Published March 20, 2015
Research
Popular Rice (Oryza sativa L.) Cultivars
Show Contrasting Responses to Heat Stress
at Gametogenesis and Anthesis
W. Shi, T. Ishimaru, R. B. Gannaban, W. Oane, and S. V. K. Jagadish*
Abstract
Studies on heat tolerance in rice (Oryza sativa L.)
have focused on a range of accessions that are
currently not grown or cultivated in small local-
ized geographic regions, while popular rice cul-
tivars occupying millions of hectares have not
been tested for their response to heat stress.
Hence, 18 popular rice cultivars grown across
tropical and subtropical regions were subjected
to 38°C for 6 h of heat stress at anthesis and
five selected contrasting entries were exposed
to six consecutive days (6 h each day) of stress
coinciding with both gametogenesis and anthe-
sis. Among the tested entries, Ciherang was
the most tolerant, after both 6 h at anthesis and
6 d of stress across both gametogenesis and
anthesis, whereas Moroberekan and KDML-105
were the most susceptible. Using a quick and
easy phenotyping protocol and by considering
all the sterile spikelets across target panicles,
the crucial physiological window “spikelets with
exserted anthers but with no ovule enlargement”
was identified as the most sensitive stage with
exposure to heat stress at both critical repro-
ductive stages. Geographic origin of rice variet-
ies was not clearly related to the degree of tol-
erance or susceptibility to heat stress because
both conditions were observed in cultivars from
each target region. Our findings complement
other ongoing efforts that are using heat-toler-
ant landraces and wild rice as donors to further
increase tolerance to sustain global rice pro-
duction under future warmer climate.
crop science, vol. 55, march– april 2015  www.crops.org
International Rice Research Institute, DAPO Box 7777, Metro
Manila, Philippines. Received 21 Jan. 2014. *Corresponding author
(k.jagadish@irri.org).
Abbreviations: IRRI, International Rice Research Institute; RH,
relative humidity.
I n response to the predicted increase in global temperatures by
up to 2°C between 2046 and 2065 (IPCC, 2013), a sustained
option envisaged for safeguarding global food security is through
the development of heat-tolerant cultivars (Battisti and Naylor,
2009). Rice is highly vulnerable to heat spikes coinciding with
critical developmental stages such as gametogenesis and flowering
(Yoshida et al., 1981; Prasad et al., 2006; Jagadish et al., 2010b,
2011). Hence, predicted increases in frequency and magnitude of
heat spikes under future climate will lead to significant reduction
in rice yield. The vulnerability of the major rice-producing regions
to these phenomena has been mapped both regionally (Wassmann
et al., 2009) and at the global scale (Teixeira et al., 2013).
A series of phenotyping studies have quantified heat toler-
ance among indica and/or japonica rice accessions (Yoshida et
al., 1981; Matsui et al., 2001; Prasad et al., 2006; Jagadish et al.,
2008). From the above studies, the most heat-sensitive develop-
mental stages identified are gametogenesis and flowering. Heat
stress during gametogenesis reduces pollen production (Prasad
et al., 2006) and pollen development and viability ( Jagadish et
al., 2013). Poor anther dehiscence, significant reduction in pollen
count, poor pollen germination on the stigma, and restricted
pollen tube growth rate are characteristic impacts of heat stress
coinciding with anthesis (Satake and Yoshida, 1978; Jagadish et
Published in Crop Sci. 55:589–596 (2015).
doi: 10.2135/cropsci2014.01.0054
© Crop Science Society of America | 5585 Guilford Rd., Madison, WI 53711 USA
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589
al., 2010b). In rice, it takes just 30 to 80 min from the
time of flower opening to closing, and within this inter-
val anther dehiscence, pollination, pollen germination,
and pollen tube reaching the ovary are completed, with
the whole process including fertilization lasting about
1.5 h to a maximum of 4 h (Cho 1956; Wassmann et al.,
2009). Heat stress alters normal reproductive processes
highlighted above, resulting in increased spikelet sterility.
Evidence that heat stress does not affect the female repro-
ductive organ negatively has been obtained using cross-
pollination techniques (Yoshida et al., 1981).
