Performance of Advanced Rice Lines Derived from a Heat Tolerance

Breeding Program in High Temperature Conditions

Chandrasekara Mudiyanselage Charitha Manoj Chandrasekara

November 2022
Performance of Advanced Rice Lines Derived from Heat Tolerance Breeding

Program in High Temperature Conditions

By

Chandrasekara Mudiyanselage Charitha Manoj Chandrasekara

A thesis

Submitted in Partial Fulfillment of the Requirements for the Degree of

BACHELOR OF SCIENCE IN AGRICULTURE

2022

Field of Specialization: Crop Science

Department of Plant Sciences

Faculty of Agriculture

Rajarata University of Sri Lanka

Puliyankulama

Anuradhapura

Sri Lanka
 DECLARATION
This is to certify that the thesis on ‘Performance of Advanced Rice Lines
Derived from Heat Tolerance Breeding Program in High Temperature Conditions’ is
completed by Chandrasekara Mudiyanselage Charitha Manoj Chandrasekara in partial
fulfillment of the requirements for the degree of Bachelor of Science in Agriculture.
The thesis has been carried out under our guidance and is a record of the bona fide work
carried out successfully.

External supervisor

…………………………….
Dr. K.G.P.B. Karunarathne
Assistant Director of Agriculture (Research)
Plant Breeding Division
Rice Research and Development Institute
(RRDI)
Bathalagoda
Ibbagamuwa
Date……………………
2022-12-31

Internal supervisor

…………………………….
Dr. D.M.D. Dissanayake
Senior Lecturer
Department of Plant Sciences
Faculty of Agriculture
Rajarata University of Sri Lanka
Puliyankulama
Anuradhapura
Date……………………

Head of the department

…………………………….
Dr. U.G.A.I. Sirisena
Senior Lecturer
Department of Plant Sciences
Faculty of Agriculture
Rajarata University of Sri Lanka
Puliyankulama
Anuradhapura
Date……………………
Affectionately dedicated to

my loving parents, teachers,

family members and

friends
 Acknowledgments

First and foremost, I offer my heartiest gratitude to Dr. D.M.D. Dissanayake,

my internal supervisor, Department of Plant Sciences, Faculty of Agriculture, Rajarata
University of Sri Lanka, who has given me this research opportunity, continuous
support, valuable advice, and supervision for the successful completion of my research.
I wish to extend my heartfelt gratitude to Dr. K.G.P.B. Karunarathne, Assistant
Director of Agriculture (Research), my external supervisor at Plant Breeding Division,
Rice Research and Development Institute (RRDI), Bathalagoda who gave me great
guidance for my research to make it successful with valuable experiences and providing
great learning opportunities. I wish to give my sincere thanks to Dr. D.M.J.B.
Senanayake, Director, Mr. W. Jayasinghe, Senior Agricultural Instructor, Ms. A.
Hathurusinghe, Research Assistant, Mrs. M.A.D. Jayasundara and Mr. H. Senavirathna,
Development Officer at RRDI, Bathalagoda for supporting me in tremendous ways to
make this research a success. Special thanks go to Mrs. C. Piyasiri, Program Assistant
for her great support in data analysis. All the staff of the breeding laboratory and
Agronomy Division, RRDI, Bathalagoda will be truly remembered forever in my heart
and I offer my sincere thanks to all the staff of RRDI, Bathalagoda for their kindness
and active cooperation given to me in any respect during the completion of the research.
My heartfelt gratitude also goes to Mr. S. Rajeshkanna, Assistant Director of
Agriculture (Research), and Mr. T. Kaviraj, Research Assistant, as well as everyone
else who helped me with my research at the Rice Research Station at Murunkan. I would
like to express my warm thanks to Dr. U.G.A.I. Sirisena, Head of the Department of
Plant Sciences, Dr. G.V.T.V. Weerasooriya, Dean of the faculty, and other academic
and non-academic staff of the Faculty of Agriculture, Rajarata University of Sri Lanka,
for supporting me to complete this study.
Finally, I offer my special thanks to my loving parents who were always there
behind me, giving me all the support I needed. I wish to extend my deepest gratitude to
all my friends for every support they have given me during my research.

i
 Abstract

The study was conducted to evaluate the performance of 12 advanced

rice lines derived from a heat tolerance breeding program during the Yala season, 2022,
inside a plant house in pots arranged in CRD with four replicates, under heat stress
(HS), in the Rice Research and Development Institute (RRDI), Bathalagoda (BG), and
in open field conditions at BG and Murunkan (MR), Sri Lanka. The 12 treatments were
transplanted in open fields as RCBDs with three replicates. Location × Variety
interaction in BG and MR open fields was significant only in filled grain percentage
(FGP) and 1000 grain weight (TGW) (p<0.05). In the pot experiment, Nona Bokra
showed significantly higher pollen viability (PV). The number of spikelets per panicle
(NSP) was significantly higher in HT07 among varieties, and a significantly higher FGP
was represented by N22. IR64EMF was recorded as significantly higher TGW in the
pot experiment and in the open field at BG. In the open-field experiment at BG, a
significantly higher PV was recorded in Bg 374. The significantly lowest NSP and
TGW were recorded by HT04 and Moroberekan. Among varieties, HT02 had
significantly higher FGP and TGW. The open field experiment at MR revealed that Bg
374 was significantly higher for NSP and significantly lower FGP was demonstrated
by Moroberekan (p<0.05). A significantly higher TGW in the pot experiment and the
highest significant NSP in the open field at BG were recorded by Bg 20-1204 with a
grain production of 4.53 t/ha. In the open field experiment at MR, even though
IR64EMF had the least PV, Bg 20-1204 was significantly greater in PV and FGP than
others, with a yield of 2.57 t ha-1 (p<0.05). All of the aforementioned results led to the
identification of Bg 20-1204 as the promising line for heat tolerance.

Keywords: Tolerance to heat, Pollen viability, Spikelet fertility

ii
 Table of contents

Acknowledgments ......................................................................................................... i
Abstract .........................................................................................................................ii
Table of contents .........................................................................................................iii
List of tables................................................................................................................. iv
List of figures ............................................................................................................... iv
Acronyms ..................................................................................................................... vi
Chapter 1. Introduction .............................................................................................. 1
1.2 Broad objective....................................................................................................... 3
1.3 Specific objectives .................................................................................................. 3
Chapter 2. Review of literature .................................................................................. 4
2.1 Climate change and agriculture ........................................................................... 4
2.2 High-temperature fluctuations in Agriculture .................................................... 4
2.3 Effects of high temperature on rice production .................................................. 6
2.4 Physiological processes in rice plant under heat stress conditions .................... 6
2.5 Rice breeding for development of heat tolerance ............................................... 8
Chapter 3. Materials and methods ........................................................................... 10
3.1 Pot Experiment..................................................................................................... 10
Location . ..................................................................................................................... 10
Experiment design....................................................................................................... 10
Rice varieties used:...................................................................................................... 11
Crop cultivation ........................................................................................................... 12
Temperature treatment ............................................................................................... 13
Sampling and measurements ...................................................................................... 13
Data analysis ............................................................................................................... 15
3.2 Open Field Experiments at Bathalagoda and Murunkan ................................. 15
Locations .................................................................................................................... 15
Crop management ....................................................................................................... 15
Sampling and measurements. ..................................................................................... 17
Experimental design .................................................................................................. 17
Data analysis . ............................................................................................................. 18
Chapter 4. Results and Discussion ........................................................................... 19
4.1 Results of pot experiment .................................................................................... 19
Thermal variation ....................................................................................................... 19
Pollen Viability and Yield Components Under HS .................................................... 20

iii
Chapter 5. Conclusions and Recommendations ...................................................... 33
References ................................................................................................................... 34

List of tables
Table 3.2.1 Fertilizer recommendations for paddy cultivation - Intermediate
zone and Dry zone (Source: Department of Agriculture) ...................................... 16
Table 4.1.1. Probability of significance difference in parameters for varieties ... 21
Table 4.1.2. PV and yield components as affected by HS (38C) .......................... 21
Table 4.1.3. Probability of significance in open field experiments at BG and MR
...................................................................................................................................... 24
Table 4.1.4. Mean separation data from both open field experiments at BG and
MR .......................................................................................................................... 25,26
Table 4.1.5. Significant differences between treatment means of parameters from
open field experiments at BG and MR ..................................................................... 29

