Identification of traits and qtls contributing to salt tolerance in rice (oryza sativa l )

Cutting part of leaf sheath between 2 cm from base to collar Chromosome Cutting method Control treatment Dry weight Electrical conductivity Genome wide association study High density ric

Identification of traits and QTLs contributing to

salt tolerance in rice (Oryza sativa L.)

Thieu Thi Phong Thu

2019

Identification of traits and QTLs contributing to

salt tolerance in rice (Oryza sativa L.)

A dissertation submitted to Graduate School of Bioscience and Bioenvironmental Science, Faculty of Agriculture, Kyushu University

in partial fulfillment of the requirement for the degree of

Doctor of Philosophy

by Thieu Thi Phong Thu

Laboratory of Plant Nutrition

Japan, 2019

Table of contents Acknowledgment

Acknowledgment

I would like to express my sincerest appreciation and deepest gratitude to the following organizations and individuals in the pursuit of my Doctoral degree:

Vietnam Government, Ministry of Agriculture and Rural Development, and Ministry

of Education and Training for granting me the scholarship; Vietnam National University of Agriculture, and Department of Cultivation Science for allowing me to leave to go on study and their kind supports;

My supervisor Assoc Prof Dr Takeo Yamakawa for his guidance, encouragement with all his heart and invaluable advices throughout my research;

Prof Dr Ken Matsuoka, Assoc Prof Dr Akiko Nakashita-Maruyama, Dr Masamichi Kikuchi from Laboratory of Plant Nutrition; and Prof Dr Hideshi Yasui, Dr Yoshiyuki Yamagata from Laboratory of Plant Breeding for their kind helps and invaluable comments and suggestions during my study;

All members of Plant Nutrition Laboratory for their kind understanding, their enthusiasm supports and helps during my time in Japan; All my Vietnamese friends and other International friends for their helps and moral supports which made my stay in Japan

a pleasant and memorable one;

Vietnam international education development (VIED) at Ministry of Education and Training and Mrs Nguyen Thi Thanh Tam, an officer of Students Affairs Division, for their kind understanding, their enthusiasm supports and help me to complete both the necessary proceeds in Viet Nam and Japan

Last, but not least to my husband Duong Thanh Huan, my little daughter Duong Hong Ngoc and my parents, my parent’s in-law, my sisters and their family, my husband’s

older brother and his family for their love and moral support

Cutting part of leaf sheath between 2 cm from base to collar Chromosome

Cutting method Control treatment Dry weight Electrical conductivity Genome wide association study High density rice array

High salt susceptible group Honesty significant difference Indica Block

Japonica Block Potassium Kyushu university cultivated rice Leaf

Linkage disequilibrium Leaf dried weight reduction Magnesium

Moderately salt tolerant group Nitrogen

Sodium Phosphorous Pearson correlation index Quantitative trait loci

Salt susceptible group

Sheath Shoot length reduction Sheath dried weight reduction Standard evaluation score Single nucleotide polymorphisms Salt overly sensitive

Salt stress treatment Salt stress treatment/Control treatment

Salt tolerant group World rice collection Yoshida solution

Thesis Summary

The global population is expected to reach to 9 billion people by 2050, increasing the pressure for agricultural production in marginal saline lands Agriculture will have to increase its crop productivity by 70-110% in 2050 to feed that world Globally, it is estimated that 45 million of the 230 million ha of irrigated lands (19.5%) have to suffer from salinization problems by various degrees This is a serious problem because irrigated areas are responsible for one-third of world food production Therefore, agriculture production has been faced to not only increase crop yields that have not been seen before but also have to

do this in a changing climate Development of the salt tolerant varieties has been considered one of the key strategies to increase rice production in the coastal area through breeding by genetic methods The important steps have been pointed out for genetic method of improving salt tolerance in rice such as evaluation of variation of genetic sources for salt tolerance in rice, identification of molecular markers associated with QTLs or genes conferred tolerance

to salt stress, discovery of genes regulating salt tolerance and development of cultivars harboring those salt tolerance genes Salinity tolerance in rice is regulated by multiple genes Expression of salinity tolerance is the result of many physiological and biochemical activities in plants Therefore, determining the representative traits for salinity tolerance is a very difficult but extremely important task The objectives of this research are to determine the optimal salinity tolerant traits, the effective sampling method, and finally to find QTLs contributing to improving salt tolerance in rice

Thirty-seven rice varieties were screened for salt tolerance using a hydroponic system with ion components similar to seawater in growth chamber Two-week-old seedlings were grown for 7 days on Yoshida hydroponic solution The treatment group then

After a 2-week period of salt stress, standard evaluation scores (SES) of visual salt injuries

were assessed The K, Na, Mg, and Ca contents were then determined for the roots, sheaths, and leaves Following the SES results, the 37 varieties were divided into four groups: salt-tolerant (STGs), moderately salt-tolerant (MSTGs), salt-sensitive (SSGs), and highly salt-sensitive (HSSGs) In the control treatment, STGs had the highest sheath K content, whereas HSSGs had the lowest Sheath K content was also highly and negatively correlated with SES This suggests that sheath K may be useful for identifying salt-tolerant varieties under non-saline conditions Plant growth was significantly affected under salt stress, but STGs had the smallest decrease in dry weight of sheath SES was significantly correlated with sheath and leaf Na, sheath K and Mg contents, and sheath and leaf Na/K and Na/Mg ratios The results suggested that sheath K content, Na/K, and Na/Mg ratios may be useful indicators for genetic analyses of salt-tolerant varieties under salt stress conditions

Another set of twenty-nine rice varieties were screened for salt tolerance using a hydroponic system with ion components similar to seawater in a green house Following the SES results, the 29 varieties were divided into three groups: salt tolerant (STG), moderately salt tolerant (MSTG), salt susceptible (SSG) Under the control treatment, salt tolerant varieties exhibited low K content in root Under the salt stress treatment, the salt tolerant varieties exhibited low SES, high N content in leaves and sheaths, low Na content in leaves and sheaths, low Mg contents in leaves and sheaths, and low Ca content in sheaths The salt tolerant varieties also showed high ratio of sheath dry matter in salt stress treatment and sheath dry matter in control treatment, high N content in leaves and sheaths, high K content

in sheaths, and low Na/K ratios in leaves and sheaths Therefore, these parameters might be useful to understand salt tolerance in rice

Four rice varieties were used in sampling method experiment in phytotron In the control, Yoshida solution was used continuously during the experiment In the salt treatment,

were tested including cutting method and three leaves method The results showed that cutting method and three leaves method are useful for experiments related to salt tolerance

in rice The mineral contents in salt tolerant cultivars was significantly different in comparison with salt susceptible variety in cutting part of leaf sheath between 2 cm and collar (CAS) and leaf blade The CAS and the leaf blade were useful for identifying salt tolerant related traits Leaving roots and shoots from the base to 2 cm might be possible to continue the growth of seedling and produce seeds which will be used for re-phenotyping of the progeny The first three leaves may not be needed for sampling to determine mineral contents As a result, it is possible to take a sample without the first three leaves and using cutting part of leaf sheath between 2 cm and collar The K content, Na content, Mg content and Na/K ratio in CAS and Na content, Na/K ratio in leaf blade should be used as salt tolerant traits in molecular and genetic analysis