The majority of the entries tested in the previously
studies are either not grown currently; if they are, it is in
relatively narrow geographic limits. Landraces are locally
grown rice accessions that have acquired over time greater
adaptation to environments they inhabit. Significant
efforts are being made to identify landraces and wild rice
progenies possessing higher tolerance or escaping char-
acteristics in response to heat stress during the sensitive
reproductive stage, with an aim to actively use them in
rice breeding programs ( Jagadish et al., 2010a; Ishimaru
et al., 2010; Ye et al., 2012). In spite of intensified efforts
and the increasing importance of overcoming the negative
impact of heat stress on rice, no attempt has been made
to systematically phenotype popular rice cultivars. More-
over, the predicted increase in temperature would have a
greater negative impact on rice yields across vulnerable
tropical and subtropical regions where temperatures are
already close to critical thresholds (Prasad et al., 2006;
Wassmann et al., 2009). Hence, cultivars that are well
adapted to major tropical and subtropical rice-growing
regions currently occupying large proportion of cultivated
area were considered as popular cultivars.
The overall objectives of our work were (i) to quan-
tify the responses of popular rice cultivars to heat stress at
two sensitive stages (i.e., gametogenesis and anthesis) and
(ii) to ascertain the applicability of a phenotyping pro-
tocol used to determine the impact of drought stress at
flowering (Oane and Bennett, 2009) as an easy and quick
way to identify the most heat-susceptible physiological
window during flowering.
Materials and Methods
The experiments were conducted during 2012 at the Interna-
tional Rice Research Institute (IRRI), Los Baños (14°11¢N,
121°15¢E, 21 m), Philippines. Seeds of representative set of
cultivars grown across more vulnerable rice-growing regions
of South and Southeast Asia, Latin America, and West Africa,
which account for >95% of global rice-growing area, were col-
lected. In addition, representative popular cultivars were chosen
based on location-specific breeder’s nominations as having high
probability of exposure to high temperatures under current cli-
mate or with a potential to be affected by stress exposure in
the near future. The selected popular cultivars were tested for
heat tolerance at two sensitive stages (i.e., gametogenesis and
590 www.crops.org
anthesis). Seeds for both the experiments were obtained from the
IRRI Genetic Resources Center (Table 1). Plants were grown
in the greenhouse in a natural condition with average daily tem-
perature of 30.8°C (SD ± 2.6°C) and relative humidity (RH)
of 71.6% (SD ± 7.4%) till they reached the targeted stage, after
which they were moved into the controlled-environment walk-
in facility to impose heat stress. At IRRI sun rises and sets at
0630 and 1830 h, respectively, during the growth period, expos-
ing plants grown in the greenhouse to 12 h of photoperiod.
Crop Husbandry
Experiment 1. Eighteen locally grown cultivars from South-
east Asia, South Asia, West Africa, and Latin America were
selected for high-temperature tolerance phenotyping. All the
entries tested were O. sativa indica grown widely across tropical
and subtropical rice-growing regions, except N22 (aus), a heat-
tolerant check, and Moroberekan and KDML-105 representing
japonica subspecies and indica cultivar from higher altitudes,
respectively (see Table 1 for additional details). Seed dormancy
was broken by exposing them to 50°C for 3 d, followed by
pregermination and sowing in seeding trays. Two 21-d-old
seedlings were transplanted into plastic pots (25 cm in height;
26 and 20 cm in diameter at the top and bottom, respectively)
filled with 5.0 kg of the same clay loam soil used for seedling
establishment with 2.0 g ammonium sulfate [(NH4)2SO4], 1.0
g single superphosphate, and 1.0 g muriate of potash (KCl). An
additional 2.5 g of (NH4)2SO4 was top dressed at 25 to 30 d
after transplanting. Plants were grown under fully flooded con-
ditions throughout the experiment, even when the plants were
moved in and out of the chambers. No pest or disease incidence
was observed throughout the experiments.
Experiment 2. Five cultivars based on two criteria—(i)
contrasting (tolerant or susceptible) for spikelet fertility with
heat stress exposure at anthesis and (ii) a representative toler-
ant entry from each region of interest—were selected from
the outcome of Experiment 1 to reconfirm their tolerance by
comparing them against a common susceptible entry. In addi-
tion, the impact at the second most heat-sensitive stage, that is,
gametogenesis (Yoshida et al., 1981; Jagadish et al., 2013), was
also quantified across all five selected cultivars. All other crop
management practices, including seeding, timing, and amount
of fertilizer application and other crop husbandry measures,
were similar to those in Experiment 1.
