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MOHAMMAD GOLAM KIBRIA. Department of Soil ..... 2007 and Ali et al. 2004). ...... Dadkhah AR, Stewart WS and Griffith H 2001: Effects of salinity on yield and.

PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF SALT-SENSITIVE AND SALT-TOLERANT RICE GENOTYPES TO SALT STRESS

MS Thesis MOHAMMAD GOLAM KIBRIA

Department of Soil Science Bangladesh Agricultural University Mymensingh

June 2015

PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF SALT-SENSITIVE AND SALT-TOLERANT RICE GENOTYPES TO SALT STRESS

A Thesis

Submitted to Bangladesh Agricultural University, Mymensingh In Partial Fulfillment of the Requirements for the Degree of Master of Science in Soil Science By MOHAMMAD GOLAM KIBRIA Examination Roll No.: 14 Ag. SS JJ 07 M Registration No.: 36196 Session: 2009-10

Department of Soil Science Bangladesh Agricultural University Mymensingh

June 2015

PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF SALT-SENSITIVE AND SALT-TOLERANT RICE GENOTYPES TO SALT STRESS A Thesis

Submitted to Bangladesh Agricultural University, Mymensingh In Partial Fulfillment of the Requirements for the Degree of Master of Science in Soil Science By MOHAMMAD GOLAM KIBRIA Approved as to style and contents by

Professor Dr. Md. Anamul Hoque Supervisor

Dr. Mahmud Hossain Sumon Co-supervisor

Dr. Mahmud Hossain Sumon Chairman, Defence Committee and Head, Department of Soil Science Bangladesh Agricultural University Mymensingh-2202

June 2015

ACKNOWLEDGEMENT All praises for "Almighty Allah" the Omnipresent, Omnipotent, Omniscient, most gracious, most merciful and the supreme ruler of the universe Who has blessed the author with life, time and energy and enable him to complete this research work and manuscript for the degree of Master of Science (MS) in Soil Science. The author would like to wish with pleasure to express his heartfelt respect and the profound gratitude and indebtedness to his respected teacher and research supervisor Dr. Md. Anamul Hoque, Professor, Department of Soil Science, Bangladesh Agricultural University, Mymensingh for his keen interest, scholastic guidance, invaluable suggestions, helpful comments, constructive criticism and constant inspiration, providing facilities and supports needed to undertake this research work throughout the entire period and constructive suggestions for the improvement of the thesis. The author also expresses deep sense of gratitude and indebtedness to his respected cosupervisor Dr. Mahmud Hossain Sumon, Associate Professor and Head, Department of Soil Science, Bangladesh Agricultural University, Mymensingh for his kind help, valuable advice, constructive criticism and encouragement to complete the research and thesis. The author is indebted to all honorable teachers of the Department of Soil Science, Bangladesh Agricultural University for their valuable advice and continuous encouragement to accomplish the research work as well as preparation of this thesis. The author is ever indebted to his younger brother Asif Ahmed for his encouragement throughout the research work. The author is pleased to extend his gratefulness to all his close friends and roommates for their continuous encouragement and cordial cooperation during the entire period of the study. Cordial appreciation and thanks are extended to all the staffs of Department of Soil Science for their cordial cooperation. Finally the author desires to express immense gratitude to his respected parents for their constant inspiration and blessing.