List of figures

Figure 2.2.1. Differentiation of annual maximum temperature in Sri Lanka........ 5

Figure 2.2.2. Monthly maximum temperature – Murunkan, Mannar (Yala
2022) (Source: Department of Meteorology, Sri Lanka) ......................................... 5
Figure 3.1.1. Psychrometer, TFA Dostmann GmbH & Co. KG, Wertheim,
Germany ..................................................................................................................... 12
Figure 3.1.2. OPTIKA B-290 microscope, OPTIKA Srl, Ponteranica, Italy ........ 13
Figure 3.1.3. Pots arranged in CRD in pot experiment at BG ........................... 14
Figure 3.1.4. Pot experiment at BG .......................................................................... 15
Figure 3.2.5. RCBD per each open field experiment .............................................. 17
Figure 3.2.6. An opened field experiment ................................................................ 17
Figure 4.1.1. Daily maximum air temperature change during flowering in pot
experiment .................................................................................................................. 19
Figure 4.1.2. Daily maximum RH change during flowering in pot experiment ... 20
Figure 4.1.3. Nona bokra pollens after crushing and stigma with poor receptivity
under HS ..................................................................................................................... 20
Figure 4.1.4. Sterile pollens of HT01 inside the anther in HS ................................ 20
Figure 4.1.5. Pollen viability of treatments under HS in pot experiment ............. 22
Figure 4.1.6. Anthers of treatments under HS in pot experiment ......................... 22
Figure 4.1.7. Pollen germination on the stigma of treatments under HS ............. 23
Figure 4.1.8. PV and FGP of treatments under HS in pot experiment ................. 23

iv
Figure 4.1.9. Early morning flowering of IR64EMF .............................................. 24
Figure 4.1.10. Pollen viability of treatments in the open field at BG .................... 26
Figure 4.1.11. Pollen viability of treatments in the open field at MR .................. 26
Figure 4.1.12. RH at flowering in the open field at BG (Source: Department of
Meteorology) ............................................................................................................... 27
Figure 4.1.13. RH at flowering in the open field at MR (Source: Department of
Meteorology). .............................................................................................................. 28
Figure 4.1.14. Monthly maximum and minimum RH at MR (Source:
Department of Meteorology). .................................................................................... 28
Figure 4.1.15. FGP of open field experiments at BG and MR ............................... 28
Figure 4.1.16. TGW of open field experiments at BG and MR ............................. 29
Figure 4.1.17. Maximum temperature (C) at flowering in open field at BG
(Source: Department of Meteorology) ..................................................................... 30
Figure 4.1.18. Maximum temperature (C) at flowering in open field at MR
(Source: Department of Meteorology) ..................................................................... 30

v
 Acronyms

Acronym Description

ANOVA Analysis of variance

BG Bathalagoda
CRD Complete randomized design
CSP Cold shock protein
DAT Days after transplanting
DMRT Duncan's multiple range test
DTR Diurnal temperature ranges
EMF Early morning flowering
FGP Filled grain percentage
HS Heat stress
HSP Heat shock protein
HT Heat tolerant
IL Low country intermediate zone
KO Knockout mutants
MR Murunkan
N22 Nagina 22
NPSM Number of panicles per square meter
NSP Number of spikelets per panicle
PV Pollen viability
RCBD Randomized complete block design
RH Relative humidity
RRDI Rice research and development institute
SAS Statistical analysis system
Srl Limited liability company (Società a responsabilità
limitata)
TGW Thousand grain weight

vi
 Chapter 1. Introduction

The biodiversity of rice is significantly appreciated with more than 140,000

cultivars distributed over a boundless scope of geographic regions and different
ecosystems worldwide. Rice has been praised twice by the United Nations for its value
as a unique source of food that contributes to food security (Mohapatra and Sahu, 2022).
Once in every century, there were periods with severe temperature increments in the
past, and such incidences of a sudden increases in atmospheric temperature can be
predicted to occur in the future with the risk of a food shortage. The decline in rice
production due to temperature increments could be avoided by identifying and selecting
tolerant genotypes, and breeding suitable rice cultivars. As a result, the physiological
and genetic mechanisms should be studied, and heat-tolerant varieties should be
developed to improve quantitative and qualitative rice production. Screening and
breeding for heat tolerance in rice is a major objective to generate resilient rice varieties
that can adapt to climate change. Replacing sensitive genotypes with heat-tolerant
varieties, modifying the sowing time, and using growth regulators are some ways of
building heat resistance in rice to reduce yield reduction (Khan et al., 2019). Early
morning flowering (EMF) reported in wild species of rice and landraces is
advantageous. Other reproductive stage-specific traits like pollen fertility, pollen
shedding percentage, stigma receptivity, and spikelet fertility were also considered to
develop heat-tolerant rice genotypes (Senguttuvel et al., 2022). An increase in spikelet
sterility due to heat stress exposure during flowering in rice is a major threat to rice
productivity in tropical regions like South Asia. Therefore, exploiting and incorporating
the early morning flowering (EMF) trait is desirable (Bheemanahalli et al., 2017). The
tolerant N22 rice variety's cold- and heat-shock proteins may be able to withstand heat
during anthesis (Senguttuvel et al., 2022). Multiple elite genes are combined in a single
genotype in the gene pyramiding breeding technique, and it may improve rice tolerance
to heat stress by incorporating multiple heat tolerance genes into existing elite
accessions. Studies had focused their analyses on the flowering stage in rice, because
decreased spikelet fertility can reduce the crop yield. Rice pollen characteristics such
as floret sterility, anther dehiscence, apical and basal pore length, pollen fertility, pollen
count per stigma, and pollen germination on stigma can be exploited as screening tools
for varietal development, but selection must be focused on those germplasm sources

1
that can tolerate high temperature conditions. By learning more about the molecular
processes governing the methods by which rice plants tolerate heat, bioengineered rice
cultivars that can withstand extreme heat stress conditions can be created (Fahad et al.,
2018). Global warming has resulted in heat-induced spikelet sterility due to the loss of
healthy pollen grains induced by metabolic heat responses in rice plants (Wada et al.,
2020). Sri Lanka has an average temperature between 27°C and 28°C, making it one of
the hottest countries on earth. The average daily maximum temperature is 31C. Over
the last century, Sri Lanka has experienced warming of around 0.8°C. Sri Lanka has
two seasons, including the Maha season, related to the northeast monsoon (from
September to March), and the Yala season, related to the southwest monsoon (from
May to August). Due to heat stress in Sri Lanka's high-temperature regions, the seasonal
maximum temperatures for both the Maha and Yala seasons have shown that the Yala
season may have a higher influence on rice output (the Dry Zone). Apart from the low
rainfall, heat stress may be the reason for Yala to become the minor cropping season in
the Dry Zone. In Northern Province, the seasonal maximum temperature (June-July-
August) increases during the Yala season (Climate Risk Country Profile: Sri Lanka
(2021): The World Bank Group and the Asian Development Bank.). As the south-west
monsoon (May-September) directs rains into the Wet Zone, the Northern and North-
Western parts of Sri Lanka remain at a high temperature during that time. Climate
resilience is going to be achieved by scientists worldwide with advanced breeding
techniques. Rice is densely cultivated in the tropical and subtropical Asian regions,
under high temperatures and it is a high-temperature sensitive crop, especially at the
booting and flowering stages. Heat-tolerant rice varieties are limited, and heat stress
conditions at the booting and flowering stages could become a reason for the loss of
pollen viability through sterile pollen, decreased pollen shedding, and poor pollen
germination. By assessing the pre-breeding performance of lines obtained from
advanced breeding programs, parental lines with efficient responses to heat stress
should be chosen. The replacement of heat-sensitive cultivars with heat-tolerant
cultivars could build climate-resilient rice cultivation systems (Ye et al., 2021).