High-temperature Treatment
Experiment 1. To impose stress, plants at anthesis were moved
into walk-in growth chambers (3.3 × 3.2 × 2.7 m; 10.6 m 2
area). Each chamber was fitted with six independent units of 1
kW high-intensity discharge lamps, providing photosynthetic
photon flux density of ³650 µmol m-2 s-1 at plant height for
11 h and 215 µmol m-2 s-1 for 2 h during temperature change
period (1730–1830 h and 0530–0630 h) providing a photope-
riod of 13 h d -1. At least three replicate pots (six plants) were
moved into the chambers when at least three to four tillers of
each plant started to flower to increase the number of anthesing
spikelets exposed to stress. The height adjustment approach was
employed by placing pots on benches with different heights to
ensure that panicles of all 18 cultivars were about 130 to 150 cm
from the ground surface. The lights in the chambers were about
crop science, vol. 55, march– april 2015
Table 1. Spikelet fertility (SF) of 18 popular rice cultivars exposed to high temperature for a day (6 h; 0900–1500 h) during anthe-
sis. Values presented are means ± SE.
Total spikelets
SF (%)‡ observed§
Cultivated Major cultivating Ecological Control Heat Control Heat
Cultivar † region countries group (30°C) (38°C)¶ (30°C) (38°C)
Tolerant
Ciherang Southeast Asia Indonesia Oryza sativa indica 93.6 ± 1.7 92.1 ± 1.5a 171 (6) 283 (9)
Samba Mahsuri South Asia India O. sativa indica 96.1 ± 2.6 88.1 ± 2.5a 275 (7) 444 (15)
BG90-2 West Africa Mali, Gambia, Ethiopia, Nigeria O. sativa indica 99.2 ± 0.8 87.2 ± 4.2ab 118 (5) 134 (7)
ADT36 South Asia India O. sativa indica 97.3 ± 1.1 84.4 ± 5.8ab 258 (8) 283 (12)
N22 South Asia India O. sativa aus 94.9 ± 1.8 84.4 ± 5.6abc 57 (4) 44 (3)
Epagri 108 Latin America South of Brazil and Venezuela. O. sativa indica 93.8 ± 2.7 83.6 ± 3.5ab 121 (6) 214 (7)
BR-IRGA 410 Latin America Southern Brazil O. sativa indica 96.0 ± 1.5 82.3 ± 2.9ab 106 (5) 551 (23)
Swarna South Asia India, Bangladesh, Nepal O. sativa indica 98.8 ± 0.8 82.1 ± 2.9abc 198 (9) 257 (8)
NSIC Rc222 Southeast Asia Philippines O. sativa indica 95.1 ± 2.1 80.6 ± 6.2abcd 122 (5) 203 (6)
Moderately tolerant
MTU1010 South Asia India O. sativa indica 95.2 ± 1.9 68.9 ± 3.1bcd 155 (6) 833 (28)
Sahel 134 West Africa Senegal, Mauritania O. sativa indica 86.9 ± 4.1 57.3 ± 4.2d 273 (10) 591 (22)
Fedearoz 50 Latin America Colombia, Venezuela, Costa Interspecific indica 92.9 ± 1.8 56.3 ± 6.3d 212 (7) 472 (13)
Rica, Panama × japonica
Bouake 189 West Africa Cote d’Ivoire, Guinea Conakry O. sativa indica 90.2 ± 2.9 52.4 ± 6.0cde 115 (6) 160 (6)
IR64 South and Bhutan, Burkina Faso (FKR42), O. sativa indica 96.6 ± 1.5 51.3 ± 8.2d 100 (5) 166 (7)
Southeast Asia Cambodia, Ecuador (INIAP11),
Gambia, India, Indonesia,
Mauritania, Philippines,
Vietnam (OM89)#
Susceptible
Sahel 329 West Africa Senegal, Mauritania O. sativa indica 85.2 ± 1.2 22.7 ± 3.5ef 231 (7) 595 (22)
Nerica L-19 West Africa Nigeria, Mali, Burkina Faso, O. sativa indica 78.4 ± 5.8 25.7 ± 4.7f 164 (6) 734 (18)
Liberia, Sierra Leone,
Cameroon, Togo
KDML-105 Southeast Asia Thailand O. sativa indica 92.0 ± 2.3 13.8 ± 3.0f 217 (8) 123 (6)
Moroberekan West Africa Cote d’Ivoire O. sativa japonica 92.7 ± 1.2 9.8 ± 4.8f 319 (6) 297 (9)
†
Cultivar abbreviations ADT(Aduthurai)36, N(Nagina)22, NSIC (National Seed Industry Council) Rc 222, MTU (Marateru)1010, KDML (Khao Dawk Mali)-105.
