Genotypic variation for tolerance to low temperature stress in rice

The objectives of this study were to identify genotypic variation for tolerance to low temperature stress in rice using a population developed from a cross between low temperature tolera

Genotypic Variation For Tolerance To Low Temperature Stress In Rice

Long Viet Ha Bachelor of Crop Science at

Thai Nguyen University of Agriculture and Forestry, Vietnam in 2004

A thesis submitted for the degree of Master of Philosophy at

The University of Queensland in 2015

School of Agriculture and Food Sciences

Abstract

Lack of irrigation water for flooded cultivation and low temperature during the reproductive phase are factors limiting rice production in the Riverina region of New South Wales, Australia The adoption of aerobic technique could be beneficial as it requires less irrigation water, but a major limitation of aerobic cultivation is that there is no insulation effect of flooded water in the field to protect young panicles from low air temperature, causing spikelet sterility Suitable varieties need to tolerate low temperature at the booting stage, grow well under aerobic condition and be adapted to other conditions such as different rates of nitrogen application Three controlled temperature glasshouse experiments were conducted at The University of Queensland, Brisbane, Australia from 2013-15 The objectives of this study were to identify genotypic variation for tolerance to low temperature stress in rice using a population developed from a cross between low temperature tolerant and susceptible parents, and to determine the genotypic consistency in spikelet sterility under different flood and aerobic conditions and levels of nitrogen application, when exposed

to low temperature during the booting stage

In all experiments two controlled temperature glasshouses were utilised The plants were grown in the warm glasshouse at 28oC/19oC except for 14 days when they were exposed to low temperature at 21oC/15oC at the booting stage Experiment 1 phenotyped 101 F5 Kyeema//Kyeema/Norin PL8 (KKN) lines alongside the parent varieties, Kyeema and Norin PL8; and the recently released Australian low temperature tolerant variety Sherpa Experiment

2 and 3 consisted of 20 and 10 genotypes respectively, which were selected from Experiment

1 In these experiments, there were also plants grown continuously in the warm glasshouse Experiment 1 was grown under flooded conditions, while Experiment 2 and 3 were grown under aerobic and flooded conditions with Experiment 3 also utilising two nitrogen application rates (0 and 150kg/ha, -N and +N treatments) The plants were exposed to low temperature at the late booting stage in Experiment 1 and the early booting stage in Experiment 2 and 3 Spikelet sterility was used as the criterion for low temperature tolerance/susceptibility

Most genotypes in this study showed a consistency in spikelet sterility under low temperature at the booting stage between experiments (r=0.83 between Experiment 1 and 2, r=0.93 between Experiment 1 and 3, and r=0.90 between Experiment 2 and 3) under standard flood conditions Genotype’s response to low temperature under different growing conditions was also similar between flooded and aerobic conditions (r=0.90) in Experiment 2, flooded +N and aerobic +N (r=0.90) and flooded -N and flooded +N (r=0.97) in Experiment 3

While there was no significant difference in mean spikelet sterility without low temperature exposure between aerobic and flooded conditions in both Experiment 2 and 3, mean spikelet sterility under low temperature was significantly higher for aerobic conditions in Experiment 2 (75.4% vs

51.7%) and Experiment 3 (70.8% vs 56.8%) than those of flooded conditions The mean low water temperature in the flooded condition (15.3oC) was 1.4oC higher than the air temperature, and this may have caused soil temperature difference, resulting in the difference in spikelet sterility.

Even if grown under warm conditions, some genotypes showed high spikelet sterility (≥37%) in Experiment 2 which was given high N application Experiment 3 showed that N application increased tillering and spikelet number per plant, leading to increased spikelet sterility under low temperature condition Furthermore, spikelet sterility was found to be correlated with total spikelet number per plant under flooded low temperature +N among 13 genotypes tested in Experiment 3 In this experiment, spikelet sterility was also significantly correlated with grain yield/plant but some genotypes which showed low spikelet sterility had low grain yield

In this study, 5 tolerant and susceptible genotypes were identified for flooded and aerobic conditions in controlled glasshouse experiments However, further research is required in the field

in order to confirm the consistency in spikelet sterility of those genotypes Other future research needs such as identifying mechanisms for the genotypic variation in low temperature tolerance is also suggested

Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis The content of my thesis

is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis

Long Viet Ha

Publications during candidature

No publications

Publications included in this thesis

No publications included

Contributions by others to the thesis

Significant contributions were made to the conception and design of the project as well as critical revision of the research data by Professor Shu Fukai, Dr Jaquie Mitchell and Dr Doug George

Statement of parts of the thesis submitted to qualify for the award of another degree

None

Acknowledgements

I sincerely thank my three supervisors from the School of Agriculture and Food Sciences, The University of Queensland, Professor Shu Fukai, Dr Jaquie Mitchell and Dr Doug George who offered continual encouragement, guidance and inspiration throughout my study

I also sincerely thank professional English editor from the School of Agriculture and Food Sciences, The University of Queensland, Dr John M Schiller who offered encouragement and motivation, particularly for editing academic English of my thesis

I would like to thank Mr Christopher Proud for his technical support - without him this study would not have been conducting well Thank you to Mr Thinh Tran who provided invaluable assistance

in the analysis of the experiments during the study I acknowledge Ms Zuziana Susanti who was willing to help and share knowledge during the conduct of the experiments

Finally, thank you to my wife, Anh Vu and my daughter, Anh Ha for their love and encouragement, especially during the final stages of the study when our beautiful daughter Chi was born

Keywords

Rice, low temperature tolerance, glasshouse, flooded condition, aerobic condition, low temperature during the booting stage and nitrogen application

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 070302, Agronomy, 80%

ANZSRC code: 070305 Crop and Pasture Improvement, 20%

Fields of Research (FoR) Classification

FoR code: 0703, Crop Pasture and Production, 80%

FoR code: 0701, Agriculture, Land and Farm Management, 20%

TABLEOFCONTENTS

CHAPTER 1.INTRODUCTION 16

1.1.GENERAL INTRODUCTION 16

CHAPTER 2.LITERATURE REVIEW 18

2.1.INTRODUCTION 18

2.2.RICE GROWTH AND DEVELOPMENT 19

2.2.1.VEGETATIVE PHASE 20

2.2.1.1 Seed germination 20

2.2.1.2 Seedling emergence 20

2.2.1.3 Tillering 21

2.2.1.4 Stem elongation 21

2.2.2.REPRODUCTIVE PHASE 21

2.2.2.1 Panicle initiation to heading stage 22

2.2.2.2 Flowering 23

2.2.2.3 Grain filling and ripening 24

2.3.RICE YIELD AND THE EFFECT OF ENVIRONMENTAL CONDITIONS 24

2.4.THE EFFECT OF LOW TEMPERATURE AT DIFFERENT GROWTH STAGES 26

2.4.1 Vegetative phase 26

2.4.1.1 Germination stage 26

2.4.2 Reproductive phase 27

2.4.2.1 Booting stage 27

2.4.2.2 Mechanisms of low temperature induced male sterility 29

Anther respiration 30

Cytological abnormalities 30

Carbohydrate metabolism 31

Enzyme activity 31

2.4.2.3 Flowering stage 32

2.7.AEROBIC RICE 32

2.5.NITROGEN EFFECT ON RICE GROWTH 33

2.6.CONCLUSIONS OF LITERATURE REVIEW AND FOCUS OF THIS STUDY 34

CHAPTER 3 EXPERIMENT 1 GENOTYPIC VARIATION IN SPIKELET STERILITY WITHIN A POPULATION EXPOSED TO LOW TEMPERATURE AT THE BOOTING STAGE 35

3.1.INTRODUCTION 35

3.2.MATERIALS AND METHODS 36

3.2.1 Genetic materials 36

3.2.2 Cultural details 36

3.2.3 Data collections and analysis 37

3.3.RESULTS 38

3.3.1 Air temperatures 38

3.3.2 Heading date 39

3.3.3 Spikelet Sterility 40

3.3.4 Other characters 43

3.4.DISCUSSION 44

3.5.CONCLUSIONS 46

CHAPTER 4 EXPERIMENT 2 THE EFFECTES OF LOW TEMPERATURE AT THE BOOTING STAGE AND AEROBIC CONDITIONS ON SPIKELET STERILITY IN 20 SELLECTED GENOTYPES AND 3 VARIETIES 48