A genome wide association study (GWAS) was performed to identify potential QTLs associated with salt stress tolerance in rice by correlating the genotyping data set with the phenotypic expression of 225 diverse rice accessions for 11 biochemical and agronomic traits GWAS was run using a mixed linear model (MLM) and population parameters previously defined (P3D) in Tassel 5.0 and predicts genomic regions associated with traits for Japonica subpopulation and Indica subpopulation To analyze single nucleotide polymorphisms (SNP) calls, a high-density rice array of 700,000 SNPs was used, which derived from the sequencing of wild and domesticated rice The kinship matrix (K) estimated from SNP genotyping data Association signals at the same QTL block were confirmed location on chromosome by analysis of linkage disequilibrium (LD) block using gPlink-2.050 and Halotypeview version 4.2 GWAS resulted in detection of numerous SNP markers scattered over the rice genome that were associated with various salt tolerance traits A QTLs region on chromosome 3 was found to contribute to the variation in salt tolerance in Indica

subpopulation and related to two traits of sheath Ca content and sheath Mg content Three QTLs regions on chromosome 2, 4, 5 were found to contribute to the variation in salt tolerance in Japonica subpopulation and related various traits of sheath Na content, sheath

Mg content, sheath Na/K ratio, leaf Na content, leaf Na/k ratio GWAS and LD analysis showed several SNP makers highly related to salt tolerant traits

The results of this thesis dressed out both the insight in traits and mechanisms related

to salt tolerance and sampling method for salt stress experiment in rice The underlying genetics as presented in this thesis is of direct use to breeders and scientists and will significantly contribute to improving the salt tolerance traits in rice

Chapter 1 General introduction

1.1 The salinization and current worldwide problems

The global population is expected to reach to 9 billion people by 2050, increasing the

pressure for agricultural production in marginal saline lands (Negrão et al 2011) Global food

production will need to increase by approximately 50% by 2050 to match population growth (Flowers 2004; Rengasamy 2006) Tester and Langridge (2010) also reported that crop productivity of agriculture has to increase by 70-110% in 2050 to feed that world However, agricultural production faces increasing challenges from salt intrusion due to global warming, ice melt, and rising sea levels The area and salinity level of salt-affected soil have been increasing in parallel with the increase of world population Globally, it is estimated that saline and sodic soils cover 397 million and 434 million ha (FAO 2016), respectively, while 45 million ha of irrigated lands (19.5%) have been affected by salinization This is a serious problem because irrigated areas are responsible for one-third of world food production (Linh

et al 2012) In addition, many factors that related with climate change like water scarcity,

elevated temperature, flooding and salinity cause abiotic stress and bad effect on crop productivity Therefore, agriculture production has been faced to not only increase crop yields to a level that have not been seen before but also have to do this in a changing climate

(Roy et al 2011)

Among abiotic stresses, salt stress is noticed as the most serious factor (Tuteja 2007)

of irrigated land And, the salinized areas are growing to 10% a year because of various problems, including low precipitation, high surface evaporation, using salty water for irrigation, and poor agricultural practices Until 2050, it is considered that more than half of

the arable land would be salinized (Jamil et al., 2011) Soil salinization widespread in humid

regions such as South and Southeast Asia (Szabolcs and Pessarakli, 2010), where rice is a

staple food crop (Kobayashi et al 2017) Many high-output rice cultivation regions have been

faced with salinization because these regions are located in coastal areas Therefore, it might cause a lot of bad impact on the productivity of rice

Salt tolerant cultivars may provide opportunities to improve the salinity tolerance of rice through breeding One of the main strategies that has been focused is producing the salt tolerant varieties in saline region through breeding by genetic methods to increase rice production The important steps have been pointed out for genetic method of improving salt tolerance in rice such as investigation of genetic sources variation for salt tolerance, determination of molecular markers related with QTLs or genes regulated tolerance to saline condition, identification of genes regulating salt tolerance and introduce those salt-tolerance

genes to elite cultivars (Sahi et al 2006)

1.2 The saline soil and adverse effects of salt stress on plant

The saline soil

The saline soil refers to a soil that contains sufficient soluble salts to impair its

and the anions (Cl-, SO42-) The most popular ions that related to soil salinity are Ca2+, Mg2+,

NaCl) (Munns and Tester 2008) The condition is assumed as salinity when its EC cause the decrease of most crop

The adverse effects of salt stress on plant

Salinity severely limited the crop production because of inducing an abiotic stress to

plant (Shannon 1998) In general, two types of stresses on plant come from salinity are osmotic and ionic stresses Osmotic stress happens when the salt concentration around the roots increase over the threshold of plant tolerance Ionic stress occurs in case of increase of

processes and cell death (Munns and Teaser 2008) There are three ways which salt stress affects on the plant growth and development The first is by reducing the soil osmotic potential and inducing the bad situation that water can not enter the roots The second is ion

absorption of other mineral elements As a consequence, these ways results the poor growth and plant death (Mc Cure and Hanson 1990)

Salt stress can induce the physiological drought problems as well as the ion toxicity

physiology and metabolism and be reasons of stresses such as hyper ionic and hyper osmotic that severely depress the plant growth Moreover, salt stress also makes the changes in K/Na

above 100 mM damage to cell metabolism and limit activities of enzymes, cell division and expansion, and membrane structure This also decreases the photosynthesis and produces reactive oxygen species

can interrupt the osmotic balance, the stomata and enzyme functions (Singh and Sengar

consequences such as the retarded plant growth (Saqib et al 2008), slowing down flowering

and poor fertility, bad development of panicle and grain in rice (Zeng and Shannon 2000,

(Fageria et al 2012), inhibition of photosynthetic activity (Netondo et al 2004, Chaum et al

2006, Chaves et al 2009, Moradi and Ismail 2007)

1.3 The salt tolerance mechanism in rice

Halophytes and glycophytes are two groups of salt tolerant plant and salt susceptible plant The halophyte plants can tolerate to high saline condition of 400 mM NaCl, while glycophytes can not (Mass and Nieman 1978) Among agriculture crops, few crops are halophytes, and large proportions of crops are sensitive to salinity stress

With in cereals, the highest tolerance to salt stress specie is rye (Secale cereale) with a

2005) The rice growth periods of germination, active tillering and towards maturity are tolerant to salt stress The seedling, flowering and early reproductive stages are considered

mechanisms were seen in rice including biosynthesis and accumulation of osmolytes for osmoprotection, ion homeostasis and compartmentation, antioxidants, reactive oxygen

species (ROS) detoxification, and programmed cell death (Hoang et al 2016)