‡
Tolerant (SF > 80%), moderately tolerant (SF > 50% to £ 80%), susceptible (£50%). Cultivar classification based on spikelet fertility percent obtained with stress exposure
at anthesis.
§
Numbers in the parenthesis indicate the numbers of panicles used for determining spikelet fertility across control and heat stress treatments.
¶
Letters following the values indicate results from the Tukey’s test.
#
Alternate country names given to IR64.

1 m from the panicles during anthesis. Hence, additional heat
load from the lights or differences in plant height would have
had minimum interference with our results. Spikelets were
exposed to high temperature (38°C) from 0900 to 1500 h (6
h, short-duration stress), gradually reduced to 24°C by 1830 h
and maintained throughout the night till 0530 h, with a diur-
nally constant RH of 70%. Simultaneously, two replicate pots
(four plants) were moved into a control growth chamber main-
tained at day/night temperature of 30/24°C and RH of 70%.
Growth chambers were replicated for most parts of the stress
exposure period but not for the control treatment. A digital
temperature sensor placed above the canopy in the middle of
the growth chamber recorded ambient air temperature and RH
to be consistently maintained at the set target. Spikelets that
flowered during the treatment hours in the growth chamber
were marked with a fine-tipped pen (0.5-mm diameter, Zebra
Co. Ltd.). The marking of spikelets was undertaken immedi-
ately after the treatment ended to ensure that marked spikelets
were completely closed, to avoid any manual interference with
spikelet fertility. After the 6 h of temperature treatment, pots
were returned to the greenhouse and plants were grown under
optimum conditions till maturity.
Experiment 2. For the five selected cultivars, two indepen-
dent sets of 10 pots each were transferred into growth chambers
to impose high-temperature treatment when the main tiller
of plants reached gametogenesis, determined by employing
an established phenotypic marker ( Jagadish et al., 2013), and
at anthesis. Briefly, identifying the right morphological stage
based on phenotypic marker (-8 to -9 cm intercollar distance
between the last fully opened leaf and the yet-to-emerge flag
leaf ) and imposing six consecutive days of heat stress ensures
that > 95% of the spikelets in a panicle are exposed to stress
during gametogenesis. The target temperature settings (heat
stress and control, day and night), photoperiod, and RH were
the same as in Experiment 1. High temperature of 38°C (actual
= 37.6°C, SD ± 0.6°C) with RH of 70% (actual = 63.7%, SD ±
5.9%) and control at 30°C (actual = 30.7°C, SD ± 0.5°C) with
70% RH (actual = 68.8%, SD ± 2.6%) were maintained. Unlike
the setup in Experiment 1, plants were continuously kept in the
walk-in chambers for six consecutive days of heat treatment

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Figure 1. Sterile spikelets at different stages of spikelet development: (A) stage 1: immature spikelets; (B) stage 2: spikelets with anthers
stuck in the middle without filament elongation; (C) stage 3: spikelets with elongated filaments, undehisced anthers just below unopened
lemma and palea; (D) stage 4: spikelets with exserted anthers but no ovule enlargement; (E) stage 5: spikelets with enlarged ovaries but
not filled with starch; and (F) stage 6, spikelets with incomplete grain filling. All photos taken using Olympus SZX-7 stereomicroscope-
magnification at ×25. (Figure modified with permission from International Rice Research Institute.)