4.1.INTRODUCTION 48

4.2.MATERIALS AND METHODS 49

4.2.1 Genetic materials 49

4.2.2 Cultural details 49

4.2.3 Water and temperature treatments 50

4.2.4 Measurements 50

4.2.5 Data Analysis 51

4.3.RESULTS 51

4.3.1 Air and water temperatures 51

4.3.2 Time of heading 51

4.3.3 Plant height 53

4.3.4 Spikelet Sterility 55

4.3.5 Spikelet number per panicle 57

4.4.DISCUSSION 59

4.5.CONCLUSIONS 60

CHAPTER 5 EXPERIMENT 3 GENOTIPIC VARIATION FOR SPIKELET STERILITY BETWEEN FLOODED AND AEROBIC CONDITIONS UNDER TWO DIFFERENT LEVEL OF NITROGEN FERTILIZATION EXPOSED TO LOW TEMPERATURE AT THE BOOTING STAGE 62

5.1.INTRODUCTION 62

5.2.MATERIALS AND METHODS 63

5.2.1 Genetic materials 63

5.2.2 Cultural details 63

5.2.3 Measurements 64

5.2.4 Data Analysis 64

5.3.RESULTS 64

5.3.1 Air temperatures 64

5.3.2 Heading date 65

5.3.3 Plant height 67

5.3.4 Spikelet Sterility 68

5.3.4.1 Spikelet sterility per main stem 68

5.3.4.2 Spikelet sterility/plant 71

5.3.5 Spikelet number 72

5.3.5.1 Spikelet number per plant 72

5.3.5.2 Spikelet number per panicle 73

5.3.6 Panicle number per plant 75

5.3.7 Grain yield per plant 76

5.4.DISCUSSION 78

5.5.CONCLUSIONS 81

CHAPTER 6.MAJOR FINDINGS AND FUTURE RESEARCH 82

6.1.INTRODUCTION 82

6.2.MAJOR FINDINGS 82

6.3.FUTURE RESEARCH REQUIREMENT 84

LIST OF TABLES Table 2.1 Response of the rice plants to varying temperatures at different growth stages (Yoshida,

1977) 20 Table 2.2 Duration of each development stage of a panicle or spikelet (days) (Matsushima, 1966) 22 Table 3.1 Days-to-heading of the 3 varieties in the warm glasshouse and exposed to low

temperatures 39 Table 3.2 The 10 most low temperature tolerant and 10 most susceptible genotypes based on

spikelet sterility (%) of 96 genotypes from Kyeema//Kyeema/Norin PL8 crosses and 3 varieties 42 Table 3.3 Ten selected tolerant and susceptible genotypes (* denotes genotypes selected from Ms

Zuziana Susanti’s experiment) 46 Table 4.1 Air and water temperatures (oC) in the low and warm temperature glasshouses 51 Table 4.2 Heading date and heading delay of 20 genotypes and 3 varieties under four growing

conditions (flooded/aerobic and warm/low temperatures) 52 Table 4.3 Plant height (cm) of 20 genotypes and 3 varieties under low and warm temperature

environments in aerobic and flooded conditions 54 Table 4.4 Spikelet sterility (%) of 20 genotypes and 3 varieties under flooded/aerobic/ and

warm/low temperature conditions 56 Table 4.5 Correlation coefficient matrix of spikelet sterility under four growing conditions

(flooded/aerobic and warm/low temperature conditions) 57 Table 4.6 Spikelet number/panicle of 20 genotypes and 3 varieties under flooded/aerobic/ and

warm/low temperature conditions 58 Table 4.7 Five most consistently low temperature tolerant and susceptible rice genotypes under

flooded condition from Experiment 2 60 Table 5.1 Air temperatures (oC) in the warm and low temperature glasshouse 64 Table 5.2 Heading date of 10 genotypes and 3 varieties in three water and N conditions (flooded -

N, flooded +N and aerobic +N) in two temperature conditions (warm and low) 65 Table 5.3 Plant height (cm) of 10 genotypes and 3 varieties under the different growing environments 68 Table 5.4 Spikelet sterility per main stem (%) of 10 genotypes and 3 varieties under the different

growing environments 69 Table 5.5 Spikelet sterility per plant (%) of 10 genotypes and 3 varieties under the different growing

environments 71 Table 5.6 Spikelet number per plant of 10 genotypes and 3 varieties under the different growing

environments 72

Table 5.7 Spikelet number per panicle of 10 genotypes and 3 varieties under different growing

environments 74 Table 5.8 Panicle number per plant of 10 genotypes and 3 varieties under different growing

environments 76 Table 5.9 Grain yield (g) per plant of 10 genotypes and 3 varieties under different growing

environments 77 Table 6.1 The average percentage of spikelet sterility of 10 genotypes and 3 varieties among 3

experiments under flooded low temperature conditions 83

LIST OF FIGURES

Figure 3.1 Minimum and maximum air temperatures in the warm glasshouse 38

Figure 3.2 Minimum and maximum air temperatures in the low temperature glasshouse 38

Figure 3.3 Average number of days required before heading 40

Figure 3.4 The effect of number of days exposed to 12oC on spikelet sterility 40

Figure 3.5 Frequency distribution of spikelet sterility of 96 genotypes and 3 varieties 43

Figure 3.6 The relationship between days-to-heading and spikelet sterility on the main stem of individual plants exposed to low (14oC) night temperature 43

Figure 4.1 Average number of days required before heading in different growing environments 53

Figure 5.1 Average number of days required before heading in the different treatments and growing environments 66

Figure 5.2 Relationship for spikelet sterility/main stem between flooded warm temperature +N and other two warm temperature conditions (a) and three low temperature conditions (b) 70

Figure 5.3 Relationship between spikelet number per plant and spikelet sterility per plant under flooded low temperature +N treatment 73

Figure 5.4 The relationships between grain yield per plant and spikelet sterility per plant in three water and N growing environments (a) flooded -N, (b) flooded +N and (c) aerobic +N, all under low temperature conditions 78

Figure 5.5 The consistancy in spikelet sterility between Experiments 2 and 3 in flooded low temperature -N (a) and aerobic low temperature (b) 80

Experiment 2 99 Appendix 4 Air temperatures in the warm and low temperature glasshouses in Experiment 3 100 Appendix 5 Correlation coefficient matrix of spikelet number/panicle and spikelet sterility/main

stem of 10 genotypes and 3 varieties under warm and low temperature conditions (flooded -N and +N and aerobic +N) in Experiment 3 101

CHAPTER 1 INTRODUCTION 1.1 General introduction

Rice (Oryza sativa L.) is grown widely as an annual crop in many countries in temperate and

tropical zones, in latitude from 53°N to 40°S (Lu and Chang, 1980, Rural Industries Research & Development Coporation, 2006) It is a staple food for over 3 billion people in the world (IRRI, 2011) Rice is the second most important cereal crop, and is planted on approximately 155 million ha world-wide (Van Nguyen and Ferrero, 2006) Rice yields are affected by a range of environmental conditions, including drought, high and low temperatures, salinity, and flooding

Low temperature is a serious problem and capable of reducing rice grain yields in many temperate zones and high altitude regions (Mackill and Lei, 1997) Throughout the world, low temperatures have been estimated to cause an annual grain loss of about 10% (Garg et al., 2003) Low temperature stress affects different growth stages of the rice plant, from germination to grain maturity (Andaya and Mackill, 2003, Ye et al., 2009) Days-in-low temperature (i.e duration) is also an important factor that determines the effects of low temperature on rice yield The most critical stage for low temperature effect is the young microspore stage, which occurs during booting (Farrell et al., 2006b, Gunawardena

et al., 2003a, Shimono et al., 2007, Ye et al., 2010) During the booting stage, a long period of exposure to low temperature results in an increase of spikelet sterility (Heenan 1984) Low temperature during the young microspore stage results in spikelet sterility, because the development of microspore under low temperatures at this time is abnormal, resulting in a decline in grain yield (Satake 1976) The cause of low yield is believed to be most likely due to the failure of the male organs resulting in spikelet sterility (Farrell et al., 2006b, Gunawardena et al., 2003b, Heenan, 1984, Shimono et al., 2007)

The level of low temperature tolerance in rice is genetically controlled, and there is wide varietal difference in the levels of low temperature tolerance Satake and Shibata (1992) exposed 19 japonica varieties to identical day/night air temperature treatments (12°C/12°C for 3 days) at the booting stage and found that spikelet sterility varied from 17% to 79% among the varieties Farrell et al (2006a) exposed 23 rice varieties to cool water (maintained at about 19°C for 25 days) at the booting stage and the resulting spikelet sterility ranged from 8% to 70% among the varieties Most studies on genotypic variation used varieties with different genetic backgrounds, and the use of populations derived from a cross for such study is limited A number of studies have been conducted to determine the physiological mechanism(s) responsible for low temperature induced spikelet sterility (Hayase et al., 1969; Satake, 1976; Satake et al., 1987, 1988) Spikelet sterility levels which are usually determined

at maturity is the most popular trait that indentifies low temperature tolerant varieties in rice (Satake and Shibata, 1992)