2
1.2 Broad objective

 To evaluate the performance of advanced rice lines derived from heat tolerance
breeding in high-temperature conditions

1.3 Specific objectives

 To measure the pollen viability of heat tolerance lines in both ambient and heat
stressed environments
 To study the heat tolerance traits in advanced breeding lines of rice
 To select the heat tolerant lines, depending on the performance of advanced rice
lines
 To study the growth and yield performance of advanced rice lines in high-
temperature conditions

3
 Chapter 2. Review of literature

2.1 Climate change and agriculture

Crop damages due to climate change have increased, causing frequent and
severe impacts of global warming on crops worldwide. Climate change will alter
agricultural productivity; one region's production will decline while global production
increases in other regions. The economies of different regions will also change (Aydinal
et al., 2008). Climate change parameters, including average temperature, energy
consumption, precipitation, CO2 emissions, and non-climatic components such as area
of crop production and fertilizer use efficiency, are expected to have a detrimental effect
on agricultural output. CO2 emissions and energy consumption can be managed
properly to increase agriculture production, but average temperature and precipitation
cause production losses in agriculture. The area used for crop cultivation and the
effectiveness of fertilizer application both boost agricultural output. The development
of new crop varieties with resistance to extreme temperatures and scarcity is required
to adapt to the changing climate (Van et al., 2022).

2.2 High-temperature fluctuations in Agriculture

The location of Sri Lanka in the Indian Ocean between latitudes 5 – 10 N
causes it to have a tropical maritime temperature level. Consequently, the temperature
variation is wider each day than it is in other tropical areas (Department of Meteorology,
Sri Lanka). The country of Sri Lanka has warmed by about 0.8C in the previous
century. There are two seasons in Sri Lanka: The Yala season, which corresponds to
the southwest monsoon, and the Maha season, which is associated to the northeast
monsoon (from September to March) (from May to August). Sri Lanka is one of the
hottest places on earth with an average temperature of 27°C and 28°C. 31°C is the
typical daily high temperature. (Climate Risk Country Profile: Sri Lanka (2021): The
World Bank Group and the Asian Development Bank). The seasonal maximum
temperatures during both Maha and Yala seasons have shown that the Yala season may
have a greater impact from heat stress on rice production in high-temperature areas of
Sri Lanka (Dry Zone). Apart from the low rainfall heat stress may be the reason for
Yala to become the minor cropping season in the Dry Zone.

4
 The seasonal maximum temperature rises in the Northern Province during the
Yala season (June-July-August) (Department of Agriculture, Sri Lanka). Mean annual
temperatures from 27°C in the coastal belt to 16°C in the central hills have a significant
variation in Sri Lanka (Department of Meteorology Sri Lanka). In the Mannar region,
at Murunkan the highest maximum temperature was recorded in the months of April

Figure 2.2.1. Differentiation of annual maximum temperature in Sri Lanka

(1961-2020)

and May. (Figures 2.2.1. and 2.2.2.). The Eastern and Northern parts of Sri Lanka have
recorded larger Diurnal Temperature Ranges (DTRs), while southern and western parts
indicate the lowest DTR. DTR is the difference between the maximum and minimum
temperature (Shelton et al., 2022).

Figure 2.2.2. Monthly maximum temperature – Murunkan, Mannar (Yala 2022)

(Source: Department of Meteorology, Sri Lanka)

5
2.3 Effects of high temperature on rice production
Heat stress conditions were the most common type of climatic factor in rainfed
rice cultivation (Mittler et al., 2001). High temperatures affect, plants at various
organizational levels, shortening the reproductive phase to cut down on flower numbers
(Chaudhary et al., 2020). The heat stress conditions cause pollen sterility and spikelet
infertility in rice. An advanced breeding program has developed heat-tolerant rice lines
to be verified for high performance under high-temperature conditions (Wada et al.,
2020). High-temperature stress can be mitigated by developing adaptations, as
concluded in research, related to the rice plant. Rice is frequently produced in regions
where the temperature is higher than what is ideal for growth. High-temperature
conditions during the critical stages of the rice plant could reduce the rice grain weight
and quality (Krishnan et al., 2011).
The impact of heat stress is different according to the stage of the rice plant.
There is an 8% yield reduction per every 1C increment of temperature, in early-rice
plants due to the heat stress conditions at the grain filling stage. In late rice plants, heat
stress normally impacts the vegetative growth stage, which is not so susceptible to high-
temperature conditions (Song et al., 2022). In rice plants under heat stress, adequate
canopy cooling by transpiration was a typical tactic to lower high spikelet temperature
(Weerakoon et al., 2008).

2.4 Physiological processes in rice plant under heat stress conditions

The capability of rice to perform significant physiological processes under

stress, such as anther dehiscence, pollination, pollen germination, and fertilization,
while still maintaining high seed filling, is known as absolute tolerance of heat-stress
(Jagadish et al., 2010). The ideal temperature for that specific rice plant's growth and
reproduction determines how well that rice variety performs under high-temperature
conditions (Hatfield, et al., 2022). The concept of tolerance often includes components
of escape, i.e., rice genotypes can escape or avoid heat stress during anthesis by heading
at cooler phases of the season (macro-escape) or by having anthesis occur during cooler
early morning hours; micro-escape (Jagadish et al., 2008). Under high temperatures,
the spikelets inside the flag-leaf sheath were completely sterile, resulting in low spikelet
fertility due to the absence of anthesis and fertilization. A substantially greater level of

6
sterility was produced by the extra heat that was contained inside the leaf sheath due to
the absence of unrestricted air movement (Cruz and O'Toole, 1984; O’Toole and
Namuco, 1983). Even through a longer blooming time in response to the imposed stress
treatments may be a viable alternate escape strategy, plants that were subjected to
independent high temperatures did not show greater spikelet fertility. Thus, in addition
to the immediate adverse effect of heat stress occurring at the same time as anthesis,
which causes spikelet sterility, the carryover effect on un-anthesized spikelets even
after stress removal also causes sterility. The cumulatively deleterious impact of
extended heat stress on successive flowering days was examined after the spikelet
marking strategy. A cumulative impact of heat stress was indicated by the linear decline
in spikelet fertility on successive blooming days, whether in the heat-tolerant N22 or
the heat-sensitive rice variety, Moroberekan (Matsui and Omasa, 2002). According to
the findings, there is a serious disruption in the pathways for metabolizing energy and
carbohydrates, and heat shock proteins may be crucial in avoiding cellular damage
brought on by heat stress. Furthermore, the relationship between high temperatures and
grain quality has been underlined in proteomic investigations of rice caryopsis
development and grain filling under heat stress (Lin et al., 2005, 2010). The expression
of high-temperature-responsive proteins varied amongst rice genotypes; the thermos-
induced accumulation of certain proteins, such as cold (CSP) and heat (HSP) shock
proteins, may contribute to the tolerant species' increased temperature tolerance
(Jagadish et al. 2010). Heat shock may significantly change cellular metabolism and
affect a cell's membrane, cytoskeleton, and nucleic acid composition (Kim et al., 2002).
Utilizing cleistogamous (closed flower) plants will physically prohibit outcrossing. For
the rice flower to open, mechanical activity of fleshy appendages known as lodicules,
which are typically thought of as the rice flower's equivalent of grass petals, is required
(Lombardo et al., 2017). Cleistogamy in rice flowers may be a characteristic for heat
tolerance as a result of its ability to prevent pollen from experiencing heat stress. The
impact of high-temperature stress during flowering in rice is reduced by cleistogamy.
Global warming has been linked to a significant agricultural issue known as rice
infertility, triggered by a high temperature at flowering. During the reproductive stage,
rice plants are particularly vulnerable to extreme temperature stress (Koike et al., 2015).
Cleistogamous flowers do not open; instead, they remain as buds. Cross-pollination
occurs often in chasmogamous flowers (and sometimes they are self-pollinated).
Cleistogamous flowers can only pollinate themselves.