(long-duration stress) to expose all the spikelets on the target

(main tiller) panicle to stress during both gametogenesis and
anthesis. Spikelets exposed to heat stress at gametogenesis or
during anthesis do not develop normally and undergo irrevers-
ible damage to developmental progress at different physiological
windows (Fig. 1), resulting in reduced spikelet fertility. The
aim of this experiment was to identify the key physiological
window affected among the selected contrasting rice cultivars
from Experiment 1. A maximum of five unopened spikelets
from plants exposed to heat stress at anthesis, were removed
manually from the base of few panicles at the end of the 6 d of
treatment and the plants were moved back to the greenhouse
and maintained under optimum conditions till maturity. The
removed spikelets were not used for any analysis. Figure 2. Micro-thermocouples pierced into the soft leaf sheath
just above the un-emerged panicle during gametogenesis (A; in-
Observations set, closer view) and placed between individual spikelet’s lemma
Tissue Temperature and palea at anthesis (B).
Panicle temperatures of the five selected cultivars at gameto-
genesis and spikelet temperatures at anthesis were measured in Spikelet Fertility and Sensitive
Experiment 2 using at least three 0.2-mm-diameter Type T Stage Characterization
copper constantan thermocouples (RS Components Corpora- In Experiment 1, the marked spikelets were collected at maturity,
tion) in each chamber. Tissue temperatures were recorded every and filled (fully and partially filled) and unfilled spikelets were
10 s and averaged over 5 min using a data logger (CR1000 data counted separately. Finally, spikelet fertility was analyzed follow-
logger, Campbell Scientific, Inc.). These tissue temperatures were ing Jagadish et al. (2007). In Experiment 2, the main tiller panicle
recorded for 6 h (from 0900 to 1500), that is for 1 d of heat treat- from each pot was harvested to determine spikelet fertility. All
ment. Micro thermocouples were carefully pierced into the soft filled spikelets were meticulously picked by hand. To identify
leaf sheath just above the unemerged panicle during gametogen- the stage at which further developmental progress was stopped,
esis and at anthesis were placed inside individual spikelets, within sterile spikelets were categorized into different sensitive stages
the lemma and palea (Fig. 2). Three replicate thermocouples using the drought stress phenotyping protocol used by Oane and
in each chamber were used to measure the sensors consistency, Bennett (2009) (Fig. 1). Spikelet fertility was determined by the
before measuring the panicle and spikelet temperature (Supple- number of fully filled spikelets divided by the total number of
mentary Table 1). spikelets on the panicle, including both filled and sterile spikelets.

592 www.crops.org crop science, vol. 55, march– april 2015

Statistical Analysis Table 2. Spikelet fertility (SF) of representative popular rice
Difference of means of spikelet fertility across the 18 acces- cultivars exposed to high temperatures for 6 d (0900–1500 h
sions exposed to heat stress was analyzed by Tukey-Kramer each day) during gametogenesis and anthesis. Values pre-
sented are means ± SE.
test installed in the R program (ver. 2.10.1). Spikelet fertility
from Experiment 2, panicle temperature and spikelet tissue SF (%)†
temperature were analyzed as a complete randomized design Cultivated Control Gametogen- Anthesis
using Genstat (GenStat 14th edition, Rothamsted Experimen- region Cultivar ‡ (30°C) esis (38°C) (38°C)
tal Station, Harpenden, UK), with pot as an experimental unit. Tolerant
Significant difference between spikelet sterility proportions Southeast Asia Ciherang 95.8 ± 0.6 83.8 ± 1.9 82.8 ± 3.4
among six sensitive stages were estimated using the contrasts Moderately tolerant
function in ANOVA framework in Genstat. South Asia ADT36 89.3 ± 1.8 82.8 ± 1.9 76.4 ± 3.0
West Africa BG90-2 86.3 ± 2.0 75.6 ± 4.3 76.4 ± 5.0
Latin America Epagri 108 83.6 ± 1.0 31.1 ± 4.7 52.1 ± 6.2
Results Susceptible
Spikelet Fertility and Tissue Temperature Southeast Asia KDML-105 78.3 ± 3.4 60.9 ± 4.3 20.4 ± 5.7
All 18 cultivars completed their daily flowering cycle LSD at 5%§

within this 6-h stress exposure period, with an average Genotype (G) (5.9)*** (7.3)***

peak of flowering at 1030 to 1100 h. Rice cultivars were Treatment (T) (3.7)*** (4.6)***

divided into three distinct heat-response groups based on G×T (8.3)*** (10.3)***
their statistical significance and percent spikelet fertility *** Significant at 0.1%.
recorded with heat stress exposure during anthesis: tolerant †
Tolerant (SF > 80%), moderately tolerant (SF > 50 to £ 80%), susceptible ( £ 50%).