The Author June, 2015

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PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES OF SALT-SENSITIVE AND SALT-TOLERANT RICE GENOTYPES TO SALT STRESS MOHAMMAD GOLAM KIBRIA ABSTRACT Salinity impairs antioxidant defense systems and causes cellular damage to plants. A pot experiment was conducted at the net-house of the Department of Soil Science, Bangladesh Agricultural University, Mymensingh to elucidate the physiological and biochemical characteristics of salt-sensitive and salt-tolerant rice genotypes in response to salt stress. The investigation was carried out with four rice cultivars viz. BRRI dhan28, BRRI dhan47, Binadhan-8 and Binadhan-10 having difference in salinity tolerance. The experiment was laid out in a complete randomized design (CRD) with four replications. Full doses of triple super phosphate, muriate of potash, gypsum and zinc oxide were added to soils during pot preparation and urea was applied in two equal splits. Thirty-days-old seedlings of all the rice varieties were transplanted into pots. At active tillering stage, plants were exposed to different levels (0, 20, 40 and 60 mM NaCl) of salinity. Salt stress caused a significant reduction in growth of all the rice cultivars. Growth reduction was higher in salt-sensitive cultivar than salt tolerant ones. Binadhan-10 showed a higher salt-tolerance in all physiological parameters of rice. Percent reduction in shoot and root biomass was also the lowest in Binadhan-10. Chlorophyll content was significantly decreased under salt stress except Binadhan-10. The intercellular proline content was significantly increased in all salt-tolerant rice cultivars with the increase in salt concentration and the highest proline content was obtained in Binadhan-10. There were remarkable differences in antioxidant enzyme (catalase, peroxidase and ascorbate peroxidase) activites of rice differing in salt tolerance. Catalase (CAT) and ascorbate peroxidase (APX) activities significantly decreased in salt-sensitive genotype whereas significant increases were observed in salt-tolerant genotypes with the increasing salt concentration. Peroxidase (POX) activity was significantly decreased in all the salt-sensitive and salt-tolerant genotypes with increasing NaCl concentration. The K+/Na+ ratio significantly decreased in shoot and root of all rice genotypes. The salt-tolerant genotype (Binadhan-10) maintained higher levels of chlorophyll and proline content as well as increased CAT and APX activities and K+/Na+ ratio under salt stress and therefore, this might be the underlying mechanism for salt tolerance. Keywords: Antioxidant enzymes, Salinity, Chlorophyll, Proline, K+/Na+ ratio v   

CONTENTS Chapter

Title

Page

ACKNOWLEDGEMENTS ABSTRACT CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF APPENDICES Chapter I

INTRODUCTION

iv v vi ix x xi 1

Chapter II REVIEW OF LITERATURE

5 2.1. Effect of salinity on Physiological parameters of plants 5 2.2. Effects of salinity on biochemical attributes of plants 7 2.2.1. Chlorophyll contents 7 2.2.2. Intracellular proline contents 8 2.3. Effects of salinity on antioxidant enzymes activity of 10 Plants 2.3.1. Catalase 11 2.3.2. Peroxidase 12 2.3.3. Ascorbate peroxidase 13 2.4. Potassium (K) and sodium (Na) ratio 13

Chapter III

MATERIALS AND METHODS 3.1. Pot experimentation 3.2. Description of the experimental site 3.2.1. Location 3.2.2. Soil 3.2.3. Climate 3.3. Treatments and design of the experiment 3.4. Description of cultivars under study 3.4.1. BRRI dhan28 3.4.2. Binadhan-8 3.4.3. Binadhan-10 3.4.4. BRRI dhan47

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15 15 15 15 15 15 16 16 16 16 17 17

CONTENTS (Cont’d.) Chapter

Title

Page

3.5. Crop cultivation 3.5.1. Pot preparation 3.5.2. Fertilizer application 3.5.3. Transplanting 3.5.4. Salinity development 3.6. Intercultural operations 3.6.1. Weeding 3.6.2. Irrigation 3.6.3. Plant protection measure 3.6.4. General observation of the experimental pots 3.7. Harvesting 3.8. Analysis of post harvest soil samples 3.9. Collection of growth related data 3.9.1. Plant height 3.9.2. Shoot fresh weight 3.9.3 Shoot dry weight 3.9.4. Root length 3.9.5. Root fresh weight 3.9.6. Root dry weight 3.9.7. Number of tillers per hill 3.10. Biochemical analysis of rice cultivars 3.10.1. Chlorophyll contents 3.10.2. Intracellular proline contents 3.11. Preparation of enzyme extracts 3.12. Antioxidant enzymes activity analysis 3.12.1. Catalase 3.12.2. Peroxidase 3.12.3. Ascorbate peroxidase 3.13. Potassium (K) and sodium (Na) ratio 3.13.1. Preparation of plant samples 3.13.2. Digestion of plant samples 3.13.3. Determination of potassium (K) 3.13.4. Determination of sodium (Na) 3.14. Statistical analysis

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17 17 17 17 18 18 18 18 18 18 18 19 19 19 19 19 19 19 20 20 20 20 20 21 21 21 21 21 22 22 22 22 22 23