Munns and Tester 2008 reported that ion exclusion and osmotic tolerance are main ways which happen in salt tolerant rice varieties These ways are also assumed ion exclusion,

osmotic tolerance and tissue tolerance (Roy et al 2014) To reduce the over leaf

in roots Ion exclusion includes two pathways, one is the retrieval of Na+ from xylem, and the other occurred in root system by returning of ions back to the soil

long distance signals and relates to drought stress which caused by salt stress to protect leaf

expansion and stomatal conductance (Rajendran et al 2009) Tissue tolerance is related to

vacuole accumulation of sodium, synthesis of compatible solutes and production of catalyzing detoxification of ROS enzymes

Intracellular cation compartmentation

preformed well, toxic effects occurred some days or weeks after, then older leaves start dying

was found (Moradi and Ismail 2007) Rice young leaf is protected from salt stress because

old leaf accumulated toxic ions Wang et al (2012) showed that under salt stress condition,

salinity, up-regulation of OsHKT1;1, OsHAK10 and OsHAK16 promotes old leaves

old leaves

Rice panicle has lower salt content in saline tolerant cultivars, and compared to husks and rachis, grains have lowest salt contents (IRRI 2018) The healthy level of flag leaf is an important parameter for good yield under salt stress condition Evaluation of mineral content

is flag leaf shows lower salt content in this leaf in salt tolerant cultivars (Ahmad et al 2013)

Thus, flag leaf should be selected for evaluating tolerance to salt stress at the reproductive stage (IRRI 2018)

Under the salt stress condition, salt tolerant varieties showed lower Na+ in leaves and

shoots in comparison with salt susceptible varieties (Dionisio-Sese and Tobita 2000, Lee et

al 2003, Moradi et al 2007, Ghosh et al 2011) Pokkali, a saline tolerant variety, showed

low accumulation of Na in cytosol because of decrease the Na uptake, temporary accumulating in the cytoplasm, and quickly extruding into vacuoles (Kader and Lindberg 2005)

The sodium ion exclusion and the regulation of sodium transporter was studied in

tolerance were found

Cell ion homeostasis and cation transporter

One of the important parameters that shows a cultivar is tolerant to salt stress is

in plant cells under salt stress condition (Maathuis and Amtmann 1999, Cuin et al 2003), and support to photosynthesis and plant growth (Rodrigues et al 2013) Maintaining low

or both phenomenon Under salt stress condition, rice varieties with different salt tolerance showed the mineral contents were different in roots, leaf sheaths and leaf blades Salt tolerant varieties showed higher K content, lower Na content, lower Mg content and lower Na/K ratio

in leaf sheath and lower Na content, lower Na/K ratio in leaf blades than salt susceptible

varieties (Thu et al 2017, Thu et al 2018)

transporters have been done Wang et al (2012) reported that under saline condition,

up-regulation of OsHKT1;1, OsHAK10 and OsHAK16 promotes old leaves accumulate Na+ ion

Moreover, expression of OsHKT1;5 and OsSOS1 is low in old leaves, it might reduce ability

of storage Na+ in old leaf cells

The expressions of many ion transporters were reported in shoot, especially shoot base

The expression of SKC1 is observed in the parenchyma cells around the xylem vessels (Ren

et al 2005) SKC1 was related to controlling K+/Na+ homeostasis under saline condition The

SKC1 was then known as OsHKT1;5 gene The OsHKT1;5 was seen in the plasma membrane

and in the phloem of vascular bundles in basal nodes Noticeably, the OsHKT1;5 transcript

nodes, especially node I (Kobayashi et al 2017) The OsHAK1 mediate the growth of rice over

the salt stress condition in both low and high K levels In deficient condition of K and saline

condition, the expression of OsHAK1 is up-regulated in root and shoot apical meristem, the epidermises and steles of root, and vascular bundles of shoot (Chen et al 2015) The GUS reporter from OsHAK5 promoter showed activity in root and root-shoot junction (Yang et al

2014)

The ion pumps like symporters, antiporters and carrier proteins present on the

protein SOS3 and cooperates with this protein in SOS pathway SOS2 is a serine/threonine

the sequestration of Na+ into cell vacuoles in case of high cytosol Na+ content happens

(Blumwald 2000) The activity of Na+/H+ antiporter rises more in saline tolerant varieties

than in saline susceptible varieties (Staal et al 1991) Rice salt tolerance was improved by

NHX1, a vacuolar transporter (Fukuda et al 2004) Bassil et al (2012) reported that rice had

compartmentation (Blumwald 2000)

1.4 Genome wide association studies (GWAS) on salinity stress in rice

An advantageous achievement in plant breeding is genomic mapping which help to

locate the positions related to certain traits (Grotewold et al 2015) Analysis of QTLs (Pandit

et al 2010) shows particularly the evidently positions on chromosomes In the past few years,

the development of GWAS brings the accurately identification of position on chromosomes

(Korte and Farlow 2013, Si et al 2016) Yano et al 2016 showed an effective approach to

identify a gene related to agronomic traits is GWAS which its performance is done by analyzing the association between genotypic and phenotypic data And this could find various natural allelic together in a sole research The strength of relationship between genotypic and phenotypic data is determined in this approaches by employing statistical formalisms In addition, molecular markers or genes and alleles related to particular traits

are identified and used in breeding (Patishtan et al 2018) Generally, rice is sensitive to salt

stress However, salt-tolerant cultivars may provide opportunities to increase the rice salinity tolerance through breeding In addition to discovering formerly found genes, the GWAS also finds new genes involved in salt tolerance The results of GWAS analysis will support to have

an insightful understanding of salt stress tolerance and deliver many useful molecular markers to breeding programs

A difficulty in analysis of association mapping is population structure A large population including many varieties which come from large geographical ranges, represent well its evolution with various localization adaptations These might lead to good familial relatedness and differences of structure types which cause association panel to be not fully

random (Pritchard et al 2000) As a sequence, marker-trait associations might be false (Zhao

et al 2007) Thus, a suitable method in statistic to solve these problems is very necessary

(Patterson et al 2006) A well-known method is integrating of clustering information, which

the association panel members classified, and molecular makers, which examined within the

evaluated subgroups, in statistic software (Pritchard et al 2000; Falush et al 2003; Balding

2006) Another method is using the mixed models to explain differences in panel individuals

genetic (kinship matrix) (Yu et al 2006; Malosetti et al 2007)

GWAS in rice is being developed by many researchers and their works One of valuable achievements is McCouch’s research in 2016 This research brings an open platform with many advantages such as using very large population of 1568 inbred rice accessions, effectively genotypic data of 700,000 SNPs with every 0.54 kb interval of 1 SNP through 12 rice chromosomes Variation of haplotype was mostly captured and non-synonymous SNPs were optimally observed in 16M SNPs panel in this study

The objectives of this research

The studies in aimed to identify traits and QTLs that related salinity tolerance in rice

In Chapter 2 and Chapter 3, the experiment was conducted in growth chamber and in greenhouse condition These study induced salt stress using artificial seawater (ASW) and different plant parts including leaf blade, sheaths, and roots to evaluate its effects on rice

growth and mineral content and to determine useful parameters for identifying salt tolerant rice varieties In Chapter 4, cutting method and three leaf method were tested using 4 rice varieties with different salt tolerance The experiment was conducted to determine the useful sampling method for salt stress experiment In Chapter 5, we performed GWAS to identify potential QTLs associated with salt stress tolerance in rice by correlating the genotyping data set generated using a high-density rice array including 700.000 SNPs with the phenotypic expression of 225 diverse rice accessions for 11 biochemical and agronomic traits The purpose is to identify QTLs conferring mineral contents relating to salt tolerance in rice