7
2.5 Rice breeding for development of heat tolerance

Gaining yield is far more significant than survival in actual crop breeding. The
reproductive stage should be given more consideration when assessing the heat
tolerance of the rice line because the flowering stage is when rice plants are most
vulnerable (Satake and Yoshida, 1978). Numerous studies on the physiological and
molecular processes of heat tolerance have made extensive use of the rice cultivar
Nagina 22 (N22), which has attracted particular interest for its exceptional resistance
to heat stress during the blooming stage. Reduced grain filling percentages, spikelet
counts, and grain weights, all of which vary depending on the rice cultivar and the
reproductive period in which heat stress occurs, are the main causes of yield losses
during heat stress. During the mid-late reproductive phase (flowering and grain filling),
the heat-tolerant rice cultivar Nagina 22 was most resilient, whereas the early
reproductive phase was extremely vulnerable; (panicle initiation) (Li et al., 2015). The
best adaptation technique for extremely changeable future climates is to make rice more
resilient to shocks such as heat stress during its most vulnerable blooming stage (Horie
et al., 1996).
During the dry season, morphological and physiological traits such as pollen
viability and spikelet fertility were studied under ambient conditions (38°C) and in heat
stress conditions (44°C). All the observed trait values under ambient conditions were
higher in mutants compared to N22 and IR64, causing the reduction of all observed
traits for all the genotypes except the increase in plant height. Significant reduction in
yield per plant was observed under heat stress conditions in both N22 and NH219, and
there were also significant differences under heat stress conditions for both pollen
viability and spikelet fertility (Poli et al., 2013). Heat tolerance had increased the
spikelet transpiration rate, resulting in strong transpirational cooling and a lower
temperature inside spikelets, especially in heat-tolerant genotypes (Wang et al., 2021).
The overexpression of some genes could increase pollen viability and seed setting rate
under heat stress conditions, suggesting that a particular gene could positively regulate
heat tolerance in rice at the heading and flowering stages (Zhou et al., 2022).
In order to understand the physiological, biochemical, and molecular
mechanisms controlling heat tolerance, a large population was screened to identify
different genotypes. This method is one of the most well-known approaches used by
breeding scientists to create crop varieties with increased heat tolerance. Heat stress is

8
often accompanied by drought stress (Sehgal et al., 2017). Reduced 100-grain weight
was the result of the reduced spikelet fertility at high temperatures, which also led a
large reduction in grain filling. The spikelet fertility, 100-grain weight and panicle
weight had declined due to high daytime heat. The decrease in spikelet fertility was
mainly associated with impaired pollen viability and pollen germination (Cheabu et al.,
2019). One aspect of rice agriculture that was especially impacted by the high
temperatures was anthrax dehiscence. N22 rice variety had shown a significantly higher
anther dehiscence. Moroberekan rice variety was observed as the least anther
dehiscence. The pollen count pollinated on the stigma was reduced due to heat stress
(72%). N22 was the highest pollen count having variety (52-70). The germinated pollen
count on the stigma was decreased by heat stress (81%). The rice variety with the most
pollen that germinated was IR64. A higher pollen germination percentage was recorded
in N22 rice variety. The spikelet fertility was reduced by 72-81% with exposure to heat
stress. A significantly higher spikelet fertility was observed in N22 rice variety (Rang
et al., 2011).
Complex reaction of rice plants to heat stress involves both protein up-
regulation and protein down-regulation, suggesting that rice's ability to withstand heat
may be explained by an increase in the abundance of some proteins and a reduction in
the abundance of others. However, the majority of the transgenic studies for rice that
could withstand heat relied on the overexpression of certain proteins. Despite the
observation that knockout (KO) mutants of rice shown better resistance to high
temperature, research on lowering protein abundance for heat-tolerant rice is scarce
(Koh et al., 2007).

9
 Chapter 3. Materials and methods
3.1 Pot Experiment
Location

A pot experiment was conducted inside a plant house at the Rice Research and
Development Institute (RRDI), Bathalagoda (BG); 7.532N longitude, 80.435E
latitude; 117 m above sea level; in Northwestern Province, Sri Lanka, which belongs to
the Low Country Intermediate Zone (IL1a), with an annual rainfall and average
temperature of >1400 mm and 28C, respectively. The soil type associated with red-
yellow podzolic was sandy clay loamy soil. Rice cultivation was conducted during the
Yala season from April to August in 2022.

Experiment design
An experiment is set up using a CRD when each treatment is applied a specified,
possibly unequal, number of times, the particular units to receive a treatment being
selected completely random. Rice seeds from 12 rice varieties were sown on dry-bed
nursery and subsequently transplanted into plastic pots in order to analyze the
performance under high temperature condition in sensitive and tolerant genotypes
(Figure 3.1.3). One plant from each cultivar in a single pot were moved to the plant
house at BG for heat stress treatment and all the 48 pots were arranged in CRD (Figure
3.1.2.).

Figure 3.1.3. Pots arranged in CRD in pot experiment at BG

10
 A daytime heat stress (38°C) was maintained during the blooming initiation
stage.

Figure 3.1.4 Pot experiment at BG

Rice varieties used: 12 treatments

Treatment Variety Treatment Variety

No. No.
T1 IR64EMF T7 HT02
T2 N22 (Nagina 22) T8 HT04
T3 Nona Bokra T9 HT05
T4 Bg 374 T10 HT06
T5 Bg 20-1204 T11 HT07
T6 HT01 T12 Moroberekan

11
 IR64EMF was created by combining genes from the wild rice species Oryza
officinalis. The heat tolerant check was N22, and the heat susceptible check was
Moroberekan. Nona Bokra was used as the salinity tolerant check. Six rice lines derived
from the advanced breeding program at RRDI, Bathalagoda (HT lines) were used. The
heat tolerant check was N22, and heat susceptible check was Moroberekan rice variety.
Nona Bokra was an indica landrace that could tolerate salt and shared genetics with
Koshihikari, a dominant but salt-sensitive japonica cultivar (Mitsuya et al., 2019). The
average yield of Bg 374 is 5.9 t/ha according to the Department of Agriculture. It was
a white pericarp with intermediate bold shape grains, which had given yields in 3 ½
months. Bg 20-1204 was an advanced heat-tolerant rice line derived from the parents
Bg 358/N22. HT01, HT02, HT04, HT05, HT06, and HT07 were advanced rice lines
derived at RRDI, Sri Lanka. A West African upland japonica variety with significant
drought tolerance was known as Moroberekan. (Champoux et al., 1995). While IR64
(48) and N22 (71) were moderately and extremely heat tolerant, respectively,
Moroberekan (18% spikelet fertility at 38C) was the most heat-sensitive genotype
(Jagadish et al., 2010).

Crop cultivation

Seeds were sown in the upland (dry-bed) nursery and allowed to be germinated.
After 21 days, the seedlings were transplanted into plastic pots (27.8 cm diameter and
24.5 cm height) filled with soil and kept inside the plant house. Four replicates were
transplanted in pots per treatment. A total of 48 pots was arranged in a randomized
complete block design (RCBD). Department of Agriculture fertilizer recommendations
of the year 2013 were followed when fertilization in the pot experiment (Table 3.2.1).
To establish the relationship between heat stress and pollen viability, the soil pot
experiment was conducted inside the plant house (controlled environment) and plants
were exposed to direct sunlight and high temperature during the daytime at flowering.
The pollen viability test was observed through the microscope (OPTIKA B-290
microscope, OPTIKA Srl, Ponteranica, Italy).

12
Temperature treatment

The psychrometer was used to measure the wet-bulb and dry-bulb temperatures
at 7.00 a.m., 9.00 a.m., 11.00 a.m., and 13.00 p.m. during the period of flowering, daily
(Figure 3.1.1.).

Figure 3.1.1. Psychrometer, TFA Dostmann GmbH & Co. KG, Wertheim,
Germany

The daytime temperature was maintained at a maximum of 38C for high temperature
treatment during the flowering period by following temperature-control methods inside
the plant house. All the rice plants in pots were exposed to the same 38C temperature.
Relative humidity was maintained in a range of 60 – 80% by following suitable
humidity control methods. Windows were opened to control the temperature when it
exceeded 38C, allowing for natural air circulation. The floor and walls were moistened
to increase the RH to the above-mentioned range. The daytime temperature of open-
fields at BG has never dropped below 35C during a Yala season.