CONTENTS (Cont’d.) Chapter Chapter IV

Title

Page

RESULTS

24

4.1. Effects of salt stress on physiological parameters of rice cultivars 4.1.1 Plant height 4.1.2 Root length 4.1.3 Shoot fresh weight 4.1.4 Shoot dry weight 4.1.5 Root fresh weight 4.1.6 Root dry weight 4.1.7 Number of effective tillers hill-1 4.2. Effects of salinity on biochemical attributes of Rice Cultivars 4.2.1. Chlorophyll contents 4.2.2. Intracellular proline contents 4.3. Effects of salinity on antioxidant enzymes activity in Rice 4.3.1. Catalase (CAT) 4.3.2. Peroxidase (POX) 4.3.3. Ascorbate Peroxidase (APX) 4.4. Potassium (K) and Sodium (Na) ratio 4.4.1. In Shoot 4.4.2. In Root

Chapter V DISCUSSION Chapter VI SUMMARY Chapter VII CONCLUSION

24 25 27 28 30 31 32 34 34 36 38 38 39 40 41 41 42

44 48 51

REFERENCES APPENDICES

52 66

viii   

24

LIST OF TABLES TABLE

TITLE

PAGE

4.1

Effect of salinity on plant height of different rice cultivars

24

4.2

Effect of salinity on root length of different rice cultivars

26

4.3

Effect of salinity on shoot fresh weight of different rice

27

cultivars

4.4

Effect of salinity on shoot dry weight of different rice

29

cultivars

4.5

Effect of salinity on root fresh weight of different rice

30

cultivars

4.6

Effect of salinity on root dry weight of different rice

31

cultivars

4.7

Effect of salinity on number of tillers per hill of different

33

rice cultivars

4.8

Effect of salinity on chlorophyll-a content in different

34

rice cultivars

4.9

Effect of salinity on chlorophyll-b content in different rice cultivars

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35

LIST OF FIGURES FIGURE

TITLE

PAGE

4.1

Effect of salinity on percent decrease in plant height of different rice cultivars Effect of salinity on percent decrease in root length of different rice cultivars Effect of salinity on percent reduction in shoot fresh weight of different rice cultivars

25

4.2 4.3 4.4 4.5

Effect of salinity on percent reduction in shoot dry weight of different rice cultivars Effect of salinity on percent reduction in root fresh weight of different rice cultivars

28 29 31

4.6

Effect of salinity on percent reduction in root dry weight of different rice cultivars

32

4.7

Effect of salinity on percent decrease in number of tillers per hill of different rice cultivars Effect of salinity on total chlorophyll content of different rice cultivars

33

Effect of salt stress on intercellular proline content in four rice cultivars differing in salt tolerance Effect of salinity on catalase (CAT) activity in different rice cultivars

37

4.11

Effect of salinity on peroxidase (POX) activity in different rice cultivars

39

4.12

Effect of salinity on ascorbate peroxidase (APX) activity in different rice cultivars

40

4.13

Effect of salinity on K+/ Na+ ratio in shoot of four different rice varieties Effect of salinity on percent decrease in K+/ Na+ ratio in shoot of four different rice varieties Effect of salinity on K+/ Na+ ratio in root of four different rice varieties Effect of salinity on percent decrease in K+/ Na+ ratio in root of four different rice varieties

41

4.8 4.9

4.10

4.14

4.15 4.16

x   

26

36

38

42

43 43

LIST OF APPENDICES APPENDIX

TITLE

I

Physio-chemical characteristics of initial soil samples

66

II

Monthly record of air temperature, relative humidity, rainfall and sunshine hours during the period from January, 2014 to May, 2014 Chemical characteristics of post-harvest soil samples Different photographs indicating the effect of salinity on shoot and root growth of rice cultivars used in the experiment