Chapter 2 Effects of salt stress on plant growth characteristics and mineral contents

in diverse rice genotypes

2.1 Introduction

Agricultural production faces increasing challenges from salt intrusion due to global warming, ice melt, and rising sea levels Globally, it is estimated that saline and sodic soils cover 397 million and 434 million ha (FAO 2016), respectively, while 45 million of the 230 million ha of irrigated lands (19.5%) have been affected by salt This is a serious problem

because irrigated areas are responsible for one-third of world food production (Linh et al

2012) Despite the advanced management technologies available today, salinization of millions of hectares of land continues to have severe effects on global crop production Moreover, global population is expected to reach to 9 billion people by 2050, increasing the

pressure for agricultural production in marginal saline lands (Negrão et al 2011) Global food

production will need to increase by approximately 50% by 2050 to match population growth (Flowers 2004, Rengasamy 2006)

Rice (Oryza sativa L.), which supplies 20% of global daily calories, is highly

susceptible to salinity Salt stress causes serious damage to many cellular and physiological processes, including photosynthesis, nutrient uptake, water absorption, plant growth, and cellular metabolism, all of which lead to yield reduction (Pardo 2010) The salinity threshold

stage (Asch et al 2000)

Salt tolerant cultivars may provide opportunities to improve the salinity tolerance of

rice through breeding Development of salt-tolerant varieties has been considered one of the key strategies to increase rice production in coastal areas Identifying the key parameters for evaluating salt tolerance in rice is a topic of considerable research worldwide Previous studies have used hydroponic systems with additional sodium chloride (NaCl) salt as a screening method for salt tolerance in rice However, all soils contain a mixture of soluble salts; the most common cations associated with soil salinity are Ca2+, Mg2+, and Na+ (Alam 1999) Salt accumulation in arable soils is derived mainly from irrigation water that contains trace amounts of NaCl from seawater (Tester and Davenport 2003, Flowers and Yeo 1995) Few studies have investigated salt stress following exposure to ion components similar to those in seawater This study induced salt stress using artificial seawater (ASW) to evaluate its effects

on rice growth and mineral content and to determine useful parameters for identifying tolerant rice varieties

salt-2.2 Materials and methods

2.2.1 Plant materials

We selected a diverse set of genotypes, including 25 varieties from the Kyushu University Cultivated Rice Collection (KCR) and 12 varieties from the World Rice Collection (WRC) The list of rice varieties are presented in Table 2.1 The seeds were supplied by the Plant Breeding Laboratory, Faculty of Agriculture, Kyushu University

2.2.2 Hydroponic system and plant growth conditions

Seedlings were screened using a hydroponic system with a salt treatment and a control The experiment used a randomized complete block design with two replications and four

seedlings in each replication Yoshida (Y) solution (Yoshida et al 1976) was used for rice

growth in the control and was a base solution in the salt stress treatment The solution was

changed twice a week The pH was measured frequently with a pH meter (pH Meter 10P, DKK-TOA Corporation, Tokyo, Japan) and maintained at 5.5–6.0 The hydroponic system was placed in a growth chamber with a constant temperature of 25°C and a photoperiod of 16 h light and 8 h dark

HM-2.2.3 Screening procedure

Rice seedlings were prepared using a commercial seedbed soil (Kokuryu Baido, Seisin Sangyo Co., Kitakyushu, Japan) Seeds of 37 rice varieties were sterilized to remove fungi using 10% ethanol for 3 minutes, followed by 30 minutes of shaking in 5% bleach (NaClO) The NaClO was then removed by rinsing the seeds five times with distilled water Rice seeds were incubated at 30°C for 24 h One seed was planted with seedbed soil in each shell and kept in a tray with tap water Seedlings in the control and salt treatments grew uniformly for 2 weeks in tap water and 1 week in Y solution In the control, Y solution was used continuously for the experiment In the salt treatment, seedlings were grown for the next 2 weeks in a 12-

electrical conductivity (EC) of the solution was measured using an EC meter (Hand Held Conductivity Meter, Model CM-31P, DKK-TOA Corporation, Tokyo, Japan) to ensure that it

were 87.478 mM, 5.759, mM, 11.186 mM, and 2.156 mM, respectively

2.2.4 Assessing salt tolerance, measuring growth parameters, and determining mineral contents

After a 2-week period of salt stress, a standard evaluation score (SES) of visual salt injury was used to assess all seedlings and categorize salt tolerance following the method

described by Gregorio et al (1997) Shoot and root length were measured, and then the root

was cut from the shoot After oven drying at 70°C for 24 h, the shoot was divided into sheath and leaf, weighed, and ground into a fine powder The K, Na, Mg, and Ca contents in each

followed by atomic absorption spectrophotometry (Z5300 Polarized Zeeman Atomic Absorption Spectrophotometer, Hitachi, Tokyo, Japan)

2.2.5 Statistical analysis

Analysis of variance was used to test for differences, followed by Tukey’s HSD test; both were conducted using STATISTIX 8 (Analytical Software, Tallahassee, FL, USA) The correlations among parameters were investigated using correlation and regression analysis

in Excel (Office Professional Plus 2016, Microsoft, Redmond, WA, USA)

2.3 Results

2.3.1 Salt tolerance of rice genotypes under salt stress

Standard evaluation score (SES) is a most popular indicator used to evaluate the salt stress-induced symptom of rice when growing under salt stress condition If SES of a variety

is in the range between 3.0 and 5.0, it is a salt tolerant one There are some popular rice varieties which were categorized into the salt tolerant variety group such as Pokkali, FL478 The SES of Pokkali and FL478 was 3.0 when growing under salt stress condition of 12 dS

variable among the 37 genotypes The SES results following the salt stress treatment are shown in Table 2.1 The 37 genotypes were divided into four groups: salt tolerant (STGs; 7 varieties; KCR 20, KCR 124 and KCR 136 had SES of 3.3, 3.4 and 3.9 respectively), moderately salt tolerant (MSTGs; 11 varieties), salt sensitive (SSGs; 18 varieties; KCR157

cultivar had a score of 7), and highly salt sensitive (HSSGs; 1 variety, KCR246, with a score

2.3.2 Salt stress effects on plant growth

The response of rice growth to salt stress varied among genotypes (Figure 2.1)

Significant differences were observed in shoot length reduction, sheath dried weight (DW)

reduction, and leaf DW reduction The percent shoot length reduction was high in STGs and

low in HSSGs Variety KCR121 had the greatest reduction in shoot length (38.93%), and

WRC 41 had the lowest (3.77%) The percent sheath DW reduction was much lower in STGs

compared with HSSGs, ranging from −46.85% (KCR20) to 73.44% (KCR157) The leaf

DW reduction percentage differed widely among varieties, from 3.85% in KCR19 to 65.26%

in KCR175 STGs had the highest root, sheath, and leaf DWs at 0.42 g, 0.73 g, and 0.92 g,

respectively, and HSSGs had the lowest DWs at 0.14 g, 0.23 g, and 0.41 g, respectively