In Australia, water is one of the most general factors which limit the production of agricultural systems Recently, agriculturalists across most regions of Australia have suffered from insufficient available rainfall (The Commonwealth Scientific and Industrial Research Organization, 2014) However, most rice varieties grown commercially in Australia were developed for full irrigation conditions, and varieties suitable for water limited conditions may be required because of reduced availability of irrigation water in recent years The development of low temperature tolerance in rice would provide further potential for a significant increase in rice yield (Rural Industries Research & Development Coporation, 2006) In addition, the adoption of aerobic techniques could be beneficial

in the Riverina district in New South Wales (Rice Growers' Association of Australia 2012) Using aerobic cultivation techniques would allow for the stabilization of yields, with less variability during periods of drought, and possibly increased total production However, a major limitation to aerobic cultivation in many rice growing regions around the world is low temperature induced spikelet sterility (Farrell et al., 2006a, Farrell et al., 2006b) It could be expected that the effects of low temperature induced sterility might be amplified even further under aerobic conditions, as low temperature exposure in shallow water during the microspore stage doubles sterility when compared with deep water (Borrell et al., 1997, Gunawardena et al., 2003b)

Another factor determining low temperature tolerance is level of plant nutrition, particularly N availability to the plants Haque (1988) found that the level of low temperature induced spikelet sterility in rice at the reproductive stage increased further under high N conditions (i.e in response to higher levels of application of N fertilizer) Under low temperature conditions at the booting stage, the application of high N rates increases spikelet sterility induced by increasing tillering and spikelet numbers per plant, leading to a reduction in the number of engorged pollen grains per anther (Gunawardena et al., 2003a)

The objectives of this study were to identify genotypic variation for tolerance to low temperature stress in rice using a population developed from a cross between low temperature tolerant and susceptible parents, and to determine the genotypic consistency in spikelet sterility under different water conditions and levels of nitrogen application, when exposed to low temperature during the booting stage

CHAPTER 2 LITERATURE REVIEW 2.1 Introduction

In Australia, rice is grown mostly in the Riverina region (35°S146°E, 120m) of the state of New South Wales (NSW) (Lewin and Heenan, 1987) In this region, it is in the Murrumbidgee, Coleambally and Murray irrigation areas where the rice is grown and where more than 90% of the crops are irrigated, with the Murrumbidgee and Murray rivers being the main water sources (Quayle

et al., 2006) This region is regarded as providing favorable conditions for rice cropping, on account of available water resources, land and climate (Kealey et al., 2000)

Rice crops planted in the Riverina region in southern NSW are generally high yielding because this temperate region provides high levels of solar radiation during the cropping period; there is also usually enough water for irrigation and low night temperatures during the ripening phase (Kelleher, 1988) The total area planted to rice annually in Australia is about 150,000 ha depending on water availability, and this produces over 1 million tons (Quayle et al., 2006) Rice crops are grown in Australia once per year, with planting taking place in middle to late spring (October-November) and harvesting during autumn (April-May)

Rural Industries Research & Development Coporation (2006) reported that the Australian rice industry has improved its water use efficiency by 60%; this means that more rice is able to be grown with less water One of the adaptation systems for water shortage used overseas is aerobic rice, and it has been possible to develop input responsive varieties with high yield potential which are adapted

to less water use when grown under aerobic, non-saturated soil conditions (Bouman et al., 2005) Aerobic rice production developed overseas is focused on a separate target environment to the traditional upland rice environment, where yields are generally low due to the shortage of water during the critical growth stages Most aerobic rice varieties have been developed by crossing traditional upland varieties with drought tolerance traits, and improved lowland varieties with high yield potential (Prasad, 2011) Hence, the aerobic cultivation technique could be adopted in the Riverina district in New South Wales (Rice Growers' Association of Australia 2012)

Low temperature during the reproductive phase is one of the major environmental constraints in the rice growing areas in the Riverina region of Australia (Lewin and Heenan, 1987) Low temperature during late January and early February can cause spikelet sterility, resulting in reduced grain yield (Farrell et al 2001b) The variation of temperature, including prolonged periods of low temperature, is one of the main production constraints in temperate rice producing countries in general, and in Australia in particular For instance, Williams (1997) found that a 4oC below average night temperatures for 30 days during the reproductive phase in 1996 resulted in yield reduction of 25% in Australia

Low temperatures during the reproductive phase reduce yield by more than 1 ton ha-1 every 4 years and more than 2 tons ha-1 every 10 years in Australia (Farrell et al., 2001a) According to a report from (Rural Industries Research & Development Coporation, 2006), low temperature damage during the reproductive phase, particularly at the young microspore stage, leads to high percentage of spikelet sterility and accounts for annual losses of up to 120 million Australian dollars

Heenan (1984) reported that high rates of nitrogen (N) application can increase the impact of low temperature on grain yield, particularly when the N is applied at the pre-flooding (PF) stage rather than at panicle initiation (PI) Furthermore, Gunawardena and Fukai (2005) found that high levels of

N application reduced grain yield by increasing spikelet sterility, particularly under low temperature conditions in the NSW rice industry Spikelet sterility in the NSW rice growing regions varies from season to season and is exacerbated by high rates of N application This variation is affected by the amount of N which is applied at pre-flooded and panicle initiation when rice exposed to low temperature during panicle development, especially at the early booting stage, which is most sensitive to low temperature effects Gunawardena et al (2003b) found that by limiting the number of tillers per plant, which were controlled by removing unwanted tillers, the total number of spikelet per plant were decreased and this reduction maintained a large number of engorged pollen grains per anther resulted in a reduction in spikelet sterility

The identification and selection of genotypes for low temperature tolerance is potentially essential for the improvement of rice production in rice growing environments under low temperature conditions

2.2 Rice growth and development

Most cultivated rice in tropical and subtropical areas has a growth period of 120 to 180 days from germination to maturity, the actual duration depends on the varieties and environmental conditions (Yoshida, 1981a) There are three main phases in rice plant growth and development: the vegetative phase (germination to panicle initiation), the reproductive phase (panicle initiation to flowering), and the grain filling phase (flowering to maturity) (Nakagawa and Horie, 1997)

Table 2.1 Response of the rice plants to varying temperatures at different growth stages

(Yoshida, 1977) Growth stage Critical temperature (

o C) Low High Optimum

by temperature and day length (IRRI, 2009) The optimum temperature for vegetative growth of rice ranges between 25 and 31oC (Table 2.1)

2.2.1.1 Seed germination

Germination of seed occurs when seed dormancy has been broken, the seed absorbs adequate water and it is exposed to temperatures varying from approximately l0 to 40oC Germination is defined as the appearance of the white tip of the coleoptile, which occurs a short time after a seed is placed in soil or water (Yoshida, 1981a) The coleoptiles begin growing in the embryo which becomes swollen after absorbing water; they then appear by forcing the ventral scale open The primordia for the first and then the second leaf also start growing insignificantly inside the coleoptiles Seminal roots protrude by breaking the coleorhiza and this completes the germination stage (Matsuo and Hoshikawa, 1993)

25-30oC, but that growth is not adversely affected for temperatures as high as 35oC

2.2.1.3 Tillering

The tillering stage begins when the 1st tiller appears, usually about 20 days after sowing at optimum temperature of 25-31oC It continues until the number of tillers is maximized which usually occurs about 40 days after sowing Tillers emerge from the axillary buds of the nodes and are replaced by leaves when they grow and develop (IRRI, 2009) Tillering commonly begins from the 2nd leaf When the 5th leaf on the main stem protrudes, the 1st leaf of the tiller appears on the axillary of the 2nd leaf on that stem Similarly, when the 6th leaf on the main stem protrudes, the 1st leaf of the tiller appears on the axillary of the 3rd leaf on that stem (Yoshida, 1981a) After emerging, the primary tillers give rise to secondary tillers In addition to the primary and secondary tillers, new tertiary tillers appear from the secondary tillers as the plant becomes taller and larger After that, the tillers successively lengthen when the plant enters the next stage which is called stem elongation (IRRI, 2009)

2.2.1.4 Stem elongation

Stem elongation may start before panicle initiation or it may occur during the latter part of the tillering stage It continues until full plant height is reached and is followed by heading Stem elongation lasts longer in varieties which have long growth duration (IRRI, 2009) In this regard rice varieties can be classified into three categories: groups: short duration varieties which mature between