66

III IV

 

xi   

PAGE

67 68

CHAPTER I

INTRODUCTION Climate change is the most serious environmental threat that causes sea level rise and affects the coastal areas of Bangladesh by developing salinity. Soil salinity is a major concern to agriculture all over the world because it affects almost all plant functions. Millions of hectares of land throughout the world are too saline to produce economic crops, and more land is becoming non-productive each year due to salinity build up. Three hectares of arable lands are lost each minute because of soil salinization. Approximately 7% of the world’s land area, 20% of the world’s cultivated land and nearly half of the irrigated land are affected by soil salinity (Szabolcs, 1994; Zhu, 2001; FAO, 2008). Soil salinization due to irrigation is becoming increasingly detrimental to agriculture (Flowers, 1999). In view of another projection, 2.1% of the global dry land agriculture is affected by salinity (FAO, 2008). Salinity is a serious problem impairing normal plant growth and limits the realization of yield potential of modern cultivars. Agriculture is the most important sector of Bangladesh’s economy. Physiological stress in plants due to salinity is the major factor reducing crop yields in coastal areas of Bangladesh. Over 30% of the cultivable area of Bangladesh lies in the coastal and offshore zones. Out of 2.86 million hectares of coastal and offshore lands, about 1.06 million hectares are affected by varying degrees of salinity (SRDI, 2010). The area under salinity is increasing with time (from 0.83 m ha to 1.056 m ha in 36 years; SRDI, 2010) due to rise in sea water level with increased global temperature. Soil salinization is a major process of land degradation that decreases soil fertility and crop productivity. There is a report that coastal regions of Bangladesh are quite lower in soil fertility (Haque, 2006). Salinity largely reduces the crop yield in the coastal areas of the country mainly in Khulna, Satkhira, Bagerhat, Barguna, Patuakhali, Noakhali and Chittagong districts. Usually 30-50% yield losses occur depending on the level of soil salinity. According to the Intergovernmental Panel on Climate Change (2007), crop production may fall by 10-30% by 2050 in Bangladesh due to climate change. 1

The most important cereal crop in the world is rice, yielding one–third of the total carbohydrate source. Three billion peoples consider rice as their staple food, accounting for 50–80% of their daily calorie intake. Rice is a very salt–sensitive monocot (Shereen et al. 2005 and Darwish et al. 2009). The total area under rice in Bangladesh is about 10.83 million hectares with a production of 33.54 million metric tons (BBS, 2011) whereas rice yield is very low in coastal areas. To increase rice yield, it is imperative to know about the physiological and biochemical changes in plants under salt stress to develop salt-tolerant rice cultivars. Plants show differential responses to salinity which is efficiently expressed into physiological attributes adopting or alleviating the shock for salt stress. Decrease in chlorophyll content becomes a first indication of responses in different plants subjected to salinity stress (Roy and Basu, 2008). In general, high level of salinity in soil causes imbalance in osmotic potential, ionic equilibrium and nutrient uptake (Niu et al. 1995; Munns, 2002). Salt stress injury in rice is mostly caused by the accumulation of Na+ more than that of Cl- (Munns and Tester 2008). The high concentration of sodium in soil solution alters the uptake of other nutrients and cause toxic effects (Loupassaki et al. 2002). In addition, it impairs a wide range of cellular metabolisms including photosynthesis, protein synthesis and lipid metabolism (Alia-Mohanty et al. 1992; Ashraf, 1994; Zhu, 2001; Parida et al. 2005; Lichtenthaler et al. 2005). Salinity imposes both ionic toxicity and osmotic stress to plants, leading to nutritional disorder and oxidative stress. Salt stress causes increased uptake of Na+ and Cl-, and decreased uptake of essential cations particularly K+ (Khan et al. 2003). The deficiency of K+ initially leads to chlorosis and then causes necrosis (Gopal and Dube, 2003). As time of exposure to salinity is prolonged, plants experience ionic stress, which can lead to premature senescence of adult leaves and mortality of plants (Amirjani, 2011). Plants have evolved a variety of adaptive mechanisms to respond to salt stress. One of the main adaptive mechanisms to salt stress in plants is the accumulation of compatible solutes (Ashraf and Foolad, 2007; Sharma and Dietz, 2006). Proline is the most common compatible solutes that occur in plants under salt stress. Proline is a proteinogenic amino acid with an exceptional conformational 2