Table 2.1 The SES at seedling stage of 37 rice varieties

KCR10A Y CHANG JU 3.9 KCR175 KATAKTARA DA2 7.1

KCR57 TA-MAO-TAO 6 KCR226 KETANGENKA RASI 8

WRC6 PULUIK ARANG 6.1 WRC41 KALUHEETANI 8.1

KCR196 BLACK GORA (NSC12) 6.8 KCR244 SILAD 8.6

EC, electrical conductivity; SES, standard evaluation score; STGs, salt-tolerant group;

MSTGs, moderately tolerant group; SSGs, sensitive group; HSSGs, highly

salt-sensitive group

Figure 2.1 The growth of 37 rice varieties under salt stress treatment

SES, Standard Evaluation Score; ShtL_R, Shoot length reduction; ShtW_R, Sheath dried

weight reduction; LW_R, Leaf dried weight reduction

0 2 4 6 8 10

2.3.3 Mineral content in root, sheath, and leaf

The Na, K, Mg, and Ca contents were measured in roots, sheaths, and leaves for all 37 varieties, and means were calculated for each salt-tolerance group The results are shown in Figure 2.2 and Table 2.2

followed by the sheaths and leaves, whereas in SSGs and HSSGs, these values were lowest in the roots and highest in the sheaths This indicated that STGs accumulated and stored Na in the roots, thereby inhibiting Na transfer to the sheath and leaves (e.g., in genotypes KCR143, KCR136, and KCR20; Figure 2.2) By contrast, Na in the roots of SSGs and HSSGs was

generally transferred to the sheath and leaves (e.g., in genotype IR29; Figure2 2)

K content

In the control (data not shown), the K content in roots ranged from 8.90 (WRC57) to

(KCR140) in the treatment group (Table 2.2) The K content in sheaths ranged from 15.88

content among the four salt-tolerance groups are shown in Figure 2.2 There were no significant differences in K content in roots or leaves among the salt-tolerance groups under

either the control or the treatment condition (Figure 2.2) However, in both the control and treatment groups, sheath K differed significantly among the salt-tolerance groups; it was highest in STGs, followed by MSTGs, SSGs, and HSSGs There was also a high negative correlation between sheath K and the SES in the control (Pearson correlation index was -0.62), whereas other correlations were not high The similar patterns of sheath K content in the control and treatment groups suggest that sheath K could be used as an indicator for identifying salt-tolerant varieties under non-saline conditions

Mg content

In the control, the sheath and leaf Mg content did not differ significantly among the four salt-tolerance groups However, Mg content in the roots of STGs and MSTGs differed significantly from those in SSGs and HSSGs (Figure 2.2) The correlation between SES and root Mg content in the control was quite low (Pearson correlation index was -0.28) In the

2.2) Leaf Mg did not differ significantly among the four salt-tolerance groups, whereas root

Ca content

In both the control and treatment groups, Ca content was the highest in leaves and lowest in the roots However, there were no significant differences among the roots, sheaths, and leaves among the four groups in the control (Figure 2.2) In the treatment group, there were differences in Ca content in sheaths and leaves, but not in root, among the four groups However, these differences were not significant

Table 2.2 The dried weight and mineral contents of 37 rice genotypes under the 12dSm-1 EC salt stress treatment

Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf KCR20 3.25 0.60 1.05 1.05 4.96 17.37 14.86 17.10 11.08 5.23 3.02 3.74 5.69 0.27 0.37 1.47 3.45 0.64 0.35 5.67 2.96 0.92 KCR143 3.38 0.23 0.49 0.78 6.20 17.96 12.95 24.06 22.85 9.93 3.65 5.21 5.10 0.28 0.51 1.30 3.88 1.27 0.77 6.59 4.39 1.95 KCR124 3.63 0.36 0.60 0.83 6.84 19.27 15.71 18.47 10.04 4.09 3.53 4.41 4.15 0.30 0.47 1.12 2.70 0.52 0.26 5.23 2.27 0.99 KCR10A 3.88 0.44 0.98 0.96 4.39 16.52 13.69 13.10 17.36 7.51 2.88 4.07 5.97 0.28 0.48 1.64 2.99 1.05 0.55 4.54 4.27 1.26 KCR136 3.88 0.51 0.70 0.94 8.68 12.13 10.21 20.67 11.17 7.23 3.68 3.68 4.37 0.26 0.43 1.47 2.38 0.92 0.71 5.61 3.03 1.65 KCR208 4.50 0.45 0.61 0.93 7.34 9.49 10.66 18.27 23.11 14.52 3.67 4.38 4.38 0.27 0.45 1.13 2.49 2.43 1.36 4.98 5.28 3.32 KCR19 4.75 0.36 0.69 1.00 7.49 19.97 15.54 16.19 16.93 8.42 3.82 6.60 5.75 0.29 0.62 1.31 2.16 0.85 0.54 4.23 2.57 1.46 KCR108 5.13 0.43 0.62 0.95 3.93 15.66 14.41 16.97 24.20 8.65 2.80 5.84 5.17 0.40 0.68 1.57 4.32 1.55 0.60 6.07 4.15 1.67 IR24 5.50 0.36 0.49 0.87 5.01 16.78 18.34 11.33 32.67 8.46 2.44 5.64 4.98 0.26 0.42 1.46 2.26 1.95 0.46 4.65 5.79 1.70 KCR12 5.88 0.61 0.91 0.98 4.79 19.07 12.35 14.93 14.60 10.73 3.28 4.26 6.74 0.25 0.52 1.85 3.12 0.77 0.87 4.55 3.43 1.59 KCR201 5.88 0.25 0.43 0.60 6.85 5.66 8.69 16.28 19.56 11.30 3.74 4.13 3.63 0.25 0.40 0.83 2.38 3.46 1.30 4.35 4.74 3.11 KCR57 6.00 0.36 0.64 0.81 3.93 21.15 18.28 15.33 20.37 13.00 3.22 5.32 6.15 0.37 0.63 1.49 3.90 0.96 0.71 4.77 3.83 2.12 KCR75 6.00 0.30 0.52 0.89 4.41 14.10 14.69 15.22 28.22 17.18 3.58 6.99 7.19 0.52 0.80 1.72 3.45 2.00 1.17 4.25 4.04 2.39 KCR22 6.13 0.24 0.65 0.51 5.87 15.88 12.83 9.85 15.45 8.87 2.49 4.19 5.52 0.35 0.43 1.50 1.68 0.97 0.69 3.96 3.68 1.61 KCR121 6.13 0.22 0.36 0.53 8.70 13.87 13.13 16.55 19.85 12.45 3.96 6.06 5.99 0.26 0.55 1.45 1.90 1.43 0.95 4.18 3.27 2.08 WRC6 6.13 0.62 0.79 1.15 6.91 9.83 15.73 14.80 28.35 11.84 2.14 5.65 4.87 0.15 0.59 1.19 2.14 2.88 0.75 6.93 5.01 2.43 KCR196 6.75 0.32 0.45 0.80 4.10 9.54 14.99 17.87 33.71 16.32 3.31 5.77 8.02 0.25 0.56 1.50 4.36 3.53 1.09 5.40 5.84 2.04 KCR222A 6.75 0.39 0.60 0.73 4.53 13.25 16.86 22.99 19.74 9.44 2.69 4.19 5.39 0.40 0.36 1.38 5.07 1.49 0.56 8.56 4.71 1.75 KCR140 7.00 0.21 0.25 0.33 11.51 12.03 19.46 21.58 30.17 16.51 3.31 4.52 5.39 0.24 0.46 1.46 1.87 2.51 0.85 6.53 6.67 3.07 KCR157 7.00 0.15 0.26 0.42 7.79 9.70 15.12 13.47 29.57 15.78 2.67 4.81 4.47 0.23 0.52 1.36 1.73 3.05 1.04 5.05 6.14 3.53 WRC57 7.00 0.47 0.65 1.07 6.31 8.00 15.95 14.67 33.53 12.26 3.16 5.65 5.23 0.23 0.45 1.08 2.33 4.19 0.77 4.64 5.94 2.35 KCR175 7.13 0.22 0.32 0.70 3.08 13.49 17.11 19.16 27.84 14.56 2.90 5.20 6.78 0.25 0.47 1.57 6.22 2.06 0.85 6.62 5.35 2.15 KCR192 7.13 0.24 0.47 0.53 9.51 13.97 12.38 14.69 13.08 6.06 3.51 4.30 4.74 0.24 0.62 1.22 1.55 0.94 0.49 4.19 3.04 1.28 WRC5 7.25 0.52 0.53 1.09 4.92 7.89 20.95 15.60 33.92 17.75 1.53 6.04 6.96 0.15 0.50 1.28 3.17 4.30 0.85 10.22 5.61 2.55 WRC17 7.38 0.43 0.68 1.02 6.30 9.78 15.41 13.87 31.18 13.99 1.96 5.90 5.01 0.15 0.45 1.11 2.20 3.19 0.91 7.09 5.28 2.80 T65 7.38 0.20 0.47 0.43 5.69 9.20 14.02 7.30 40.11 15.71 1.63 6.01 5.38 0.22 0.51 1.60 1.28 4.36 1.12 4.47 6.67 2.92 KCR225 7.63 0.25 0.36 0.51 6.59 5.68 11.11 17.52 28.86 14.96 3.44 5.21 5.92 0.35 0.57 1.08 2.66 5.08 1.35 5.10 5.54 2.53 WRC15 7.63 0.51 0.64 1.02 5.78 9.97 18.88 12.29 34.63 17.53 2.18 6.70 6.73 0.17 0.51 1.34 2.13 3.48 0.93 5.65 5.17 2.61 WRC48 7.88 0.59 0.69 1.24 5.71 6.20 12.82 12.55 42.03 18.15 2.58 6.10 5.66 0.16 0.44 1.72 2.20 6.78 1.42 4.86 6.90 3.21 KCR226 8.00 0.28 0.55 0.67 8.24 8.51 16.78 16.77 26.80 10.85 3.59 5.20 4.56 0.35 0.53 1.12 2.03 3.15 0.65 4.67 5.16 2.38 WRC29 8.00 0.42 0.55 0.76 9.20 9.69 16.16 12.23 52.26 20.81 2.13 6.11 6.59 0.22 0.37 1.31 1.33 5.39 1.29 5.73 8.55 3.16 WRC37 8.00 0.31 0.46 1.00 3.31 7.95 16.92 15.05 57.83 25.27 1.71 7.71 7.73 0.22 0.50 2.03 4.55 7.28 1.49 8.82 7.51 3.27 WRC49 8.00 0.36 0.54 0.66 8.42 7.75 12.82 9.71 28.35 12.12 2.24 5.16 3.97 0.26 0.41 1.58 1.15 3.66 0.95 4.33 5.49 3.05 WRC41 8.13 0.35 0.50 1.08 4.35 6.24 17.60 14.20 51.37 22.80 2.36 6.97 7.23 0.19 0.60 1.76 3.26 8.23 1.30 6.02 7.37 3.15 KCR244 8.63 0.15 0.26 0.44 9.31 5.59 10.36 13.02 41.05 23.82 2.81 5.70 5.20 0.39 0.77 1.20 1.40 7.34 2.30 4.64 7.20 4.58 KCR233 8.88 0.19 0.29 0.51 2.49 6.91 19.74 18.59 45.79 17.96 3.48 7.18 4.64 0.43 0.93 1.47 7.46 6.62 0.91 5.33 6.38 3.87 KCR246 9.00 0.14 0.23 0.41 5.18 5.66 12.79 10.41 51.42 29.51 1.80 7.36 5.73 0.16 0.65 1.22 2.01 9.08 2.31 5.78 6.99 5.15

SES, standard evaluation score; DW, dry weight

Regression of Na against K, Mg, and Ca contents under the salt stress treatment

To understand which elements are related to Na absorption and accumulation, we conducted regression analyses to determine the relationship of Na with K, Mg, and Ca contents in each plant part in the salt-stress treatment group (Table 2.3) The results showed

that root Na was positively and significantly correlated with root Mg (coefficient = 3.560, P

< 0.0001) Sheath Na was positively and significantly correlated with sheath Mg (coefficient

= 7.520, P < 0.0001), but negatively correlated with sheath K Leaf Na was positively and

were low for roots and leaves (0.384 and 0.215, respectively), but high for sheaths (0.817)

2.3.4 Correlation of parameters related to salinity tolerance

Relationships among all parameters were analyzed to understand the physiological traits that characterize salinity tolerance (Table 2.4) SES was positively and highly correlated with sheath and leaf Na, sheath Mg, sheath and leaf Na/K, sheath and leaf Na/Mg, and leaf Na/Ca ratio, and negatively correlated with sheath DW, sheath K, sheath K/Mg, and sheath K/Ca Additionally, sheath K was positively and highly correlated with sheath and leaf Na Root Mg was positively correlated with root Na, but negatively correlated with sheath Na Sheath Mg was highly and positively correlated with sheath and leaf Na These results indicated that salt tolerance in rice was related to mineral concentrations in the sheath rather than that in the roots and leaves

Table 2.3 The regression of Na against K, Mg, Ca contents (mg g-1 DW)

under salt stress treatment

DW, Dry weight; Na (mg g -1 DW) = C’ * K + C’’ * Mg + C’’’ * Ca + Intercept

Figure 2.2 The differences of mineral contents (mg g-1 DW) in roots, sheaths and leaves among 4 salt tolerance groups in the control and the salt stress treatment

DW, Dry weight; STGs, salt-tolerant group; MSTGs, moderately salt-tolerant group; SSGs, salt-sensitive group; HSSGs, highly salt-sensitive group

ns, not significant

a d c

bc b

b b c

ab ns ns

bc

b c

b

0 10 20 30 40

Root Sheath Leaf Root Sheath Leaf

ab a

c

0.0 0.5 1.0 1.5 2.0

Root Sheath Leaf Root Sheath Leaf

Ca content

Table 2.4 Pearson correlation matrix of growth parameters and nutrient contents of seedling in response to salt stress at 12 dS m-1 EC