Sampling and measurements

The yields from each line and cultivar were measured. Yield components of
rice, including the number of panicles per square meter, number of spikelets per panicle,
filled grain percentage, and 1000 grain weight, were recorded. A scientific procedure
was followed to measure anthers’ dehiscence (Wu et al., 2016). The pollen collection
process was repeated four times for each replicate. A dehisced anther had opened apical
and/or basal pores. Measuring pollen viability was conducted by counting stained
pollen grains using a 1% iodine-potassium iodide solution (Gunawardena et al., 2003).

13
Pollen grains from undehisced anthers were examined at 10× magnification using the
OPTIKA B-290 microscope (Figure 3.1.2).

Figure 3.1.2. OPTIKA B-290 microscope, OPTIKA Srl,

Ponteranica, Italy

PV was calculated as the ratio of the number of fertile pollen grains to the total
number of pollen grains. Pollen germination was observed under the microscope.
Anthers were collected using forceps at flowering (at 11.00 a.m.) into the sample bottles
of 10 ml total volume, filled with 70% ethanol (4-5 spikelets per experimental unit),
and anthers were placed on the glass slide to be crushed and spread on the glass slide
using the spoon spatula. The staining of fertile pollens into a blue-black color took place
with the addition of 1% iodine-potassium iodide solution (I2-KI), and the stained fully
fertile pollens, number of partially fertile pollens, and sterile pollens were counted. The
number of fertile pollens was taken as the sum of the fully fertile (fully stained) and
partially fertile (half-stained) numbers of pollen. The total number of pollens was
calculated by adding the numbers of fertile and sterile (non-stained) pollens together.
The pollen viability percentage (PV) was calculated using the following equation.

PV = (Number of fertile pollens)/ (Total number of pollens) ×10

The number of panicles per square meter (NPSM) was obtained as the number
of panicles per unit area. The number of spikelets per panicle (NSP) was measured by
counting the total number of spikelets in a rice plant and dividing by the number of
panicles in that particular plant. The filled grain percentage (FGP) was measured after
detaching grains from harvested panicles from plants grown in soil pots, FGP was
calculated as the ratio of the total number of filled and partially filled grains to the total
number of grains.

FGP = (Number of filled grains)/ (Total number of grains) ×100

14
 The weight of 1000 filled rice grains were detected as the 1000-grain weight
(TGW). PV and FGP were used to evaluate the performance and tolerance under heat
stress conditions. Spikelet fertility was calculated as a ratio of number of grains filled
to total number of florets as a percentage. Fertile grains (completely or partially filled
grains) were detected by applying pressure on to each floret using both forefinger and
thumb, after 20 days from anthesis completion (Prasad et al., 2006).

Data analysis

The data was analyzed by analysis of variance (ANOVA) using a statistical

computer package, SAS Ver. 9.1 (SAS Institute Inc., SAS Campus Drive, Cary, North
Carolina, 27513). It was analyzed as per the CRD in the pot experiment. The significant
differences were estimated using Duncan's Multiple Range Test (DMRT).

3.2 Open Field Experiments at Bathalagoda and Murunkan

Locations
Two experiments were conducted as open field experiments at BG and the Rice
Research Station, Murunkan (MR). Open field experiment at MR was conducted during
the peak period of the highest daily temperature in Yala season, 2022. The nursery
preparation steps were followed as in the pot experiment. After 21 days, seedlings in a
dry bed nursery were hardened enough to be transplanted in paddy field plots. A yield
trial of Bg 20-1204 was transplanted in 6×3 m2 plots. Other seedlings were transplanted
in RCBD, as 3 row progenies of 99 plants each. 33 plants were there in a single row.

Crop management

Screening of rice plants for heat tolerance traits was done at the flowering stage
so that the performance of each cultivar could be evaluated. According to their heading
times, cultivars were classified and planted so that flowering times coincide with the
heat stress period (mid-April to mid-May 2022). Spikelet fertility was assumed to be
corresponded when evaluating the performance of each variety under heat stress
conditions (Tenorio et al., 2013). Fertilizer recommendations by the Department of
Agriculture, for paddy cultivation in 2013 to the Intermediate Zone and Dry Zone were
applied in the research (Table 3.2.1).

15
Table 3.2.1. Fertilizer Recommendations for Paddy Cultivation - Intermediate
Zone and Dry Zone 2013
(Source: Department of Agriculture)

Age Group Time Fertilizer Quantity (kg/ha)

Urea TSP MOP
For 3 ½ month age group Basic 55
2 weeks 50
4 weeks 75 25
6 weeks 65 35
8 weeks 35
Total 225 55 60

16
Sampling and measurements

The sampling procedure for the pollen viability test was carried out similarly to the pot
experiment, and the same yield components were measured using the same procedure.

Experimental design

Open field experiments at BG and MR were designed in RCBD as three replicates per
treatment. The experimental units were divided into smaller groups (blocks or
replicates) so as to reduce the effect of environmental variability. Every treatment
occurred at least once within every block (Figures 3.2.4. and 3.2.5.).

Figure 3.2.5. RCBD per each open field experiment

Figure 3.2.6. An open-field experiment

17
Data analysis

The data were analyzed by Analysis of Variance (ANOVA) using a statistical computer
package, SAS version 9.0 as per the RCBD in both open fields at BG and MR. The
significant differences were estimated using Duncan's Multiple Range Test (DMRT).

18
 Chapter 4. Results and Discussion

4.1 Results of pot experiment

Thermal variation

In heat stressed (HS) experiment, the transplanted seedlings were fully exposed
to high-temperature stress inside the plant house for about 91 days after transplanting
(DAT) from 17/06/2022 to 16/09/2022 (harvesting date). A maximum air temperature
(daytime) ranging from 35C to 40C was maintained for 10 days before the initiation
of 50% flowering. However, mild heat stress was occurred at 7.00 a.m. and 9.00 a.m.
according to the temperature data captured. The daily maximum temperature was
observed at 11.00 a.m. and 1.00 p.m. (Figure 4.1.1.). The relative humidity (RH) at 7.00
a.m., 9.00 a.m., 11.00 a.m. and 1.00 p.m. was also recorded using the psychrometer
(Figure 4.1.2).

Figure 4.1.1. Daily maximum air temperature change during flowering in pot
experiment

19
 Figure 4.1.2. Daily maximum RH change during flowering in pot experiment

Pollen Viability and Yield Components Under HS

In the pot experiment, 50% flowering was initially completed by the varieties
Bg 374, HT01, HT02, HT04, HT05, HT06, and Moroberekan on 10/08/2022
(55DAT). IR64EMF, N22, and HT07 reached 50% flowering stage on 15/08/2022

Figure 4.1.3. Nona Bokra pollens after Figure 4.1.4. Sterile pollens of
crushing and stigma absent of received HT01 inside the anther in HS
pollens in HS
(60DAT) and Nona Bokra (4-month variety) achieved 50% flowering on 16/08/2022
(61DAT). Pollens that were collected on 50 flowering dates of each treatment
discovered that the highest pollen-viable variety was Nona Bokra and the lowest was
HT01 after taking the pollen count through the compound light microscope (Figure
4.1.4.). All the treatments were significantly different from each other in the pot
experiment (p<0.05) (Table 4.1.1.). Even though the PV is highest in Nona bokra, its
stigma receptivity was poor (Figure 4.1.3). Nona Bokra variety was shown the highest
20
PV while HT01, the lowest. The number of spikelets per panicle (NSP) was higher in
HT07 among varieties and a higher FGP was represented by N22. IR64EMF and Bg
20-1204 obtained the highest TGW (Table 4.1.2.).
Table 4.1.1. Probability of significance in parameters for varieties in pot
experiment
Pollen No. of No. of Filled grain 1000-grain
viability panicles per spikelets percentage weight
m2 per panicle
Probability 0.003 <0.001 0.0024 0.004 0.03
of
significance
Parameters of p<0.05 are significantly different