rigidity, low molecular weight and usually non toxic at high cellular concentrations. Increased levels of endogenous proline accumulation in plants correlate with enhanced salt tolerance (Hasegawa et al. 2000; Sharma and Dietz, 2006; Siripornadulsil et al. 2002). Proline accumulation was found to occur when plants subjected to high salt stress (Boscaiu et al. 2012; Sripinyowanich et al. 2013). Proline accumulates in the cytosol or chloroplast whereas proline degradation in plants occurs in mitochondria (Krasensky and Jonak, 2012). Proline accumulation might also play regulatory roles during plant growth under salt stress (Mattioli et al., 2008, 2009a). Proline can act as a signaling molecule to modulate mitochondrial functions, which can be essential for plant recovery from stress and (Shulaev et al. 2008; Gill and Tuteja, 2010) endogenous increase in proline induces oxidative stress tolerance by modulating the activities of antioxidant enzymes (Saiema et al. 2012; Saeedipour, 2013). Salt stress also induces the accumulation of reactive oxygen species (ROS) such as superoxide radical (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH-) in plant cells (Guo et al. 2009; Xu et al. 2009). These ROS are necessary for inter and intracellular signaling (Foyer and Noctor, 1999) but at high concentrations they seriously disrupt normal metabolism of plants (Hernandez et al. 2001). The excess production of ROS is toxic to plants and causes oxidative damage to cellular constituents, leading to cell death (Banu et al. 2009, 2010). To prevent the ROS from

damaging

cellular

components,

plants

have

developed

multiple

detoxification mechanisms, including synthesis of various enzymes and antioxidant molecules (Yamane et al. 2009, Hoque et al. 2007a, 2007b). Alleviation of oxidative damage and increased resistance to salinity may result from the presence of efficient antioxidant systems (Vaidyanathan et al. 2003; Munns and Tester 2008). To mitigate the deleterious effect of salinity due to accumulation of ROS, plants possess enzymatic and non-enzymatic antioxidant defense systems (Ashraf and Harris, 2010). The major ROS scavenging antioxidant enzymes include catalase 3

(CAT: EC 1.11.1.6), peroxidase (POX: EC 1.11.1.7) and ascorbate peroxidase (APX: EC 1.11.1.11) (Alscher et al. 2002; Arora et al. 2002). A decreased activity of antioxidant enzymes has been associated with higher salt stress in plants. Imposition of plants under higher salinity leads to an inhibition of CAT and POX activity. Catalase (CAT) converts H2O2 into water and molecular oxygen; whereas peroxidase (POX) decomposes H2O2 by oxidation of co-substrates such as phenolic compounds and/or antioxidants and ascorbate peroxidase (APX) helps to detoxify other ROS including H2O2. According to different studies it can be said that endogenous increase in proline induces oxidative stress tolerance by modulating the activities of antioxidant enzymes (Saiema et al. 2012; Saeedipour, 2013). In view of the above stated facts, it was presumed that salt stress has a significant effect on physiological and biochemical attributes of plants. So, better understanding of physiological and biochemical characteristics of plants are vital for improving salt tolerance mechanism in plants. Comparison of these attributes could be useful in identifying differences related to the ability of each cultivar to cope with salinity. This has led to the present investigation, which investigated the effects of salinity on some physiological and biochemical responses in four rice varieties (viz. BRRI dhan28, BRRI dhan47, Binadhan-8 and Binadhan-10. Results from this study can provide information on the possible involvement of ROS in the damage by NaCl stress in rice plant and also could allow deeper insights into the molecular mechanisms of tolerance to salt-induced oxidative stress. That’s why, a proper understanding of the pathways attributing tolerance to salinity and the selection of better plant types is the aim of the study. Therefore, the present study was undertaken with the following objectives: i.

To investigate the effects of salt stress on the root and shoot growth of saltsensitive and salt-tolerant rice genotypes

ii.