105 and 120 days; medium duration varieties which mature between 120 and 150 days; and long duration varieties which mature after about 150 days For the short and medium duration varieties, stem elongation often begins about the time of initiation of panicle primordia In the long duration varieties, stem elongation usually begins before the initiation of panicle primordia In photoperiod sensitive varieties, the number and total length of stems increase in response to the lengthening of the photoperiod For photoperiod insensitive varieties, there is no effect of day length on stem elongation (Yoshida, 1981a)

2.2.2 Reproductive phase

The duration of the reproductive phase and the ripening phase are about the same for different rice varieties (IRRI, 2009)

2.2.2.1 Panicle initiation to heading stage

Table 2.2 Duration of each development stage of a panicle or spikelet (days) (Matsushima,

1966) Development stages

Duration of each stage

1953 (bad weather conditions)

1954 (normal weather conditions)

Differentiating stage of the neck node……….II 2.0 2.0

Early differentiating stage of primary rachis branches………IV 1.5 1.0

Middle differentiating stage of primary rachis branches……… V 2.0 2.0

Late differentiating stage of primary rachis branches……….VI 2.2 1.0

Duration of differentiating stage of primary rachis branches…… 5.7 4.0

Early differentiating stage of secondary rachis branches……… VII 1.2 1.0

Late differentiating stage of primary rachis branches……… VIII 2.0 1.5

Duration of differentiating stage of secondary rachis branches 3.2 2.5

Initial stage of spikelet differentiation……… IX 1.0 1.0

Early differentiating stage of spikelets……… X 3.0 3.0

Middle differentiating stage of spikelets………XI 1.0 1.2

Late differentiating stage of spikelets……… XI 4.0 3.2

Duration of differentiation of spikelets……… 9.0 8.4

Differentiating stage of pollen mother cell……… XIII 2.5 1.8

Reduction division stage……… XIV-XVIII 2.0 1.8

Early stage of extine formation……… XVIII 1.2 1.6

The initiation of panicle primordium begins approximately 30 days before heading, coinciding with the time that the 4th leaf from the top starts elongating The growth and development of the panicle begins with the differentiation of the neck-node and ends when the pollen is fully mature (Yoshida, 1981a) The differentiation of the neck-node stage takes approximately 2 days and the ripening stage

of pollen takes about 5.5 days (Table 2.2) (Matsushima, 1966) During the development of the panicle,

it becomes visible to the naked eye when the young panicle increases in length to approximately 1 mm (IRRI, 2009) The panicle keeps developing slowly and when it is 5cm long (approximately 7 days after the panicle becomes visible), the spikelet primordia is differentiated and the number of spikelets

is determined (De Datta, 1981) The duration of spikelet differentiation is approximately 9 days (Table

2.2) According to Yoshida (1981a), panicle growth and development ranges from 27 to 46 days, depending on variety and temperature conditions With medium duration varieties it takes approximately 33 days for the completion of panicle development under normal temperature conditions

Booting is most likely to starts first in the main culm At this stage, the senescence (ageing and dying) of leaves and non-bearing tillers, are visible at the base of the plant (IRRI, 2009) On the 8th day after the differentiation of the stamen and about 16 days before heading, the number of pollen grains starts increasing as a consequence of the mitosis of pollen mother cells (Table 2.2) At about 12 days before heading, each mother cell starts dividing from the callose wall and becomes irregular in shape The callose wall mostly disintegrates and the cell free space formed in the centre of the anther locule becomes filled with anther fluid (Matsuo and Hoshikawa, 1993)

About 10 days before heading, the 4 daughter cells are partitioned from each other in order to form microspores, and they line the inner surface of the tapetum Then exines and germ pores start forming

on the surface of the microspore cells (Table 2.2) Approximately 9 days before heading, the vacuoles

in the microspore cell grow larger and the cytoplasm turns into a net-like state (Matsuo and Hoshikawa, 1993)

Approximately 7 days before heading, the nucleus separates equally to a position opposite the germ pore The partition takes place in a radial direction in the microspore Approximately 6 days before heading, the starch of the grains starts accumulating near the germ pore of the cytoplasm and then steadily spreads out over the cytoplasm During this time, each of the generative cells divides into 2 wedge-shaped male nuclear cells near the vegetative nucleus (Matsuo and Hoshikawa, 1993)

A useful mode for identifying the reduction division stage (one of the most critical stages affected

by temperature stress) is the auricle distance between the flag leaf and penultimate leaf (the second last leaf) The strongest activity of the reduction division stage begins when the auricle distance between the flag leaf and penultimate leaf is -3 cm (when the flag leaf auricle is below the penultimate leaf auricle) and ends when they are +10 cm (when the flag leaf auricle is above the penultimate leaf auricle) (Yoshida, 1981a) In other words, when the auricle of the flag leaf protrudes from the penultimate leaf sheath, this coincides with the reduction division stage (Yoshida, 1981a)

Heading is also known as the panicle exertion stage It is identified when the tip of the panicle emerges from the flag leaf sheath The panicle keeps developing until it partially or fully emerges from the sheath (IRRI, 2009) Generally, heading date is also recognized as the time when 50% of the panicles have exerted The heading date is different both within a plant and among plants in a plot It usually takes 10-14 days for all the plants to complete heading (Yoshida, 1981a)

2.2.2.2 Flowering

Flowering is identified as a chain of events between the opening and closing of the spikelet At the

early stage of flowering, tip portions of the lemma and palea start opening (when floral parts mature), filaments begin elongating (when spikelets are well watered), and anthers begin appearing from the lemma and palea When the spikelet opens widely, the stigma tip becomes visible (Yoshida, 1981a)

At the same time as flowering, the filaments and stigma elongate from the spikelet and pollination occurs During the 10 to 20 minutes of flowering, filaments elongate rapidly more than 10 times This

is because of the rapid longitudinal elongation of each filament cell The capillary tubes at the centre also lengthen, and they change from spiral rings of the secondary wall into ring shapes Consequently, the function of conducting water of the capillary tubesis lost, which causes anthers to wither Anther walls become dry, marking the beginning of the pollen discharging mechanism After approximately

30 minutes, the lodicules begin to wither Therefore, the lemma gradually return to its original position, closing the spikelet (this occurs between 50 to 80 minutes, or sometimes up to 2.5 hours under some conditions, after opening) (Matsuo and Hoshikawa, 1993) Flowering often occurs before

or at the time when the lemma and palea open; hence, a number of pollen grains fall onto the stigma This explains why rice is regarded as a self pollinating plant The completion of flowering of all spikelets in a panicle takes 7-10 days and it takes about 5 days for most of the spikelets to complete flowering Therefore, it takes approximately 15-20 days for full flowering of spikelets in a rice crop (Yoshida, 1981a) The time of flowering of the rice plant during the day is dependent on temperature conditions For example, most of rice varieties in tropical zones often start flowering at about 8:00

am and finish at about 1:00 pm Temperatures in the range between 30-33oC are the most suitable for flowering in rice (Table 2.1)

2.2.2.3 Grain filling (ripening)

The grain filling (or ripening) stage begins at flowering (when the floret is fertilized) and ends at physiological maturity, usually taking 30 days (IRRI, 2009) The ripening stage follows ovary fertilization and is characterized by grain growth This period is recognized by an increase in grain size and weight (fully accumulated carbohydrate), changes in grain color and ageing of the leaves The grains are green at the beginning of ripening stage and when the grains are mature, they change to yellow (Yoshida, 1981a) Generally, the ripening duration is calculated from the start of heading to when the maximum grain weight is reached at physiological maturity Weather conditions may affect the grain filling stage For example, rainy days or low temperatures can prolong this stage, while sunny and warm days can shorten it (IRRI, 2009) According to Yoshida (1977), the optimum temperature for grain filling ranges between 20-25oC (Table 2.1)

2.3 Rice yield and the effect of environmental conditions

Rice yield is determined by the sink and source size (Lubis et al., 2003) The sink size is known as yield capacity (Yoshida, 1972) or the potential yield (Takami et al., 1990), and is determined by the number of spikelets/unit area and the single grain weight Fujita and Yoshida (1984) showed that

during rice growth, the sink size is determined before heading The source size is determined by structural carbohydrates what is full heading and dry matter production during the grain filling stage (Lubis et al., 2003, Tsukaguchi et al., 1996) During the growth of rice, the source size is identified around the time of heading (Yoshida, 1981a) The hull size, which determines the weight of single fully ripened grains, is also determined before heading (Horie, 2001) In their studies, Saitoh et al (1993) found that varieties which had stems with the highest carbohydrate concentration at heading and largest sink size, gave the highest yields

non-Grain yield (t/ha) = spikelet number/m2 × % filled spikelets × 1,000-grain weight (g) × 10-5