Dried weight (g) K (mg g -1 DW ) Na (mg g -1 DW) Mg (mg g -1 DW) Ca (mg g -1 DW) Na/K Na/Mg Na/Ca K/Mg K/Ca SES Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf Root Sheath Leaf

SES 1.00

Dried weight (g)

Root -0.34 1.00 Sheath -0.57 0.83 1.00 Leaf -0.32 0.86 0.72 1.00

K (mg g -1 DW)

Root 0.01 -0.16 -0.19 -0.37 1.00 Sheath -0.73 0.18 0.47 0.17 -0.12 1.00 Leaf 0.24 0.09 -0.06 0.19 -0.32 0.10 1.00

Na (mg g -1 DW)

Root -0.40 0.01 -0.05 0.04 0.01 0.24 0.08 1.00 Sheath 0.76 -0.21 -0.47 -0.04 -0.19 -0.68 0.32 -0.36 1.00 Leaf 0.75 -0.30 -0.55 -0.16 -0.12 -0.67 0.15 -0.29 0.89 1.00

Mg (mg g -1 DW)

Root -0.47 -0.15 0.00 -0.15 0.24 0.36 -0.33 0.60 -0.60 -0.49 1.00 Sheath 0.57 -0.18 -0.37 0.12 -0.33 -0.37 0.39 -0.31 0.79 0.72 -0.40 1.00 Leaf 0.22 0.12 0.03 0.29 -0.46 0.03 0.37 -0.06 0.39 0.45 -0.27 0.48 1.00

Ca (mg g -1 DW)

Root -0.15 -0.31 -0.09 -0.28 -0.17 0.29 -0.08 0.29 -0.27 -0.22 0.51 -0.09 -0.08 1.00 Sheath 0.29 -0.36 -0.35 -0.21 -0.22 -0.09 0.09 -0.01 0.24 0.33 0.18 0.53 0.13 0.48 1.00 Leaf 0.04 0.14 0.17 0.22 -0.45 0.21 0.21 -0.16 0.23 0.18 -0.25 0.26 0.57 0.08 0.10 1.00

Na/K

Root -0.04 -0.02 -0.05 0.14 -0.75 0.13 0.38 0.54 0.07 0.03 0.15 0.17 0.30 0.38 0.32 0.31 1.00 Sheath 0.78 -0.23 -0.48 -0.10 -0.12 -0.82 0.07 -0.35 0.91 0.88 -0.49 0.67 0.23 -0.26 0.28 0.06 0.00 1.00 Leaf 0.59 -0.35 -0.52 -0.28 0.06 -0.69 -0.33 -0.31 0.67 0.87 -0.28 0.47 0.20 -0.12 0.29 -0.01 -0.17 0.80 1.00

Na/Mg

Root 0.13 0.22 -0.04 0.29 -0.27 -0.17 0.49 0.36 0.29 0.27 -0.49 0.20 0.31 -0.29 -0.17 0.09 0.39 0.20 0.00 1.00 Sheath 0.73 -0.22 -0.49 -0.15 -0.02 -0.74 0.23 -0.31 0.92 0.82 -0.57 0.52 0.24 -0.29 0.04 0.11 -0.03 0.84 0.66 0.27 1.00 Leaf 0.73 -0.40 -0.63 -0.35 0.10 -0.77 -0.05 -0.30 0.77 0.87 -0.36 0.52 -0.02 -0.17 0.32 -0.10 -0.11 0.85 0.87 0.09 0.78 1.00

Na/Ca

Root -0.06 0.37 0.06 0.40 0.11 -0.16 0.21 0.39 0.06 0.09 -0.14 0.00 0.11 -0.70 -0.31 -0.18 0.05 0.06 -0.05 0.63 0.08 0.02 1.00 Sheath 0.60 0.00 -0.27 0.08 -0.03 -0.57 0.28 -0.36 0.86 0.68 -0.65 0.52 0.32 -0.45 -0.26 0.17 -0.11 0.70 0.48 0.34 0.88 0.56 0.17 1.00 Leaf 0.70 -0.33 -0.58 -0.27 0.08 -0.75 -0.01 -0.25 0.74 0.89 -0.36 0.55 0.18 -0.23 0.27 -0.27 -0.13 0.82 0.88 0.19 0.74 0.90 0.13 0.56 1.00

K/Mg

Root 0.36 -0.03 -0.17 -0.22 0.68 -0.37 -0.04 -0.46 0.28 0.26 -0.52 0.04 -0.21 -0.51 -0.29 -0.25 -0.75 0.26 0.27 0.11 0.40 0.38 0.18 0.45 0.37 1.00 Sheath -0.78 0.25 0.56 0.11 -0.02 0.91 -0.06 0.29 -0.80 -0.77 0.38 -0.67 -0.12 0.24 -0.27 0.14 0.08 -0.85 -0.70 -0.17 -0.76 -0.80 -0.12 -0.64 -0.79 -0.33 1.00 Leaf 0.05 -0.04 -0.10 -0.09 0.08 0.07 0.60 0.13 -0.03 -0.25 -0.04 -0.03 -0.50 0.04 0.02 -0.25 0.13 -0.12 -0.48 0.14 0.00 -0.03 0.06 -0.03 -0.19 0.10 0.04 1.00

K/Ca

Root 0.18 0.15 -0.07 -0.02 0.74 -0.30 -0.08 -0.19 0.10 0.11 -0.22 -0.05 -0.26 -0.71 -0.33 -0.37 -0.71 0.12 0.13 0.07 0.20 0.24 0.55 0.28 0.26 0.81 -0.25 0.10 1.00 Sheath -0.74 0.29 0.55 0.18 -0.05 0.88 0.08 0.24 -0.66 -0.71 0.21 -0.57 -0.06 0.11 -0.48 0.14 0.06 -0.79 -0.71 -0.05 -0.62 -0.78 -0.05 -0.38 -0.75 -0.22 0.92 0.11 -0.19 1.00 Leaf 0.17 0.05 -0.11 0.07 0.05 -0.07 0.70 0.16 0.10 -0.02 -0.12 0.15 -0.10 -0.16 -0.02 -0.54 0.07 0.01 -0.29 0.37 0.10 0.01 0.34 0.13 0.17 0.16 -0.15 0.70 0.22 -0.04 1.00