21
 High temperature, had caused abnormal pollen development (obscure outline of
the pollen exine with a collapsed bacula, disordered tectum, and no nexine of the pollen
walls) as well as abnormal anther development (fewer vacuolated microspores, un-
degraded tapetum, unequally distributed Ubisch bodies), which may have decreased
pollen viability. Additionally, Heat stress had resulted in a compact knitted anther
cuticle structure of the epidermis, an un-degraded septum, dense anther walls,
disproportionate Ubisch bodies, and suppression of the confluent locule. These severely

Table 4.1.2. Pollen viability and yield components as affected by heat stress (38C -
40C) in pot experiment
Treatment Variety Pollen No. of No. of Filled grain 1000-
no. viability panicles spikelets percentage grain
(PV%) per m2 per (FGP) weight
(NPSM) panicle (TGW)
(NSP) (g)
1 IR64EMF 76.93a 288a 117d 77.12ab 20.37a
2 N22 66.23ab 132d 105d 87.2a 17.18cd
3 Nona Bokra 84.53a 112d 198cd 79.35ab 16.48d
4 Bg 374 71.53ab 210bc 283a 68.9bc 17.8cd
5 Bg 20-1204 82.58a 120d 280b 66.65bc 19.93a
6 HT01 22.03d 192bc 194cd 59.53d 17.75bc
7 HT02 82.2a 240b 182d 65.33bc 18.78ab
8 HT04 24.53cd 246ab 172d 78.3ab 17.45cd
9 HT05 64.8ab 168cd 217c 64.83bc 17.58cd
10 HT06 78.38a 168cd 234b 65.83bc 18.45ab
11 HT07 39.43bc 248ab 297a 57.7d 18.97ab
12 Moroberekan 55.43ab 136d 221c 52.28d 15.8d

Values followed by the same letter in a column are not significantly different at p<0.05

deformed structures may be partially attributable to the lower incidence of anther

dehiscence and decreased pollen shedding of the anthers (Hu et al., 2021). Observations
of a lesser number of the total pollen count in HT02 and a higher number of the sterile
pollen count in HT05, were visible through the microscope, with no change of color.

22
The blue-black color conversion of fertile pollens and partial fertile pollens with half-
blue-black color was also demonstrated (Figure 4.1.5.).
Figure 4.1.5. Pollen viability of treatments under HS in pot experiment

Microscopic observations were conducted to understand the anther dehiscence

of treatments under HS. Due to heat stress, the opening of apical pores in some
treatment was prevented with a vast number of pollens stuck inside anthers, even
though some treatments had shown high pollen viability (Figure 4.1.6.).

Figure 4.1.6. Anthers of treatments under HS in pot experiment

Pollen germination, pollen tube elongation and, stigma receptivity of

treatments, which were the traits related to the pistil of a rice flower, that could have

23
been reduced under HS, were detected (Figure 4.1.7.).

Figure 4.1.7. Pollem germination on stigma of treatments under HS

The capacity of a stigma to support the germination of viable and compatible

pollen is known as stigma receptivity (Gupta et al., 2015). Confocal microscopy has
greatly advanced the area of flower research by being utilized to study flowers (McInnis
et al., 2006). Even though some varieties had shown a higher PV, their FGP was low
(HT02 and HT06). Poor anther dehiscence, poor stigma receptivity and stigma exertion/
damage were the reasons for these fluctuations (Figures 4.1.6. and 4.1.7.). However, in
the varieties which had a few number of fertile pollens, the fully and partially fertile
pollens had shed securely onto healthy stigmas and germinated demonstrating a higher
FGP (N22, HT01, HT04 and HT07) (Figure 4.1.8.).

Figure 4.1.8. PV and FGP of treatments under HS in pot experiment

24
 The flowering of IR64EMF was recorded at 6.52 a.m. in the morning (Figure
4.1.9.).

Figure 4.1.9. Earl morning flowering of IR64EMF

Results of open field experiments at BG and MR

All the treatments were significantly different in open fields at BG and MR.
Location × Variety interaction was significantly different at BG and MR open fields,
only on FGP and TGW (p<0.05) (Table 4.1.3.).

Table 4.1.3. Probability of treatment difference in open field experiments at BG and

MR
PV (%) NPSM NSP FGP () TGW (g)
Variety 0.0001 <0.0001 0.0004 0.0006 <0.0001
Location 0.5450 0.1154 0.3297 0.0135 <0.0001
Variety × 0.3667 0.2020 0.1335 0.0388 <0.0001
Location
Parameters of p<0.05 are significantly different

In the open field experiment at BG, the highest PV was recorded in Bg 374. Bg
20-1204 was the highest in NSP, whereas HT04 and Moroberekan were the least. HT02
showed a greater FGP among varieties, and the least were HT04 and Moroberekan. The
highest TGW was recorded by HT02, while N22 had the lowest. Open field experiment
at MR revealed that Bg 20-1204 achieved the greatest PV among others. However,
IR64EMF had the lowest. Bg 374 was recorded as the highest NSP,

25
 N22
MR

HT06
HT05
HT04
HT02
HT01
Bg 374
Variety

IR64EMF

Bg 20-1204
Nona Bokra
89.67a 91.4a 91.2a 86.65bc 82.33c 92.2a 98.07a 83.87c 85.23bc 77.03c
BG
PV (%)

88.87a 88.53a 88.2ab 87.33ab 93.67a 96.2a 92.23a 88.47ab 80.37cd 71.85d
MR

272b 232bc 256bc 232bc 240bc 208c 168c 224c 328a 360a
BG
NPSM

416a 328ab 252bc 320ab 224bc 184cd 208bc 200cd 432a 372ab
MR

26
161bc 155bc 86d 171bc 147bc 366a 221ab 170bc 257ab 117cd
BG
NSP

188ab 224a 154ab 108c 94d 227a 256a 106c 145ab 105c
MR

86.97b 79.8c 57.5d 89.2a 78.0c 83.9b 78.77c 84.93b 79.23c 88.07a
BG
FGP ()

79.13ab 88.95a 62.3cd 58.73cd 62.33cd 89.35a 87.53a 71.33bc 56.63cd 88.65a
MR

28.95a 22.7b 18.9d 32.85a 27.1a 18.03d 21.13c 21.53c 17.7d 25.33a
BG
TGW (g)

21.73a 13.37d 21.8a 20.17bc 13.3d 17.83cd 20.53bc 21.77a 18.5cd 24.15a
MR
Table 4.1.4. Mean separation data from both open field experiments at BG and
 HT07

112c

81.43ab
85.83bc

85.1bc

272b

82.8b

22.5b

16.0d
116cd
224bc
Moroberekan

70.67d
85.95bc

184c

20.7c

22.1a
78.7d

120d

92d

145ab

48.5d
Values followed by the same letter in a column are not significantly different at p<0.05

although HT01 had the lowest. The FGP was higher in Bg 20-1204 across varieties
and lowest in Moroberekan. IR64EMF demonstrated the highest TGW and HT05 the
lowest (Table 4.1.4.). The grain production of Bg 20-1204 was 4.53 t/ha in the open
field at BG and 2.57 t/ha in the MR.

Figure 4.1.10. Pollen viability of treatments in the open field at BG

Figure 4.1.11. Pollen viability of treatments in the open field at MR

27
 Figure 4.1.12. RH at flowering in open field at BG
(Source: Department of Meteorology)

Figure 4.1.13. RH at flowering in open field at MR

(Source: Department of Meteorology)

In open-field experiments at BG and MR, a higher total pollen count was

shown by IR64EMF, N22 and Bg 20-1204 (Figure 4.1.10 and 4.1.11). The total
pollen number had reduced in MR than BG. The high RH in the atmosphere at MR
than of BG, during the flowering period, was the reason for the reduction of PV.

The monthly maximum RH at MR (86.8) during August was higher than that of the
BG (78.9) (Figure 4.1.14). When combined with high RH (85%–90%) at heading, a

28
high midday temperature of 35 °C caused almost total grain sterility in rice
(Abeysiriwardena et al., 2002).

Figure 4.1.14. Monthly maximum and minimum RH at MR

(Source: Department of Meteorology)

Figure 4.1.15. FGP of open field experiments at BG and MR

FGP of N22, HT01, HT02, and Moroberekan at MR was significantly lower

than that of BG. The higher RH at flowering was the cause for the induction of grain
sterility in the open field at MR (Figure 4.1.15).