The spikelets of a plant consist of a mixture of those filled, partially filled and unfilled The filled spikelets are identified as grain The 1,000-grain weight is the mean weight of 1,000 grains (Yoshida, 1981a) Generally, the percentage of filled spikelets in rice is about 85% in optimum environments One of the best ways for improving the percentage of filled spikelets is by increasing the photo-assimilates during the period the spikelets are filling (Yoshida, 1981a)

Fageria (2007) found that panicle number is determined at the vegetative phase, the number of spikelets/panicle is determined at the reproductive phase, and grain weight and spikelet sterility at the spikelet filling stage

In general, the increase or decrease of rice yield is determined by weather conditions, cultivation and management practices (Yoshida, 1981a) A high level of spikelet sterility can be the result of a range

of unfavorable conditions, including low or high temperatures, drought, nitrogen deficiency and low solar radiation (Fageria, 2007) For example, Islam and Morison (1992) found that low irradiance during the ripening phase caused a 35% decrease in grain yield This reflected a combination of a reduction in the percentage of filled grains and a reduction in individual grain weight The influence of 22% and 52% shading levels during the reproductive and ripening phases was the same, but when 77% of shading was applied at the ripening phase, the influence was much more significant than at the reproductive phase Shimono et al (2005) showed that the number of sterile spikelets is high under low solar radiation at the grain filling stage This may be because of inadequate carbohydrates to support the growth of all spikelets Consequently, there may be an increase in the number of unfilled spikelets Evans and De Datta (1979) showed that there was a significant correlation between irradiance level and grain yield, particularly within the period 30 days before and after flowering, with the greatest effect being within the 20 day period before and after flowering

Yoshida (1981a) and Mae (1997) reported that nitrogen is the most essential fertilizer for rice Using

an appropriate level of N at approximately 20 days before heading has greatest potential for increasing final grain yield This is because the growth and development of young panicles before heading occur during this period Under field conditions after the panicle initiation stage, N is usually applied (top dressing) as the young panicles reach approximately 1-2 mm in length (about 23-25 days before

heading) The application of fertilizer N at this time is an effective way to increase the number of spikelets Fageria and Baligar (2001) found that the application of a suitable level of N accounted for approximately 91% of the variation in the number of panicles/m2, 75% of the variation in spikelet sterility and approximately 73% of the variation in 1000 grain weight In addition, N is an important factor for increasing the total number of spikelets/panicle, and spikelet fertility/panicle (Gunawardena

et al., 2003a) However, Gunawardena et al (2003a) found opposite results under low temperature (18/13oC) conditions during the booting stage, especially in microspore development, with the application of pre-flooded N (150 kg N/ha) significantly increasing spikelet sterility While there was

a statistically insignificant 19% decrease in the total number of spikelets/panicle with the N application, there was a statistically significant 51% increase in the percentage spikelet sterility/panicle under low temperature conditions Under pre-flooded N application, the more N that was applied, the greater the level of spikelet sterility under low temperature, reaching in excess of 60% at the highest level of N applied Adding N during both the pre-flooded period and at panicle initiation reduced the total pollen grain number and engorged pollen grain number/anther, because of increasing tillering

In addition to N, water also plays an essential role in rice plant development Rice is very sensitive

to water deficits, particularly about the time of flowering, with potential effects on the level of spikelet sterility (Cruz, 1984, Ekanayake et al., 1989) Water deficiency (when the amount of soil water drops below saturation) affects rice plant growth and development, and ultimately yield, due to the effects in relation to a reduction in leaf surface area, photosynthetic rate, and sink size (Bouman and Tuong, 2001) Leaf area expansion of most rice varieties decreases when the water content of the soil drops below saturation, and therefore, farmers always try to keep the water level at 5-10 cm above soil surface (flooded conditions) (Bouman et al., 2007)

2.4 The effect of low temperature at different growth stages

Low temperature affects both the vegetative and reproductive phases of rice plant development, and the ability of rice varieties to tolerate low temperature can vary during the crop’s growth cycle (Zhi-Hong et al., 2005) Low temperature at the reproductive phase induces spikelet sterility and this is the main factor responsible for low rice yields in cool climates (Shimono et al., 2005)

2.4.1 Vegetative phase

Depending on the timing, low temperature during the vegetative phase can result in poor germination, reduced seedling growth rates, leaf discoloration and reduced plant height and tiller number (Ye et al., 2009) Yoshida (1977) showed that the minimum critical temperature for the vegetative phase ranges between 10-16oC (Table 2.1)

2.4.1.1 Germination stage

Bertin et al (1996) found that low temperature stress (10 to 15oC for 15 days) slowed germination in

all varieties Bertin et al (1996) conducted an experiment with 15 varieties at 10oC for 10 days and

15oC for 5 days at the germination stage Some varieties treated at 10oC for 10 days germinated but had a very low germination (under 20%) while others failed to germinate At 15oC for 5 days, most varieties germinated slowly but with a low germination rate (most under 40%); only one variety had good germination (approximately 75%) Yoshida (1981a) also reported that when the temperature was as low as10oC, rice did not germinate Therefore, the minimum critical temperature for rice germination may be identified as 10oC (Table 2.1)

As a result of exposure to a constant low temperature of 15oC for 14 days, Ye et al (2009) indicated that the percentage of germination of low temperature susceptible varieties, such as Azucena, Pelde, YRL 39, WAB160, was low, with germination rates of 8, 14.7, 68.7 and 46.7%, respectively, which were significantly below the germination rates at 28oC (78.7, 77.3, 95.3 and 84.7%, respectively) Furthermore, the mean germination time of about 8.4 days for these varieties at low temperature was 6 days longer than that at 28oC (2.4 days)

2.4.2 Reproductive phase

Rice is highly sensitive to low temperature during the reproductive phase, including panicle initiation, booting and flowering stages; during panicle initiation and booting stages, low temperature delays heading and causes incompletion of panicle exsertion, irregular maturity and increased spikelet sterility (Takeoka, 1992, Yoshida, 1981a) Low temperature at booting and flowering can directly affect spikelet sterility The minimum critical temperatures for different stages of the reproductive phase range between 15-22oC (Table 2.1)

2.4.2.1 Booting stage

Booting is the most sensitive growth stage to low temperature, particularly the early pollen microspore stage (approximately 10-12 days prior to heading), because this stage coincides with pollen development (Satake and Hayase, 1970) Pollen development is significantly influenced by low temperatures and spikelet sterility is caused by abnormal pollen, resulting in a yield reduction (Farrell

et al., 2006a, Nakamura et al., 2000)

In general, each rice plant has 10 panicles with approximately 100 spikelets/panicle (Yoshida, 1981a) Varieties can be considered as low temperature tolerance when they achieve 90% fertilization, and this may require 650 pollen grains/anther and the length of the anther needs to be 1.85-2.00 mm (Satake, 1991) For example, Satake (1991) grew two varieties, Hayayuki (a low temperature tolerant variety) and Norin-20 (a susceptible variety) Every three days, he exposed plants of each variety to 3 days of low temperature (12oC) and then returned them to warm conditions (24/19oC); he did this continuously from 21 to 2 days before heading The results showed that the variety Hayayuki maintained around 600 pollen grains/anther, and anther length at about 1.85 to 2.00 mm, and achieved slightly higher than 90% fertilization For the variety Norin-20, pollen grains/anther were reduced to

between 400 and 500, anther length to 1.6 to 1.7 mm, and fertilization to 50-60% In his experiment Satake (1991) determined that the reduction in the number of pollen grains/anther, the length of anther and spikelet fertility were most sensitive at 11 days before heading for both varieties In order to evaluate low temperature tolerance of rice varieties at the booting stage under low day/night temperatures (20.8/15oC for 14 days), Ye et al (2009) measured the reduction of morphological characters, including plant height, panicle length, number of grains per panicle and spikelet fertility The results showed that these characters all significantly declined when exposed to low temperature treatment However, there were no significant correlation between the reductions in these characters and spikelet fertility, except for plant height There were also negative correlations between the reduction of plant height and panicle length, between plant height and number of grains per panicle, and between panicle length and number of grains per panicle For example, in their experiment, Ye et

al (2009) showed that under low day/night temperatures (20.8/15oC for 14 days), plant height of low temperature tolerant varieties, such as B55 and Banjiemang, declined by 10.3 and 9.7%, resulting in a reduction in spikelet fertility of 7.0 and 6.5%, respectively Meanwhile, for the susceptible varieties, Pelde and WAB38, there was a reduction in plant height of 17.2 and 24.1%, with a reduction in spikelet fertility of 78.2 and 86.4%, respectively Hence, it was suggested that the reduction of plant height and spikelet fertility can be used for the identification and evaluation of low temperature tolerant rice varieties at the booting stage