‡
Cultivar classification based on spikelet fertility percent obtained with stress expo-
(with spikelet fertility > 80%), moderately tolerant (> 50% sure at anthesis.
to £ 80%) and susceptible (£ 50%). Nine out of 18 cultivars §
Numbers in parentheses are least significant difference (LSD) of means.
tested were categorized as tolerant, with four susceptible
entries after 6 h of heat stress exposure. Ciherang, with
>90% spikelet fertility, was the most tolerant, and Moro-
berekan and KDML-105, with <15% fertility, were the
most sensitive ones (Table 1). Four cultivars from South
Asia and two from Southeast Asia were categorized as tol-
erant, whereas, from West Africa, three among the tested
six were susceptible. With extended stress duration of 6 d,
a similar trend in fertility was observed but with a higher
decline in fertility even with the tolerant entries (Table 2).
Among the five cultivars, Ciherang maintained its high tol-
erance at anthesis and a similar response was documented at
gametogenesis, followed by ADT36 and BG-90-2. Epagri
was more sensitive to gametogenesis compared to anthe-
sis while KDML-105 resulted in greater susceptibility at
anthesis (Table 2). Both panicle and spikelet tissue tempera-
tures varied significantly across genotypes and treatment (P
< 0.001), and both these factors interacted significantly (P
< 0.01) compared to their respective ambient temperatures.
On exposure to heat stress, the tissue temperature close to
the unemerged panicle was lower compared to ambient
conditions, except in ADT36 (Fig. 3A), while spikelet tissue
temperatures were higher than the ambient during anthesis
(Fig. 3B). A significant negative relationship (R 2 = 0.48; P
< 0.05) was observed between spikelet tissue temperature
and spikelet fertility (Fig. 4).

Sensitive-stage Characterization
The highest proportion of sterile spikelets across all five Figure 3. Panicle temperature minus ambient temperature at ga-
tested cultivars was mainly due to their sensitivity at stage metogenesis (A); spikelet tissue temperature minus ambient tem-
perature at anthesis (B), exposed to control and heat stress. Bars
4 (anthers exserted with/without dehiscence with no ovary
indicate ±SE.
enlarged) under control conditions, which further increased

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 Table 3. Spikelet sterility categorized across different sen-
sitive stages among five selected rice cultivars at control
(30°C) and heat stress (38°C) exposure for 6 h a day (0900–
1500) extended over six consecutive flowering days during
gametogenesis and anthesis.
Sterile spikelets (%)†
Cultivar Treatment S1 S2 S3 S4 S5 S6
Ciherang Control 20.8 1.7 6.4 41.1 24.7 5.3
Gametogenesis 4.7 0.0 4.2 67.4 13.2 10.5
Anthesis 3.9 0.7 12.9 69.9 9.5 3.3
ADT36 Control 6.7 0.4 8.0 41.9 15.5 27.5
Gametogenesis 0.0 0.3 10.0 56.5 13.6 19.6
Anthesis 1.0 1.7 8.4 74.3 6.7 8.0
BG90-2 Control 4.5 0.0 10.9 75.2 5.9 3.6
Gametogenesis 2.0 0.0 9.0 72.2 9.2 7.7
Anthesis 0.8 0.0 26.0 60.4 8.9 4.0
Figure 4. Increase in spikelet tissue temperature decreases spike- Epagri 108 Control 9.7 1.5 19.0 50.4 7.8 11.6
let fertility. Y = 0.068x + 41.09; R2 = 0.48; n = 10. Gametogenesis 3.3 1.6 8.8 58.5 19.4 8.4
Anthesis 3.7 1.6 16.1 61.7 12.0 5.1
KDML-105 Control 14.9 4.1 17.5 59.1 2.2 2.2
under heat stress exposure across both gametogenesis and Gametogenesis 5.4 2.6 8.2 78.8 4.1 1.0
anthesis (Table 3). Across all four tolerant entries, stage 5 Anthesis 3.2 3.8 12.5 74.6 5.6 0.2
(with enlarged ovary but with no starch filling) was gener- †
“S” indicates stage as illustrated in Fig. 1.