To characterize the salt-sensitive and salt-tolerant rice genotypes at physiological and biochemical levels under salt stress

4

CHAPTER II

REVIEW OF LITERATURE Soil salinity is a prevalent abiotic stress that limits the productivity and geographical distribution of plants. The impact of salinity is most serious in countries where all or most of agricultural production is based on irrigation. As irrigated agriculture expands, more salinity problems will develop because there are millions of hectares of potentially irrigable land that could become saline. More research has been conducted on this issue and it is found that it has many detrimental effects on plant growth and development. There are many factors that influence plant response to salinity. Salinity or excessive soluble salt concentrations affects plant growth and production. The effects of salt stress can also be observed in different physiological and biochemical parameters of plants. 2.1 Effects of salinity on physiological parameters of plants Salt stress is a key constraint reducing plant growth and productivity. Salinity can affect plant growth in a number of ways. All the plant parts are not affected in the same way. Some parts are highly affected compared to some other parts of the plant when exposed to salinity. The rate of plant growth depends upon a number of important events such as cell division, enlargement and cell differentiation as well as genetic, morphological, physiological, ecological events along with their complex interactions that can be severely affected by salt stress. It has been reported that the typical symptom of salinity injury to the plant is growth retardation due to the inhibition of cell elongation (Bandeoglu et al. 2004). Shoot and root growth inhibition is a common response to salinity as indicated by certain studies (Ruiz et al. 2005 and Koca et al. 2007). Leaf area and shoot fresh weight are relatively more affected and the magnitude of reduction varies between salt-sensitive and salt-tolerant rice cultivars (Alam et al. 2004). A greenhouse experiment was conducted by Miah et al. (1992) also reported that salinity decreases straw weight of both salt-sensitive and salt-tolerant rice cultivars.

5

Another important parameter, plant height also decreases with the increasing salinity level (Islam et al. 2011). The physiological effects of excess salinity are many but visual symptoms generally do not become evident until salinity conditions are extreme and prolonged. The reduction in plant height will be higher if the plants are exposed to excess salinity for longer period. Salt stress decreases plant height at seven weeks after transplanting of rice (Miah et al. 1992). Besides rice other cereals like wheat and maize also show the similar trend. Significant reduction in plant height was observed in wheat with the increasing salinity level (Talat et al. 2013). The adverse effects of salt stress takes place at all levels of plant life, ranging from morphological to molecular levels that are observable at all phenological growth stages during plant life cycle. Changes in morphological characters are the ultimate determinants of stress effects on plants. Sumithra et al. (2006) and Kumar et al. (2009) demonstrated that the salt tolerant cultivars produce greater biomass than salt sensitive mungbean and rice cultivars respectively, when irrigated with NaCl dominated waters. Roots are more sensitive to salt stress compared to shoots. Salinity induces injuries to plant growth by causing physiological water stress (Calatayud et al. 2003), so it mainly damages plant roots (Zheng et al. 2012). Root length decreases with the increasing salinity level (Momayezi et al. 2010). Root weight also decreases as salinity retards the growth and development of plant roots (Talat et al. 2013). Salt tolerant as well as salt sensitive rice cultivars if exposed to salt stress formulate the same findings. Even root dry weight of rice also decreases significantly as the levels of salinity increased from 50-75 mM NaCl (Shereen et al. 2007) and Root length and weight of rice greatly reduces without appreciable reduction in shoot growth (Hakim et al. 2010). Along with rice root weight of wheat also decreases if the plants are exposed to higher level of salinity (Talat et al. 2013). Other than cereals pulse crops like lentil exhibits the same. When lentil crop is exposed to salt stress root weight significantly decreases with the increasing salinity level (Islam 2004). Kumar et al. (2007) concluded that the 6

severity of salinity antagonism to the germination and normal growth of plant as indicated by germination percentage, shoot length, root length, and root/shoot ratio of seedlings was higher in the salt-tolerant rice cultivar compared to saltsensitive ones. Number of tillers per hill is one of most important yield contributing characters among all the physiological parameters. Under salt stress, yield decreases due to less number of tillers in rice plants. Linghe and Michael (2000) conducted a study to determine salinity effects on rice seedlings and yield components. The results showed that number of tiller per hill decreases significantly as the salinity level increases. 2.2 Effects of salinity on biochemical attributes of plants Salinity stress involves changes in various biochemical and metabolic processes, depending on severity and duration of the stress, and ultimately inhibits crop production (James et al. 2001). Excessive soluble salt concentrations affects biochemical attributes of plants by increasing the osmotic potential of the soil solution as well as specific ion toxicities in soil. Different biochemical attributes like chlorophyll content and intercellular proline content are greatly influenced under salt stress. 2.2.1 Chlorophyll contents The decline in productivity has observed for many plant species subjected to excess salinity is often associated with a reduction in photosynthetic capacity. Chlorophyll is the most important component in the photosynthesis process. That’s why the rate of photosynthesis depends on the amount of chlorophyll content in plant leaves. Photosynthetic capacity in plant reduces under salt stress due to the presence of lower amount of chlorophyll in plant leaves. Chlorophyll content becomes a first indication of responses in different plants subjected to salinity stress (Roy and Basu, 2008). Amirjani (2011) showed that the reduction of chlorophyll-a and chlorophyll-b was detected after NaCl treatment

