wheat under heat stress. Crop Science, 47, 2067–2073.
Rodriguez‐Garay, B., & Barrow, J. R. (1988). Pollen selection for heat toler-
ance in cotton. Crop Science, 28, 857–859.
Sakoda, K., Tanaka, Y., Long, S. P., & Shiraiwa, T. (2016). Genetic and phys-
iological diversity in the leaf photosynthetic capacity of soybean. Crop
Science, 56, 2731–2741.
Salem, M. A., Kakani, V. G., Koti, S., & Reddy, K. R. (2007). Pollen‐based
screening of soybean genotypes for high temperature. Crop Science,
47, 219–231.
SAS Institute (2008). The SAS users guide, Version 9.3. NC: SAS Inst, Cary.
Schauberger, B., Archontoulis, S., Arneth, A., Balkovic, J., Ciais, P., Deryng,
D., … Frieler, K. (2017). Consistent negative response of US crops to
high temperatures in observations and crop models. Nature Communi-
cations, 8, 13931.
Schlenker, W., & Roberts, M. J. (2009). Nonlinear temperature effects indi-
cate severe damages to U.S. crop yields under climate change.
Proceedings of the National Academy of Sciences of the United States of
America, 106, 15594–15598.
Seddigh, M., & Jolliff, G. D. (1984). Night temperature effects on morphol-
ogy, phenology, yield and yield components of indeterminate field‐
grown soybean. Agronomy Journal, 76, 824–828.
Shi, W., Li, X., Schmidt, R. C., Struik, P. C., Yin, X., & Jagadish, S. V. K.
(2018). Pollen germination and in‐vivo fertilization in response to high
temperature during flowering in hybrid and inbred rice. Plant, Cell &
Environment, 1–11. https://doi.org/10.1111/pce.13146
Singh, V., Nguyen, C. T., van Oosterom, E. J., Chapman, S. C., Jordan, D. R.,
& Hammer, G. L. (2015). Sorghum genotypes differ in high temperature
responses for seed set. Field Crops Research, 171, 32–40.
Singh, V., Nguyen, C. T., Yang, Z., Chapman, S. C., van Oosterom, E. J., &
Hammer, G. L. (2016). Genotypic differences in effects of short epi-
sodes of high‐temperature stress during reproductive development in
sorghum. Crop Science, 56, 1561–1572.
Sita, K., Sehgal, A., Hanumantha, R. B., Nair, R. M., Prasad, P. V. V.,
Kumar, S., … Nayyar, H. (2017). Food legumes and rising tempera-
tures: Effects, adaptive functional mechanisms specific to
reproductive growth stage and strategies to improve heat tolerance.
Frontiers in Plant Science, 8, 1658. https://doi.org/10.3389/
fpls.2017.01658
Slaton, M. R., Hunt, E. R. Jr., & Smith, W. K. (2001). Estimating near‐infra-
red leaf reflectance from leaf structural characteristics. American
Journal of Botany, 88, 278–284.
Smith, J. R., Mengistu, A., Nelson, R. L., & Paris, R. L. (2008). Identification
of soybean accessions with high germinability in high‐temperature
environments. Crop Science, 48, 2279–2288.
Tewari, A. K., & Tripathy, B. C. (1999). Acclimation of chlorophyll biosyn-
thetic reactions to temperature stress in cucumber (Cucumis sativus
L.). Planta, 208, 431–437.
Urban, J., Ingwers, M. W., McGuire, M. A., & Teskey, R. O. (2017). Increase
in leaf temperature opens stomata and decouples net photosynthesis
 13653040, 2019, 1, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/pce.13421 by Nat Prov Indonesia, Wiley Online Library on [23/03/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
336 DJANAGUIRAMAN ET AL.

from stomatal conductance in Pinus taeda and Populus deltoides x nigra. four independent estimates. Proceedings of the National Academy of Sci-
Journal of Experimental Botany, 68, 1757–1767. ences of the United States of America, 114, 9326–9331.
Wiebbecke, C. F., Graham, M. A., Cianzio, S. R., & Palmer, R. G. (2012). Day
temperature influences the male‐sterile locus ms9 in soybean. Crop Sci- SUPPORTI NG INFORMATION
ence, 52, 1503–1510.
Additional supporting information may be found online in the
Yao, X., Zhu, Y., Tian, Y. C., Feng, W., & Cao, W. X. (2010). Exploring
hyperspectral bands and estimation indices for leaf nitrogen accumula- Supporting Information section at the end of the article.
tion in wheat. Journal of Applied Earth Observation and Geoinformation,
12, 89–100.
Yin, X., Kropff, M. J., McLaren, G., & Visperas, R. M. (1995). A nonlinear
model for crop development as a function of temperature. Agriculture How to cite this article: Djanaguiraman M, Schapaugh W,
and Forest Meteorology, 77, 1–16. Fritschi F, Nguyen H, Prasad PVV. Reproductive success of
Zhang, L., Zhu, L., Yu, M., & Zhong, M. (2016). Warming decreases photo- soybean (Glycine max L. Merril) cultivars and exotic lines under
synthates and yield of soybean [Glycine max (L.) Merrill] in the North
high daytime temperature. Plant Cell Environ. 2019;42:
China Plain. The Crop Journal, 4, 139–146.
321–336. https://doi.org/10.1111/pce.13421
Zhao, C., Liu, B., Piao, S., Wang, X., Lobell, D. B., Huang, Y., … Asseng, S.
(2017). Temperature increase reduces global yields of major crops in