29
 Figure 4.1.16. TGW of open-field experiments at BG and MR

The causes for the poor TGW and FGP may be the lack of water from the Giant
tank (Yoda wewa), the only water tank at Murunkan area, during the flowering period
(Figure 4.1.17.). However, the TGW was a significant parameter because the 12
varieties had different grain sizes and shapes (e.g. IR64EMF grain is long-medium and
Bg 20-1204 was short-round).

Table 4.1.5. Differences between the data from open field experiments at BG
and MR

Location PV (%) NPSM NSP FGP (%) TGW (g)

80.35ns 228.67ns 171.86ns a a
BG 80.63 23.01

86.63ns 254.67ns 155.57ns b b

MR 72.48 19.16

ns – not significantly different

Means with different letters in a column are significantly different at 0.05 probability
level

The highest treatment means of FGP and TGW were significantly demonstrated
by the opened field at BG than that of MR (Table 4.1.5.). The high RH during the time
of flowering that prevailed at MR was the reason for the poor grain production (Figures
4.1.12, 4.1.13 and 4.1.14). The effect of temperature on PV and grain yield was

30
considered equal at both locations (Figures 4.1.17. and 4.1.18.)

Figure 4.1.17. Maximum temperature (C) at flowering in the open field at BG

(Source: Department of Meteorology)

Figure 4.1.18. Maximum temperature (C) at flowering in open field at MR

(Source: Department of Meteorology)

Cleistogamy in rice flowers of heat-tolerant varieties could reduce the effect of

31
high temperatures. Upon flowering at 38°C, the cleistogamous rice plants demonstrated
resistance to severe temperature stress. These results contributed to the conclusion that
cleistogamy, due to glume cooling, was beneficial for rice pollination and fertilization
at high temperatures (Koike et al., 2015). Under general conditions, an enclosed stigma
increases spikelet fertility and heat tolerance in high-temperature circumstances during
rice flowering (Wu et al., 2019)

32
 Chapter 5. Conclusions and Recommendations

Bg 20-1204 was identified as a promising heat-tolerant line. The IR64EMF

variety had proven that early morning flowering was an effective heat tolerance trait.
Heat-tolerant traits at the flowering stage were supportive of the positive results of the
research. PV and rice yield could be reduced by a higher temperature above 35C. The
high relative humidity in the open field at MR could reduce rice grain production
compared to the open field at BG. Combined research using both heat-tolerant and
drought-tolerant rice varieties and further observation of rice flower-related parameters
such as anther dehiscence rate, stigma receptivity, and spikelet temperature along with
the day/night temperature records should be made in future research. The effect of light
intensity should also be taken into consideration and observed.

33
 References

1. Abeysiriwardena, D.S., Ohba, K. and Maruyama, A., 2002. Influence of

temperature and relative humidity on grain sterility in rice, 30(2), pp.35-37.
2. Bheemanahalli, R., Sathishraj, R., Manoharan, M., Sumanth, H.N., Muthurajan, R.,
Ishimaru, T. and Krishna, J.S., 2017. Is early morning flowering an effective trait
to minimize heat stress damage during flowering in rice? Field crops research, 203,
pp.238-242.
3. Champoux, M.C., Wang, G., Sarkarung, S., Mackill, D.J., O'Toole, J.C., Huang, N.
and McCouch, S.R., 1995. Locating genes associated with root morphology and
drought avoidance in rice via linkage to molecular markers. Theoretical and
Applied Genetics, 90(7), pp.969-981.
4. Chaudhary, S., Devi, P., Bhardwaj, A., Jha, U.C., Sharma, K.D., Prasad, P.V.,
Siddique, K.H., Bindumadhava, H., Kumar, S. and Nayyar, H., 2020. Identification
and characterization of contrasting genotypes/cultivars for developing heat
tolerance in agricultural crops: Current status and prospects. Frontiers in Plant
Science, 11, p.1505.
5. Cheabu, S., Panichawong, N., Rattanametta, P., Wasuri, B., Kasemsap, P., Arikit,
S., Vanavichit, A. and Malumpong, C., 2019. Screening for spikelet fertility and
validation of heat tolerance in a large rice mutant population. Rice Science, 26(4),
pp.229-238.
6. Climate Risk Country Profile: Sri Lanka (2021): The World Bank Group and the
Asian Development Bank. Available from:
https://climateknowledgeportal.worldbank.org/sites/default/files/2021-05/15507-
WB_Sri%20Lanka%20Country%20Profile-WEB.pdf [Accessed 28 August, 2022].
7. Cruz, R.T. and O'toole, J.C., 1984. Dryland rice response to an irrigation gradient
at flowering stage 1. Agronomy Journal, 76(2), pp.178-183.
8. Department of Agriculture. Available from:
https://doa.gov.lk/rrdi_climate_rainfallseasons/ [Accessed 29 May 2022].
9. Department of Meteorology Sri Lanka, “Climate of Sri Lanka,” 2022. Available
from:http://www.meteo.gov.lk/index.php?option=com_content&view=article&id
=94&Itemid=310&lang=en&lang=en. [Accessed 28 August, 2022].
10. Department of Meteorology. Available from:

34
 http://www.meteo.gov.lk/index.php?option=com_content&view=article&id=94&I
temid310&lang=en&lang=en [Accessed 28 August, 2022].
11. Fahad, S., Ihsan, M.Z., Khaliq, A., Daur, I., Saud, S., Alzamanan, S., Nasim, W.,
Abdullah, M., Khan, I.A., Wu, C. and Wang, D., 2018. Consequences of high
temperature under changing climate optima for rice pollen characteristics-concepts
and perspectives. Archives of Agronomy and Soil Science, 64(11), pp.1473-1488.
12. Gupta, R., Sutradhar, H., Chakrabarty, S.K., Ansari, M.W. and Singh, Y., 2015.
Stigmatic receptivity determines the seed set in Indian mustard, rice and wheat
crops. Communicative & Integrative Biology, 8(5), p.e1042630.
13. Horie, T., Matsui, T., Nakagawa, H. and Omasa, K., 1996. Effects of elevated CO
2 and global climate change on rice yield in Japan. In Climate change and plants in
East Asia (pp. 39-56). Springer, Tokyo.
14. Jagadish, S.V.K., Craufurd, P.Q. and Wheeler, T.R., 2008. Phenotyping parents of
mapping populations of rice for heat tolerance during anthesis. Crop Science, 48(3),
pp.1140-1146.
15. Jagadish, S.V.K., Muthurajan, R., Oane, R., Wheeler, T.R., Heuer, S., Bennett, J.
and Craufurd, P., 2010. Physiological and proteomic approaches to address heat
tolerance during anthesis in rice (Oryza sativa L.). Journal of experimental botany,
61(1), pp.143-156.
16. Khan, S., Anwar, S., Ashraf, M.Y., Khaliq, B., Sun, M., Hussain, S., Gao, Z.Q.,
Noor, H. and Alam, S., 2019. Mechanisms and adaptation strategies to improve heat
tolerance in rice. A review. Plants, 8(11), p.508.
17. Kim, H.J., Song, E.J. and Lee, K.J., 2002. Proteomic analysis of protein
phosphorylations in heat shock response and thermotolerance. Journal of Biological
Chemistry, 277(26), pp.23193-23207.
18. Koh, S., Lee, S.C., Kim, M.K., Koh, J.H., Lee, S., An, G., Choe, S. and Kim, S.R.,
2007. T-DNA tagged knockout mutation of rice OsGSK1, an orthologue of
Arabidopsis BIN2, with enhanced tolerance to various abiotic stresses. Plant
molecular biology, 65(4), pp.453-466.
19. Koike, S., Yamaguchi, T., Ohmori, S., Hayashi, T., Yatou, O. and Yoshida, H.,
2015. Cleistogamy decreases the effect of high temperature stress at flowering in
rice. Plant Production Science, 18(2), pp.111-117.
20. Krishnan, P., Ramakrishnan, B., Reddy, K.R. and Reddy, V.R., 2011. High-
temperature effects on rice growth, yield, and grain quality. Advances in agronomy,