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in leaves. Chlorophyll formation is inhibited in citrus by specific ion toxicity that is due to accumulation of Na+ or Cl- resulting from salt stress (Carter et al. 1963). Ghosh et al. (2010) showed that a relatively salt resistant variety however recorded less loss of chlorophyll than a relatively sensitive variety. The decrease in chlorophyll content under stress is a commonly reported phenomenon in other plants and may be due to the membrane deterioration (Mane et al. 2010; Tantawy et al. 2009). Usually there is dominance of chlorophyll-a over chlorophyll-b in plants but their values become closer with increasing salinity (Mane et al. 2010). Many studies have conducted on rice to explore the effect of salinity on total chlorophyll content in rice leaves. When rice plants are exposed to salt stress, total chlorophyll content decreases with the increase in levels of salinity compared to non-saline soil (Islam et al. 2007 and Ali et al. 2004). Chlorophyll a and chlorophyll b content in rice leaves show wide variation in different rice cultivars differing in salt tolerance. Salinity decreases chlorophyll a and chlorophyll b content in salt sensitive genotypes of rice (Djanaguiraman et al. 2004). The salt tolerant genotypes of rice maintained higher levels of chlorophyll a and b under higher salinity stress which could have contributed to their salt tolerance (Zheng et al. 2012). Salt treatments decrease chlorophyll content significantly in the salt treated plants compared to control plants in pea (Ozturk et al. 2012). Oil crops like soybean also response in the same way. Dogan et al. (2011) conducted an experiment on soybean under salt stress and the result revealed that total chlorophyll content decreases with the increasing salt concentration. The salt tolerant genotypes of rice maintained higher levels of chlorophyll a and b under higher salinity stress which could have contributed to their salt tolerance (Zheng et al. 2012). 2.2.2 Intracellular proline contents Plants have evolved a variety of adaptive mechanisms to respond to abiotic stresses including salinity. One of the main adaptive mechanisms to salt stress in plants is the accumulation of compatible solutes (Okuma et al. 2004; Ashraf and Foolad, 2007). Proline is the most common compatible solute that occurs in a 8

wide variety of plants. Increased levels of proline accumulated in plants correlate with enhanced salt tolerance (Okuma et al. 2004; Ashraf and Foolad, 2007).Salinity index of leaf proline showed strong positive relationship with salinity index of yield and is thus a promising index for deploying in breeding programmes for evolving salt tolerant rice (Summart et al. 2010). Proline is a proteinogenic amino acid with an exceptional conformational rigidity, and is essential for primary metabolism. Proline accumulates in many plant species in response to environmental stress. Proline accumulation in stressed plants has a protective function, which has been emphasized in numerous reviews (Hare et al. 1997; Kavi et al. 2005 and Verbruggen et al. 2008). The amino acid proline accumulates (normally in cytosol) under stress and is correlated with osmotic adjustment to improve plant salinity tolerance (Vinocur and Altman, 2005). So, it can be clearly understood that proline plays a significant role for providing salt tolerance in plants. That’s why if intercellular proline content increases it may protect plants from osmotic stress. Proline consistently increased under salinity significantly in all the tolerance groups which in accordance with Summart et al. (2010) who showed that salt stress caused an increase in the accumulation of proline; hence proline was thus a robust indicator of plant stress even at low salinity. Salt stress causes an increase in the accumulation of proline in rice which may provide protection from osmotic damage of plants (Summart et al. 2010). Some studies on rice also show that salt stress increases proline level and due to upregulation of proline synthesis genes (Nounjan et al. 2012). In barley, both salt sensitive and tolerant cultivars tend to accumulate proline with increasing concentration of NaCl (Reza et al. 2006).Accumulation of proline is enhanced with the increase in salt concentrations in pea (Ozturk et al. 2012). The same trend is also observed in other crops besides rice. Soybean also exhibits the increasing trend of intercellular proline content with the increasing salinity level (Dogan et al. 2011 and Weisany et al. 2012). In case of spices like clover, proline content increases with the increase in salinity level (Abdalla, 2011). It is clear that, 9