Farrell et al (2006a) determined that anther length, width and area, stigma area, number of engorged pollen grains and number of pollen grains intercepted by the stigma at flowering, are reduced at a mean low temperature of 13.7oC, and that these effects are related to an increase in spikelet sterility Among these traits, anther area and stigma area are considered the two important characteristics related to the variation in spikelet sterility in low water temperature Hence, varieties which are considered to have low temperature tolerance have long and wide anthers containing large numbers of pollen grains (Farrell et al., 2006a) For example, Farrell et al (2006a) found that three low temperature tolerant varieties, M103, Jyoudeki and HSC55 had large numbers of engorged pollen grains/anther (1310, 1328 and 1525), large anther area (0.82, 0.73 and 0.82 mm2), large stigma area (0.30, 0.47 and 0.37 mm2), which were all related to a low percentage of spikelet sterility (30, 31 and 34%, respectively) Meanwhile, three susceptible varieties, Paragon, Sasanishiki and YRL39, had a small number of engorged pollen grains/anther (176, 192 and 290), small anther area (0.41, 0.35 and 0.47 mm2), small stigma area (0.24, 0.27 and 0.24 mm2), and high percentages of spikelet sterility (78,

89 and 92%, respectively) Consequently, the reduction of these characteristics at low temperature can

be used to identify and evaluate rice varieties for low temperature tolerance

Shimono et al (2005) indicated that a daily average water temperature of less than about 22.5oC during a period of reproductive growth, substantially increases spikelet sterility In their experiments,

Shimono et al (2005) examined the Kirara 397 variety under average water temperature ranging from

17 to 25oC and minimum water temperatures ranging from 14 to 21oC, for between 34 to 40 days The results showed that spikelet sterility decreased from around 90% (daily minimum water temperature <20oC) to around 10% (daily minimum water temperature >22oC)

Gunawardena et al (2003b) found that both panicle and root temperatures affect spikelet sterility in rice when plants are exposed to low temperatures during the microspore development stage Root and panicle temperature treatments were imposed using warm (28/23oC) and low air (18/13oC) temperature regimes for a period of 7 days during microspore development The results showed that protecting the young panicles by deep-low water temperature reduced spikelet sterility from 48.4 to 30.6% compared to shallow-low water temperature treatment, while shallow-warm water and deep-warm water showed much lower levels of spikelet sterility than the low water temperature treatments, with 25 and 11% levels of sterility, respectively These results indicate the importance of panicle and root temperatures in determining the level of spikelet sterility

The application of a high rate of N under low temperature at the reproductive stage in rice results in

an increase in tillering and spikelet numbers per plant, leading to the reduction in the number of engorged pollen grains per anther, resulting in high spikelet sterility (Gunawardena et al., 2003a) In their experiment, Gunawardena et al (2003a) examined two rates of N (0 and 150 kg N/ha) as urea at each of pre-flood (PF) and reproductive stages under low temperature (18/13°C) for 5 days The results showed that the effect was greatest when N was applied at PF The percentage of spikelet sterility increased significantly from 14.6% (0 kg N/ha) to 46.3% (150 kg N/ha)

2.4.2.2 Mechanisms of low temperature induced male sterility

The underlying mechanisms that cause low temperature induced male sterility during the young microspore stage are being continually investigated This is because of a decrease in the number of differentiated microspores (Satake, 1991) Low temperature during the booting stage reduces the number of dehisced anthers and pollen grains on the stigma This reduction results in an increase in spikelet sterility Sawada (1978) Nishiyama (1984) observed abnormalities in the anther following low temperature damage These abnormalities included the cessation of development, unripe pollen, poor extrusion of anthers, partial or no dehiscence of anthers, pollen grains remaining in anther loculi, little or no shedding of pollen grains on stigmas, and the failure of pollen germination on stigmas Further, low temperature during the young microspore stage reduced the engorged pollen grain diameter (Gunawardena et al., 2003b) Nishiyama (1984) suggested that the main cause of spikelet sterility is the lack of anther dehiscence because of unripe pollen Spikelet sterility is primarily due to the failure of male organs, particularly anthers (Ito 1980) In order to achieve less than 10% spikelet sterility, there needs to be approximately 640 engorged pollen grains per anther (Nishiyama, 1983, Satake, 1991) Kim et al (1989a) found a similar result, with a requirement of

anther length between 1.85 to 1.00 mm, and 650 to 800 engorged pollen grains per anther, to obtain less than 10% spikelet sterility This suggests that the number of engorged pollen grains per anther at anthesis is an important factor related to the success or failure of fertilization Recent studies by Gunawardena et al (2003b) showed that low temperature treatment not only increases the total number of damaged pollen and microspores, but also decreases the engorgement, interception and efficiencies of the pollen grains

Anther respiration

Nishiyama (1970) examined anther development from the meiotic stage and measured respiratory activity, in order to investigate male injuries caused by low temperature The results indicated that protein content and respiratory activity of anthers were reduced at anthesis under low temperature compared to the controls Toriyama and Hinata (1984) found that low temperature tolerant cultivars had higher levels of anther respiration compared to susceptible cultivars, when exposed to low water temperature (20°C) for approximately one month from PI to heading They also indicated that anther respiration is a dominant heritable characteristic, and hence the critical temperature of meiotic anthers may be taken as an indicator of the degree of low temperature tolerance in rice genotypes Active respiration is essential for the development of fertile anthers This is not surprising, given the high density of mitochondria found in tapetal tissues (Lee and Warmke 1979 cited in Levings 1993), as well as the prevalence of male sterile lines related to mitochondrial dysfunction Consequently, the observations suggest that the reduction in anther respiration is an important mechanism for inducing spikelet sterility in response to low temperature conditions, either through damage to mitochondrial

function, failure of starch catabolism, or blockages in the supply and transport of sugars

Cytological abnormalities

Low temperature cytological abnormalities are more common in anthers than in pistils or other organs of the rice flower (Hayase et al 1969) Matsuo et al (1995) found that the callose wall of the tetrad dissolves and releases microspores from the tetrad into the anther locule during the young microspore stage As a result, breakdown products of callose can contribute to the rapid growth and metabolic activity of the microspores at this time Abnormalities in the immunocyto chemistry of the callose wall following exposure to low temperature have been observed and can be a result of tapetal dysfunction (Mamun 2002) The tapetum and transitory tissue play an important role in the process of pollen development by supplying the necessary nutrients (Satake 1969) During the young microspore stage, many cytological abnormalities occur in the tapetum and microspores in anthers cooled, such as tapetal hypertrophy, microspore degeneration and a delay in pollen ripening (Satake 1976) He indicated that it is unclear whether the microspores or tapetal cells, or both, are the tissues with the initial sensitivity to low temperature injury Mamun (2002) found that abnormal development of the tapetum and surrounding anther wall tissues that follows exposure to low temperature can have

consequences for the transportation of nutrients to the microspores However, the blocking of nutrients can primarily be a result of the inability of microspores to receive nutrients, or the inability

of the tapetum to supply them The total effect of these factors is a reduction in the number of engorged pollen grains at anthesis through the indehiscence of anthers that lead to spikelet sterility Consequently, an understanding of the interaction between the tapetum and microspores is important for nutrient transport (Wada et al., 1992)

Carbohydrate metabolism

The outer wall layers of the anther and the connective tissue act as a physiological buffer to sugar transport in Lilium, resulting in the accumulation of supplementary carbohydrates as starch grains (Clement and Audran, 1995) Starch accumulation and periodic starch dissolution are normal features

of the anther wall cell layers and the maturity of microspores in rice (Matsuo et al 1995) However, it has been reported that excessive accumulation of starch granules in the transitory and parenchyma tissues of anthers takes place after 2 to 4 days of low temperature treatment at the young microspore stage (Koike and Satake, 1987) During the exposure to low temperature, they also observed gradual and abnormal digestion of starch in the pollen grains When the low temperature treatments were continued, abnormalities in starch accumulation and dissolution ultimately resulted in spikelet sterility However, when the treatment was discontinued, the starch was digested rapidly throughout the inside of the pollen grain and spikelet sterility was not induced This suggests that the initial damage caused by low temperature to anther cells is the result of a disturbance to carbohydrate metabolism, perhaps induced by a change in carbohydrate distribution or hydrolysis of the starch granules At least in the initial stages of the disturbance, the damage seems to be reversible Kawaguchi et al (1996) found that during low temperature treatment (12°C for 4 days) oligosaccharide accumulation mediated by arabinogalactan-protein in the rice anther played an important role in the development of the anther