EC, electrical conductivity; SES, standard evaluation score; DW, dry weight

2.3.5 Regression between SES and mineral contents in roots, sheaths, and leaves in the salt-stress treatment

Table 2.5 shows results for the regressions of SES against Na, K, Mg, and Ca

contents in roots, sheaths, and leaves The relationship between SES and root Na was not

imply that salt tolerance was related to Na, K, and Mg contents of the sheath and Na content

of the leaves

2.3.6 Relationship between SES and Na/K and Na/Mg ratios in roots, sheaths, and leaves

in the salt-stress treatment

Figure 2.3 and Tables 2.2 and 2.6 show the relationships between SES and the Na/K and Na/Mg ratios of roots, sheaths, and leaves in the salt-stress treatment Significant differences in Na/K were observed among the four salt-tolerance groups Sheath and leaf Na/K were lowest in STGs and highest in HSSGs By contrast, the root Na/K ratio was lowest in HSSGs and highest in MSTGs, followed by STGs (Figure 2.3) The Na/Mg ratios differed significantly among groups in sheaths and leaves, but not in roots STGs had the lowest sheath and leaf Na/Mg ratios, and HSSGs had the highest

The regression analyses (Table 2.6) showed that salt tolerance was significantly and

respectively)

Table 2.5 Regressions of SES against Na, K, Mg, Ca contents (mg g-1 DW)

in the salt-stress treatment

SES, standard evaluation score; DW, dry weight; *** , P<0.001; ns, not significant

y = -1.0629x + 9.5696 R² = 0.219 ***

y = -2.8018x + 7.2583 R² = 0.023 ns

Sheath y = 0.0962x + 3.7218

R² = 0.5711 ***

y = -0.2494x + 9.4104 R² = 0.5349 ***

y = 0.8495x + 1.8628 R² = 0.3295 ***

y = 3.676x + 4.5609 R² = 0.0821 ns

Leaf y = 0.2055x + 3.6591

R² = 0.5606 ***

y = 0.1307x + 4.5567 R² = 0.0571 ns

y = 0.3214x + 4.7021 R² = 0.0468 ns

y = 0.2883x + 6.0956 R² = 0.002 ns

Figure 2.3 The differences of Na/K and Na/Mg ratio among 4 salt tolerance groups in roots, sheaths and leaves in the salt-stress treatment

STGs, salt-tolerant group; MSTGs, moderately salt-tolerant group; SSGs, salt-sensitive

group; HSSGs, highly salt-sensitive group

Table 2.6 Regressions of SES against Na/K, Na/Mg ratio under salt stress treatment

2.4 Discussion

2.4.1 Concentration of K in sheaths in the control

The results showed that sheath, but not root or leaf, K content in the control and salt stress treatment were useful as indicators to identify salt tolerance Most authors have divided plant samples into only root and shoot sections to understand the distribution of

minerals or record DWs when testing salt tolerance in rice (Ren et al 2005; Thomson et al 2010; Zheng et al 2014; Sakina et al 2015; De Leon et al 2015) In this study, we divided

plants into three parts, i.e., roots, sheath, and leaves, to gain further insights into tolerance mechanisms The results showed that the most significant differences in K content

salt-were in the sheaths in both the control and treatment groups De Leon et al (2015) reported

that salinity tolerance is most likely controlled in the shoot In the present study, the significant differences in sheath K among the four salt-tolerance groups in the control indicated that under non-saline conditions, sheath K offers a possible means to identify salt-tolerant varieties (the sheath K contents of salt-tolerant KCR20, KCR 124, KCR136 were

from that of SSGs

2.4.2 Na and K contents and Na/K ratios in roots, sheaths, and leaves in the salt stress treatment

De Leon et al (2015) found no significant differences in shoot Na uptake among rice

genotypes and no significant correlation between visual SES and shoot Na concentration These authors also suggested that salt tolerance among tolerant varieties is likely a function not of restricting Na uptake but rather of compartmentalizing Na to reduce its toxic effects

However, our results were consistent with the findings of many previous studies suggesting

that salt stress toxicity may be due to Na accumulation in the shoot (Lin et al 2004) The

root Na content in the sensitive group was about one-fifth that in the sheath and one-third that in the leaves The root Na content of the salt-tolerant group was the highest among the four groups, nearly the same as that in the sheath and about 2.5 times that in leaf Munns and Tester (2008) reported that the most significant plant adaptation to salinity is the ability to restrict the transportation and accumulation of Na in leaves Additionally, in a study of

wheat, El-hendawy et al (2009) suggested that low Na in leaves offered the best indicator

for salt-tolerance screening under both greenhouse and field conditions In our study, the salt-tolerant genotypes (KCR136, KCR20, and KCR124) had low sheath and leaf Na, while most sensitive cultivars had low root and high sheath and leaf Na (KCR246, WRC49, and T65) The low sheath and leaf Na and high root Na of salt-tolerant varieties may be affected

by high sheath K, which causes high concentrations of intracellular fluid and high osmotic pressure in sheath cells As a result, Na cannot be transferred from the roots to the sheath and leaves, and thus remains in the roots Additionally, in salt-sensitive varieties, the low root and high sheath and leaf Na may be due to low osmotic pressure in sheath cells caused

by low sheath K, which results in extreme transfer of Na from the roots to the sheath and

leaves (Deinlein et al 2014)

K is important under saline conditions because of its involvement in osmotic regulation and its competitive effect against Na Excessive Na decreases essential cations contents, especially K in rice and tomato plants, and the addition of K improves their growth under salt-stress conditions (Alam 1999) Our study showed that most genotypes grown under salt-stress conditions had higher Na and Ca and lower K in the roots, sheaths, and leaves than did the control This was consistent with the finding that all genotypes grown in saline medium had increased Na and decreased K in the roots and shoots compared with contents

under non-saline conditions (De Leon et al 2015) On the other hand, many authors have

concluded that K content in the shoot (including the sheath and leaf blade), which results in low Na/K ratios, possibly due to effective compartmentalization of Na in the roots, is an

important indicator for salt-tolerance screening (Ren et al 2005, De Leon et al 2015, Wang

et al 2012) We found significant differences in K content only in the sheath The STGs

(KCR124 and KCR19) had the highest sheath K content, while HSSGs (KCR246, KCR244, and KCR225) had the lowest Additionally, we found that SES and sheath K were highly correlated, whereas SES and root or leaf K were not, indicating that sheath K plays a more important role in salt tolerance than root or leaf K does

Na ions are harmful to plants, and K ions are essential for reducing the uptake of Na

(Wu et al 2009) Therefore, Na and K concentrations and ion balance play important roles

in rice salt tolerance (Zheng et al 2014) Shabala and Cuin (2008) reported that the intracellular K/Na ratio was a key determinant of salt tolerance Furthermore, Zhang et al

(2010) found that some genotypes (FL478 and IR651) were more salt tolerant than others

may maintain a low Na/K ratio because of higher K accumulation (Gregorio and Senadhira

1993, Koyama et al 2001, Ren et al 2005) or restriction of Na transport (Wang et al 2012,

Tester and Davenport 2003) Our results indicated that salt-tolerant varieties had low Na/K ratios in the sheath and leaves (1.1 and 0.65, respectively), whereas salt-sensitive cultivars had higher ratios (9.08 and 2.31, respectively) Salt-tolerant varieties had high K but low Na content in the sheaths Additionally, significant differences in K were found only in the sheath Therefore, in salt-tolerant varieties, the low leaf Na/K ratio was due to low leaf Na rather than to high leaf K, whereas low Na and high K in the sheath were responsible for low sheath Na/K