35
 111, pp.87-206.
21. Li, X., Lawas, L.M., Malo, R., Glaubitz, U., Erban, A., Mauleon, R., Heuer, S.,
Zuther, E., Kopka, J., Hincha, D.K. and Jagadish, K.S., 2015. Metabolic and
transcriptomic signatures of rice floral organs reveal sugar starvation as a factor in
reproductive failure under heat and drought stress. Plant, Cell & Environment,
38(10), pp.2171-2192.
22. Lin, C.J., Li, C.Y., Lin, S.K., Yang, F.H., Huang, J.J., Liu, Y.H. and Lur, H.S.,
2010. Influence of high temperature during grain filling on the accumulation of
storage proteins and grain quality in rice (Oryza sativa L.). Journal of Agricultural
and Food Chemistry, 58(19), pp.10545-10552.
23. Lin, S.K., Chang, M.C., Tsai, Y.G. and Lur, H.S., 2005. Proteomic analysis of the
expression of proteins related to rice quality during caryopsis development and the
effect of high temperature on expression. Proteomics, 5(8), pp.2140-2156.
24. Lombardo, F., Kuroki, M., Yao, S.G., Shimizu, H., Ikegaya, T., Kimizu, M.,
Ohmori, S., Akiyama, T., Hayashi, T., Yamaguchi, T. and Koike, S., 2017. The
superwoman1‐cleistogamy2 mutant is a novel resource for gene containment in
rice. Plant biotechnology journal, 15(1), pp.97-106.
25. Matsui, T. and Omasa, K., 2002. Rice (Oryza sativa L.) cultivars tolerant to high
temperature at flowering: anther characteristics. Annals of botany, 89(6), pp.683-
687.
26. McInnis, S.M., Desikan, R., Hancock, J.T. and Hiscock, S.J., 2006. Production of
reactive oxygen species and reactive nitrogen species by angiosperm stigmas and
pollen: potential signalling crosstalk?. New Phytologist, 172(2), pp.221-228.
27. Mittler, R., Merquiol, E., Hallak‐Herr, E., Rachmilevitch, S., Kaplan, A. and Cohen,
M., 2001. Living under a ‘dormant’canopy: a molecular acclimation mechanism of
the desert plant Retama raetam. The Plant Journal, 25(4), pp.407-416.
28. Mohapatra, P.K. and Sahu, B.B., 2022. Importance of Rice as Human Food. In
Panicle Architecture of Rice and its Relationship with Grain Filling (pp. 1-25).
Springer, Cham.
29. O'Toole, J.C. and Namuco, O.S., 1983. Role of Panicle Exsertion in Water Stress
Induced Sterility 1. Crop Science, 23(6), pp.1093-1097.
30. Poli, Y., Basava, R.K., Panigrahy, M., Vinukonda, V.P., Dokula, N.R., Voleti, S.R.,
Desiraju, S. and Neelamraju, S., 2013. Characterization of a Nagina22 rice mutant
for heat tolerance and mapping of yield traits. Rice, 6(1), pp.1-9.

36
31. Rang, Z.W., Jagadish, S.V.K., Zhou, Q.M., Craufurd, P.Q. and Heuer, S., 2011.
Effect of high temperature and water stress on pollen germination and spikelet
fertility in rice. Environmental and Experimental Botany, 70(1), pp.58-65.
32. Satake, T. and YOSHIDA, S., 1978. High temperature-induced sterility in indica
rices at flowering. Japanese Journal of Crop Science, 47(1), pp.6-17.
33. Scirp.org. 2022. Hatfield, J., Takle, G., Grotjahn, R., Holden, P., Izaurralde, R.C.,
Mader, T., Marshall, E. and Liverman, D. (2014) Agriculture. In Melillo, J.M.,
Richmond, T.C. and Yohe, G.W., Eds., Climate Change Impacts in the United
States The Third National Climate Assessment, U.S. Global Change Research
Program, 150-174. - References - Scientific Research Publishing. [online]
Available
at:<https://www.scirp.org/(S(351jmbntvnsjt1aadkposzje))/reference/referencespap
ers.aspx?referenceid=2552161> [Accessed 28 August 2022].
34. Sehgal, S.K., Kaur, S., Gupta, S., Sharma, A., Kaur, R. and Bains, N.S., 2011. A
direct hybridization approach to gene transfer from Aegilops tauschii Coss. to
Triticum aestivum L. Plant breeding, 130(1), pp.98-100.
35. Senguttuvel, P., Jaldhani, V., Raju, N.S., Balakrishnan, D., Beulah, P., Bhadana,
V.P., Mangrauthia, S.K., Neeraja, C.N., Subrahmanyam, D., Rao, P.R. and
Hariprasad, A.S., 2022. Breeding rice for heat tolerance and climate change
scenario; possibilities and way forward. A review. Archives of Agronomy and Soil
Science, 68(1), pp.115-132.
36. Shelton, S., Pushpawela, B. and Liyanage, G., 2022. The long-term trend in the
diurnal temperature range over Sri Lanka from 1985 to 2017 and its association
with total cloud cover and rainfall. Journal of Atmospheric and Solar-Terrestrial
Physics, 227, p.105810.
37. Song, Y., Wang, C., Linderholm, H.W., Fu, Y., Cai, W., Xu, J., Zhuang, L., Wu,
M., Shi, Y., Wang, G. and Chen, D., 2022. The negative impact of increasing
temperatures on rice yields in southern China. Science of The Total Environment,
820, p.153262.
38. Tenorio, F.A., Ye, C., Redoña, E., Sierra, S., Laza, M. and Argayoso, M.A., 2013.
Screening rice genetic resources for heat tolerance FA Tenorio1, C. Ye1, E.
Redoña1, S. Sierra1, M. Laza1 and MA Argayoso1. Sabrao J. Breed. Genet, 45(3),
pp.371-381.
39. Van Huong, N., Minh Nguyet, B.T., Van Hung, H., Minh Duc, H., Van Chuong,

37
 N., Do Tri, M., Van Hien, D. and Van Hien, P., 2022. Economic impact of climate
change on agriculture: a case of Vietnam.
40. Wada, H., Hatakeyama, Y., Nakashima, T., Nonami, H., Erra-Balsells, R., Hakata,
M., Nakata, K., Hiraoka, K., Onda, Y. and Nakano, H., 2020. On-site single pollen
metabolomics reveals varietal differences in phosphatidylinositol synthesis under
heat stress conditions in rice. Scientific reports, 10(1), pp.1-11.
41. Wang, W., Cui, K., Hu, Q., Wu, C., Li, G., Huang, J. and Peng, S., 2021. Response
of spikelet water status to high temperature and its relationship with heat tolerance
in rice. The Crop Journal, 9(6), pp.1344-1356.
42. Weerakoon, W.M.W., Maruyama, A. and Ohba, K., 2008. Impact of humidity on
temperature‐induced grain sterility in rice (Oryza sativa L). Journal of Agronomy
and Crop Science, 194(2), pp.135-140.
43. Wu, C., Cui, K., Hu, Q., Wang, W., Nie, L., Huang, J. and Peng, S., 2019. Enclosed
stigma contributes to higher spikelet fertility for rice (Oryza sativa L.) subjected to
heat stress. The Crop Journal, 7(3), pp.335-349
44. Yang, Y., Zhang, C., Zhu, D., He, H., Wei, Z., Yuan, Q., Li, X., Gao, X., Zhang,
B., Gao, H. and Wang, B., 2022. Identifying candidate genes and patterns of heat-
stress response in rice using a genome-wide association study and transcriptome
analyses. The Crop Journal.
45. Ye, C., Li, X., Redoña, E., Ishimaru, T. and Jagadish, K., 2021. Genetics and
Breeding of Heat Tolerance in Rice. Rice Improvement, p.203.
46. Zhou, H., Wang, Y., Zhang, Y., Xiao, Y., Liu, X., Deng, H., Lu, X., Tang, W. and
Zhang, G., 2022. Comparative Analysis of Heat-Tolerant and Heat-Susceptible
Rice Highlights the Role of OsNCED1 Gene in Heat Stress Tolerance. Plants, 11(8),
p.1062

38