Enzyme activity

Some researchers have tried to describe low temperature induced male sterility as an enzyme dysfunction (Koike and Satake, 1987) However, under low temperature treatments, the activity of three enzymes that participate in the metabolism of carbohydrate and phosphate (amylophosphorylase, acid phosphatase and esterase) were found to remain relatively unchanged and did not affect spikelet sterility (Koike and Satake, 1987) More recently, the focus has switched to the activity of key enzymes of the sugar transport pathway Invertase in particular has become recognised as a likely weakness in active sugar transport within the anther at low temperatures For example, sugars and starch accumulate in the anther tissues of wheat and rice under water stress during meiosis, similar to their accumulation under low temperatures (Saini, 1997) This also results in down regulation of invertase within the anthers (Saini, 1997) In addition, it has been suggested that certain

other enzymes have an impact on low temperature tolerance, these enzymes including omega-3-fatty acid desaturase (Shoji et al 1998 cited in Nakamura et al 2000) and beta-1,3-glucanase (Yamaguchi et al 1998 cited in Nakamura et al 2000)

2.4.2.3 Flowering stage

While the booting stage is the most sensitive stage to low temperature, the flowering stage is the second most sensitive stage (Matsuo et al 1995) Yoshida (1977) found that the minimum critical temperature at the flowering stage is 22oC (Table 2.1)

Ye et al (2009) measured the reduction in morphological characters, including plant height, panicle neck length, grain weight and spikelet fertility at the flowering stage, to evaluate low temperature tolerant rice varieties In their experiment Ye et al (2009) found that plant height (PH), panicle neck length (PNL), grain weight (GW) and spikelet fertility (SF) showed a remarkable reduction in response to the low temperature treatment (20.8/15oC for 14 days) For example, all varieties in the treatment showed a considerable decrease in these traits in response to low temperature Among the varieties, only a small number, such as Banjiemang and HSC55 (low temperature tolerant varieties), showed a low decline in these characters (4.9% PH, 36.7% PNL, -0.2% GW and 14.6% SF for Banjiemang and 5.5% PH, 88.7% PNL, 4.0% GW and 10.5% SF for HSC55) Meanwhile, the two susceptible varieties, Amaroo (14.6% PH, 135.1% PNL, 7.5% GW, 66.7% SF) and Azucena (12.4%

PH, 93.3% PNL, 5.4% GW, 79.6% SF) showed a significant reductions in the characters in response

to the low temperature treatment In summary, low temperature (around 18oC) during the flowering stage decreased plant height, panicle neck length, grain weight and spikelet fertility

Aerobic rice system (ARS) is a new production system in which rice is grown under non-puddled, non-flooded and non-saturated soil conditions (Prasad, 2011) Irrigation can be applied through flash-flooding, furrow irrigation (or raised beds), or sprinklers, with the goal in all cases to keep the soil under supplementary irrigation but not flooded or saturated ARS can reduce water inputs by 11.5 to

50.7% in the Philippines (Bouman et al., 2005), by 29.2 to 65.3% in northern China (Bouman et al.,

2005, Xiaoguang et al., 2005), and by 62.5 to 70.8% in Japan (Matsuo and Mochizuki., 2009) in comparison to traditional flooded conditions One important goal of ARS is to increase water use

efficiency while maintaining high yields at about 70 to 80% of the yield in flooded rice (Belder et al., 2005) ARS has the potential for reducing the water requirement for rice cropping more than other techniques that have been developed A review of data from studies in many countries such as the USA, Australia, Japan, China and the Philippines shows that the yield penalty of ARS grown in

temperate regions is generally lower compared to the tropics (Kato et al., 2009) In the tropic

regions, the yield penalty of rice crops is lower in wet-season in comparison to dry-season (Bouman

et al., 2005) Agronomic traits including shoot growth, yield, and yield components are most likely

to use in most studies of ARS to compare to those of traditional flooded rice (Bouman et al., 2005, Peng et al., 2006, Xiaoguang et al., 2005)

The yield difference between aerobic and flooded rice is contributed more by sink size (spikelet numbers per m2) than the percentage of grain filling and 1000-grain weight among the yield components (Peng et al., 2006) In their experiment, Peng et al (2006) found that factors such as varieties, dry or wet seasons and the number of seasons that aerobic rice has been continuously grown caused 8 to 69% of the yield difference between aerobic and flooded rice However, the yield gap between aerobic and flooded rice was 8-21%, due to higher biomass production, more panicles, better grain filling and greater 1000-grain weight in the flooded rice Some studies have examined the effects of water depth on spikelet sterility when exposed to low temperature during the booting stage (Borrell et al., 1997, Gunawardena et al., 2003b) However, studies of the impact of low temperature on aerobic rice have not previously been reported

2.5 Nitrogen effect on rice growth

Yoshida (1981a) and Mae (1997) reported that N is the most essential fertilizer nutrient for rice Applying an appropriate rate of fertilizer N at approximately 20 days before heading commonly increases the final rice yield This is because the growth and development of young panicles before heading occur simultaneously during this period Under field conditions, N is usually applied (top dressing) during the panicle initiation stage, as the young panicles reach approximately 1-2 mm long

at about 23-25 days before heading Applying N at this period is an effective way to increase the number of spikelets Fageria and Baligar (2001) found that the application of a suitable level of N accounted for approximately 91% of the variation in the number of panicles/m2, 75% of the variation

in spikelet sterility, and approximately 73% of the variation in 1000 grain weight

Murata and Matsushima (1975) showed that the panicle number/unit area is determined within a period of approximately 10 days after maximizing the number of tillers Furthermore, there is competition for assimilates between the development of panicles and young tillers during rice plant growth, beginning with panicle development (Dofing and Karlsson, 1993) Therefore, Gunawardena

et al (2003a) concluded that N application during the pre-flooded condition under low temperature increases tillering and spikelet number per plant, which in turn reduces the number of engorged pollen

grains per anther, leading into increased spikelet sterility

2.6 Conclusions of literature review and focus of this study

There have been a number of studies on genotypic variation for low temperature tolerance at the booting stage in rice, particularly studies related to the effects of water depth and water temperature on spikelet sterility However, these studies used mostly commercial varieties which have different genetic background This study has examined genotypic variation for spikelet sterility under flooded conditions using a population developed from a cross between tolerant and susceptible varieties In addition, there appears to be little research done on the effects of low temperature, especially for the microspore stage on spikelet sterility under aerobic conditions Compared to deep water, shallow water can result in a significant increase in sterility due to the absence of the buffering ability of water that protects the panicle, rather than it being a physiological response to difference in water level (Gunawardena et al., 2003b) Under the aerobic conditions of no standing water, low temperatures may have even stronger effect on spikelet sterility Therefore, the focus of this research is an examination of consistency in genotypic variation in susceptibility to low temperatures at the booting stage under aerobic and flooded conditions

CHAPTER 3 EXPERIMENT 1 GENOTYPIC VARIATION IN SPIKELET STERILITY WITHIN A POPULATION EXPOSED TO LOW TEMPERATURE AT THE BOOTING STAGE 3.1 Introduction

Low temperature during the booting and flowering stages causes spikelet sterility that reduces grain yield in many temperate rice growing countries (Farrell et al., 2001b) Although da Cruz et al (2006b) found the flowering stage to be more sensitive, the most sensitive stage for low temperature damage in rice is the booting stage, especially the early pollen microspore stage, which occurs approximately 10-

12 days before heading (Satake,1976) There is a wide range of genotypic differences for tolerance to low temperature among rice varieties (Matsuo et al 1995; Nakamura et al 2000) Genetic resources for tolerance to low temperature have been identified and used as donor material for new varieties (Abe et al., 1989)

Da Cruz et al (2006b) found that the use of the reduction in spikelet fertility is appropriate for distinguishing genotypic variation for low temperature tolerance at both the booting and flowering stages They examined six rice genotypes using a constant temperature of 17oC with different lengths

of exposure to low temperature (3, 5, 7 and 10 days) at the booting and flowering stages and found that a minimum of 7 days exposure to a temperature of 17oC was required to differentiate the genotypes for low temperature tolerance The results showed that the reduction in spikelet fertility percentage ranged from 2 to 14% for the three low temperature tolerant genotypes and 70 to 95% for the three susceptible genotypes, based on 7 days exposure to low temperature (17oC) while the reduction was from 6 to 27% and 89 to 99%, respectively for the two groups, following 10 days of low temperature exposure They also found that there was genotypic consistency in low temperature

tolerance at these two stages (booting and flowering) among the 6 genotypes, and that japonica varieties were more low temperature tolerant than indica varieties