ally the second most sensitive stage, with stress coinciding
with gametogenesis, except for ADT 36. However, with
Table 4. Comparative proportion of spikelet sterility
stress coinciding with anthesis, stage 3 (elongated filaments accounted for by six different sensitive stages (see Fig. 1)
and un-dehisced anthers) was identified as the second most with stage 1 as the reference (A) and across three stages
sensitive after stage 4. Similarly, the sensitive KDML-105 accounting larger proportions of sterility (stages 3, 4, and 5;
was most sensitive to stage 4, followed by stage 3, at both B). “S” indicates stage and “vs” versus. Values in the table
gametogenesis and anthesis. Spikelet sterility percentage are the P values indicating the level of significance.
varied significantly across the different stages (P < 0.001). A B
Significance of contrasts between the stages, with stage S1 vs. S1 vs. S1 vs. S1 vs. S1 vs. S3 vs. S3 vs.
1 as reference resulted in stage 4 as the most consistent, S2 S3 S4 S5 S6 S4 S5
accounting for a greater proportion of sterility at gameto- Ciherang 0.154 0.124 <0.001 0.012 0.338 <0.001 0.317
genesis and anthesis (Table 4). Further, comparing stages BG 90–2 0.670 <0.001 <0.001 0.004 0.156 <0.001 0.060
accounting for larger proportions of sterility (stages 3, 4, Epagri 0.308 <0.001 <0.001 <0.001 0.083 <0.001 0.079
and 5), with stage 3 as reference, stage 4 accounted for the ADT36 0.904 0.023 <0.001 0.012 <0.001 <0.001 0.804
significantly highest spikelet sterility across all the cultivars
KDML-105 0.416 <0.001 <0.001 0.645 0.006 <0.001 <0.001
except KDML-105 (P < 0.001), but not stage 5 (Table 4).

Discussion
Geographic origin of rice varieties was not clearly related
to the degree of tolerance or susceptibility to heat stress
as both conditions were observed in cultivars from each
target region and hence cannot be generalized. A repre-
sentative set of 18 popular cultivars selected had contrast-
ing responses to heat stress during anthesis. Among these
cultivars, an elite indica line from Indonesia, Ciherang,
was highly heat tolerant, recording the highest spikelet
fertility with stress exposure of 1 d (6 h of heat stress)
at anthesis and 6 d (6 h of stress daily for six consecu-
tive flowering days) at both gametogenesis and anthesis.
In addition, a few other cultivars such as Samba Mahsuri,
BG90-2, and ADT36 were categorized as tolerant with
6 h of stress, the latter two resulting in increased suscep-
tibility with extended duration of stress. In comparison
to the heat stress response at anthesis (references in the
Introduction section), genetic response during gameto-
genesis has not been quantified due to the lack of a pheno-
typic marker. Recently a phenotypic marker was identi-
fied ( Jagadish et al., 2013), and through its use, Ciherang
and ADT36 were identified to be tolerant. Interestingly,
Ciherang, like N22, was tolerant at heat-sensitive flow-
ering and gametogenesis stages, a promising cultivar to
be included in ongoing heat tolerance breeding programs.
On the other hand, a number of other popular cultivars
were sensitive to both these stages, indicating the need
for additional efforts in this direction to provide options
to reduce heat-induced rice yield losses across identified
vulnerable regions (Wassmann et al., 2009).

594 www.crops.org crop science, vol. 55, march– april 2015

 Physiological mechanisms of heat stress induced
spikelet sterility and phenotyping protocols have been
standardized under controlled environments using cul-
tivars that are not widely grown under current climatic
conditions (Prasad et al., 2006; Jagadish et al., 2008,
2010b). This is the first report wherein popular rice culti-
vars occupying a significant arable area globally have been
tested for their potential to tolerate heat stress at key sen-
sitive stages—gametogenesis and flowering. The advan-
tage of such an exercise would be to identify farmer- and
consumer-preferred cultivars (for example Ciherang) that
can be recommended for locations where high tempera-
tures are limiting rice production under current climates.