Ye et al (2009) also used the reduction in spikelet fertility as the main criterion for the evaluation of low temperature tolerance at the booting stage In their experiment, Ye et al (2009) examined 17 rice varieties under low day/night temperatures (20.8/15oC for 14 days); the reduction in spikelet fertility varied from 3.5 to 7% for low temperature tolerant varieties and from 51 to 86.4% for low temperature susceptible varieties However, this research was conducted with only a limited number

of varieties and not much is known about genotypic variation in low temperature tolerance for a large number of lines derived from crossing parents with contrasting responses to low temperature at the booting stage Therefore, in the current study, 104 genotypes derived from a cross between two contrasting parents were used which had differences in low temperature tolerance, flowering date and other agronomic characteristics

This experiment was conducted with the following objectives

1 To identify genotypic variation for spikelet sterility within 104 genotypes when exposed to low

temperature at the booting stage, and to select 10 tolerant and 10 susceptible progeny genotypes for further study

2 To identify whether low temperature tolerance is related to any agronomic characteristics

3.2 Materials and methods

3.2.1 Genetic materials

The experiment used 104 genotypes including 101 F5 lines derived from Kyeema//Kyeema/Norin PL8 cross which had not undergone any selection, the two parents and a standard Australian variety, Sherpa

Kyeema (YRF9 - considered as susceptible variety) was selected from the progeny of the cross YR83110 (Pelde//Della/Kulu) Pelde is a high quality non-fragrant long-grain variety, while Della is a fragrant Texas variety and Kulu is another long-grain variety which was developed at the Yanco Rice Research Station in the Riverina area of NSW

Norin PL8 is a low temperature tolerant Japanese parental line that was developed by using a low temperature tolerant variety, Silewah (originally from Indonesia) as a donor and backcrossed with a low temperature sensitive Japanese breeding line Hokkai 241 as the recurrent parent (Satake and Toryama 1979)

3.2.2 Cultural details

The experiment was conducted in a controlled temperature glasshouse at the St Lucia Campus of The University of Queensland, Brisbane, Australia, between 13 August 2013 and 8 January 2014 One litre ANOVApots® (125 mm deep, 125mm wide at top and 95mm bottom) designed to minimise root escape with only one hole in middle of the pot base (Hunter et al., 2012) were filled with soil collected from The University of Queensland Research Farm at Gatton (2733'S 15217'E)

in South-east Queensland The soil was an alluvial Lockyer prairie soil (USDA Soil Taxonomy: Fluventic Haplustoll (Isbell, 1996) and consisted of light, black clay Soil pH tends to be alkaline, around 8.2 Powell (1982) has described the soil physical and chemical characteristics in detail Five grams of slow release fertilizer (Osmocote plus 3-4 months: 16 N - 9 P - 12 K) were mixed into soil towards the middle of each pot In order to prevent the escape of the roots via the central hole of ANOVA pots, an inverted Petri dish was placed 3-4 cm above the central hole

Ten seeds were planted directly into each pot Each of 101 genotypes and 3 varieties was planted in

3 pots with the exception of Kyeema, Norin PL8 and Sherpa, for which there were 6 pots The experiment was designed in a Randomized Complete Block Design (RCBD) Twenty one days after planting, the two healthiest plants were chosen to be grown and the others were removed from the pots

Plants were grown at 28/19oC under natural light and sprinkled with water to achieve good plant establishment Over a 25 day period commencing 26 days after sowing (DAS) and ending 51 DAS, the water level in the pots was gradually raised from 2 cm at the bottom of the pots to a final water depth of 4 cm above the soil surface (flooded conditions) During the growth period, the pots were rotated within each tank every week There was a problem of poor plant growth in general as water level was raised, because the plants were subjected to water saturated conditions Most plants overcame the problem but some did not and eventually died This problem was thought to be related

to iron-deficiency and plants were therefore sprayed with iron sulphate

At the booting stage (from 51 to 90 DAS depending on the plant), all genotypes in 3 replications were moved into a low temperature glasshouse (21/15oC day/night) for two weeks Another set of three replications of three varieties (Norin PL8, Kyeema and Sherpa) were kept under warmer conditions The appropriate time for the transfer to the low temperature glasshouse to expose the plants at booting stage to low temperature stress was determined by the auricle distance: when the auricle distance between the flag and penultimate leaf of the main stem was from -7.5 and +5 cm, Ye

et al (2009) suggested to move the pot to the low temperature glasshouse However, the auricle distance of plants used to move them to low temperature glasshouse in this experiment ranged from -1.5 to +5 cm After 14 days exposure to low temperature, the pots were returned to the warmer glasshouse until maturity

3.2.3 Data collections and analysis

Monitoring of auricle distance was conducted every 4 - 6 days in the low temperature glasshouse until day 14, when the plant was then returned to the warm temperature glasshouse

Days-to-heading was defined as the number of days from seeding until the first part of the panicle

on the main stem had emerged Heading delay of 3 varieties was calculated as the difference in days

to heading between the plants in the low and warm temperature glasshouses

Dead spikelets were collected from dead branches on which all the spikelets were empty and white The number of filled grains and unfilled grains were counted for the main stem; they were also counted from tiller 1 (the tiller that appeared first) when the main stem of the plant had less than 50% spikelet sterility This was considered necessary as, in some cases, the main stem panicle was too advanced at the time of transfer to the low temperature glasshouse and it may not have been exposed

to low temperature at the critical growth stage, resulting in low spikelet sterility

Total spikelet number per main stem was calculated as the sum of fertile spikelets (filled grains), sterile spikelets (unfilled grains) and dead spikelets

One-way ANOVA was calculated using SAS software to determine probability of significance between means of the different genotypes

3.3 Results

3.3.1 Air temperatures

Daily maximum temperature in the warm temperature glasshouse during the experimental period ranged from 22.0oC to 34.9oC, while the minimum temperatures were fairly stable at around 20 - 22oC for the whole period (Fig 3.1)

Figure 3.1 Minimum and maximum air temperatures in the warm glasshouse

Daily minimum temperature in the low temperature glasshouse during the treatment period from 8 October 2013 to 24 November 2013 ranged from approximately 12.0oC to 14.3oC During 7 days from

7 to 13 October 2013 the minimum temperature was around 12oC, while from 15 October to 24 November the temperature was fairly stable at around 14oC In the same period the maximum temperature ranged from 18.5oC to 24.3oC (Fig 3.2)

Figure 3.2 Minimum and maximum air temperatures in the low temperature glasshouse

During the first two weeks of the low temperature exposure of the rice genotypes, the maximum temperature declined from 24.3oC to around 20oC and then fluctuated around 21.0oC The minimum temperature in the first week was lower than the other weeks, and hence all genotypes were not

exposed to the same low temperature (14oC), and one group of 33 out of 100 genotypes and 3 varieties were exposed to lower temperatures (12oC) from 2 to 7 days Among the genotypes, five were exposed to 12oC in all three replications, six genotypes in two replications and the remaining 22 genotypes in one replication

3.3.2 Heading date

There was a highly significant (p<0.01) variation in days-to-heading among 96 genotypes and 3 varieties (excluding the 5 genotypes where all three replications were exposed to 12oC) (Appendix 2) Days-to-heading of the genotypes ranged from 81 to 102 days-after-sowing (DAS) Heading date of

93 genotypes and 3 varieties was more skewed towards the heading date of Kyeema (99 DAS) This was probably a result of backcrossing with Kyeema as the recurrent parent

Table 3.1 Days-to-heading of the 3 varieties in the warm glasshouse and exposed to low temperatures Auricle distance, date at the start and end of low temperature treatment and the number of days before heading in warm glasshouse from the date of low temperature

exposure are also shown

(DAS: Days after sowing)

Varieties

Auricle distance

at the time of transfer

(cm)

Days from transfer to low temperature, to heading in warm temperature

Low temperature start

(DAS)

Low temperature end

Figure 3.3 Average number of days required before heading 3.3.3 Spikelet Sterility

Figure 3.4 The effect of number of days exposed to 12 o C on spikelet sterility

Of the 48 plants exposed to 12oC in the first 7 days of low temperature exposure, there was a highly significant relationship (r=0.43**) between the level of spikelet sterility and the number of days of exposure to 12oC (Fig 3.4)

Among those early heading genotypes for which some replications were exposed to 12oC and others

to 14oC, the results of the t-test showed a highly significant difference (t=5.5, p<0.01, df=20) in