Although 50% of the cultivars tested (9 out of 18) docu-
mented tolerance to short duration heat stress, it was not
sustained for longer durations in a tested subset, indicating
the need for incorporating greater tolerance with antici-
pated increase in temperature under future climate. Alter-
natively, landraces such as the most heat-tolerant donor,
N22 (Yoshida et al., 1981; Jagadish et al., 2008), have been
exploited to identify and introgress interesting genes using
molecular markers to develop heat-tolerant rice cultivars,
which is a relatively longer term strategy. Hence, both the
above approaches have their advantages, with the former
facilitating growing tolerant popular cultivars in areas
with immediate heat stress pressure, while the latter will
allow further upgrading of the tolerance of these rice cul-
tivars to sustain production under future warmer climate.
A lower panicle temperature during gametogenesis
(Fig. 3A; panicle yet to emerge) under high-temperature
exposure could reflect transpiration contributed by sur-
rounding whorl of leaves facilitating reduction in panicle
microclimate temperature. A similar phenomenon of
lower panicle temperature among improved rice cultivars
with panicles surrounded by plant leaf canopy benefitted
by effective transpiration cooling over traditional varieties
with panicle well above the crop canopy has been high-
lighted (Wassmann et al., 2009). However, at the later
anthesis stage, the panicles are at the canopy or above the
plant canopy, thereby exposing the spikelets to higher ambi-
ent temperature. In addition, reduced transpiration cooling
due to high temperature and high RH (Weerakoon et al.,
2008; Wassmann et al., 2009, Julia and Dingkuhn, 2013),
could have contributed to increased spikelet tissue tempera-
tures at anthesis. Restricted wind circulation under con-
trolled conditions, difference in wax deposition on leaves
contributing to heat stress impact cannot be ruled out. In
spite of higher spikelet tissue temperature over the target
heat stress, Ciherang consistently maintained significantly
high spikelet fertility (Fig. 4). This indicates a greater role
of resilient reproductive physiological traits such as pollen
viability and physiological processes such as pollen germi-
nation and pollen tube length, resulting in higher fertility
as demonstrated in our earlier work (Jagadish et al., 2010b).
crop science, vol. 55, march– april 2015  www.crops.org
Using a phenotyping protocol developed by Oane
and Bennett (2009), we recorded the highest proportion
of sterile spikelets that were damaged and stopped fur-
ther progress at stage 4 (anthers exserted and ovules not
enlarged) across all the five tested entries exposed to both
gametogenesis and anthesis. This indicated that heat stress
at gametogenesis and anthesis had the same physiologi-
cal failure, ultimately leading to spikelet sterility. With
microscopic analysis ( Jagadish et al., 2010b), representative
spikelets collected can be analyzed and the results extrapo-
lated to substantiate the cause behind percentage sterility
while this quick approach facilitates determining actual
mechanistic failure across the entire set of sterile spikelets
in a targeted number of panicles. Both techniques men-
tioned above complement each other, with the whole-
panicle sterility approach from the present study allowing
the identification of the most critical sensitive window
(stage 4). However, precise dissection at the spikelet level
using aniline blue staining protocol is needed to further
identify the actual physiological process, leading to steril-
ity considering that stage 4 includes a range of processes
such as anther indehiscence, pollen germination, pollen
tube growth, and so forth ( Jagadish et al., 2010b).
In summary, our work provides evidence for contrast-
ing high-temperature responses across currently grown
popular cultivars, with most of the tested cultivars signifi-
cantly affected by heat stress. Out of the tested 18 popu-
lar cultivars, Ciherang from Indonesia was identified to be
truly heat tolerant at both gametogenesis and anthesis. Only
the most popular and representative cultivars nominated
by breeders were tested in our study, but a more thorough
investigation using a larger set of cultivars grown locally
could lead to identifying additional sources of heat toler-
ance similar to Ciherang. In the absence of locally adapted
cultivars with greater heat tolerance, progress achieved
through ongoing efforts to introgress tolerance from land-
races and wild rice will facilitate increasing heat tolerance
among widely grown vulnerable popular cultivars to safe-
guard rice production under future volatile climates.
Supplemental Information Available
Supplemental information is available with this article.
Acknowledgments
The Federal Ministry for Economic Cooperation and Devel-
opment, Germany, is thanked for supporting the first author’s
extension as a master’s student, along with the IRRI-Japan proj-
ect for providing partial support toward funding the research
expenses. Dr. B. Manneh from Africa Rice and Dr. E. Torres
from CIAT are thanked for providing information on popular
cultivars in West Africa and Latin America, respectively. Bill
Hardy is thanked for editing the manuscript.
595
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