increased levels of proline accumulated in plants correlate with enhanced salt tolerance (Okuma et al. 2004; Ashraf and Foolad, 2007). 2.3 Effects of salinity on activity of antioxidant enzymes of plants To survive under salt stress plants use a number of mechanisms.One of the biochemical changes occurring when plants are subjected to biotic or abiotic stresses is the production of reactive oxygen species (ROS) (Allen, 1995).Reactive oxygen species (ROS) are highly reactive and in the absence of any protective mechanism they can seriously disrupt normal metabolism through oxidative damage to lipids, protein and nucleic acids(Asada, 1999).Antioxidant enzymes are the most important components in the scavenging system of ROS (Rout and Shaw, 2001). Reactive oxygen species including H2O2 are elevated by increased salinity. Plants possess an array of enzymatic and non-enzymatic antioxidant defense systems to protect their cells against the damaging effects of ROS (Noctor and Foyer, 1998; Apel and Hirt, 2004). The major H2O2 scavenging antioxidant enzymes include catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APX). The increase in antioxidant enzyme activities is involved in elimination of H2O2 in salt-stressed roots (Kim et al. 2005). It has been reported that antioxidant enzyme activities decrease under salt stress (Shalata et al. 2001; Khedr et al. 2003; Mishra and Das, 2003; Mittova et al. 2004). Cell membranes are one of the first targets of many plant stresses and it is generally accepted that the maintenance of their integrity and stability under salt stress condition is a major component of salt tolerance in plants. As cell membranes are the first targets of many plant stresses, reactive oxygen species (ROS) may destroy normal metabolism through peroxidation of membrane lipids (Arora et al. 2002). Lipid peroxidation of biological membranes might lead to structural alterations such as denaturalization of proteins and nucleic acids in salt stressed plants. Experimental evidences suggest that lipid peroxidation reactions of cellular membranes may play an important role in

10

radical mediated cell injury (Zhang et al. 2007). Therefore, activity of antioxidant enzymes may act as efficient determinant criteria in the toxic degree to salt stressed plants (Arora et al. 2002; Aslam et al. 2006). Cell membranes are one of the first targets of many plant stresses and it is generally accepted that the maintenance of their integrity and stability under salt stress condition is a major component of salt tolerance in plants. Crucial changes in soil salinity can lead to osmotic stress, which are primary effects of salt stress. Such free radicals and other active derivatives of oxygen may produce inevitably as by-products of physiological redox reactions. It has been reported that salt stress caused elevated levels of reactive oxygen species (ROS) (Arora et al. 2002), and the production of active oxygen exceeded the capacity of scavenging systems, resulting in oxidative damage (Taiz and Zeiger, 2010). The increased levels of ROS can inactivate enzymes, damage important cellular components, which induced plant growth arrest, and even death finally (Arora et al. 2002). To mitigate the deleterious effect of salinity on regular metabolism, plants have evolved various strategies to contend with this problem. Superoxide dismutase (SOD) is a major scavenger of superoxide and its enzymatic action results in the formation of H2O2 and O2. The hydrogen peroxide produced is then scavenged by catalase (CAT) and a variety of peroxidase like Guaiacol peroxidase (POX) and ascorbate peroxidase (APX). Catalase converts H2O2 into water and molecular oxygen, whereas peroxidase decomposes H2O2 by oxidation of cosubstrates such as phenolic compounds and/or antioxidants. Differential responses have been observed from the previous studies on the activities of antioxidant enzymes of plants under salt stress. 2.3.1 Catalase

Plants exhibit an increase in catalase activity with increasing the magnitude of NaCl stress.An experiment was conducted by Nounjan et al. (2012) on rice during salt stress and showed that salt stress increased the activity of antioxidant enzyme i.e. catalase (CAT). Besides rice soybean also shows differential response 11

to salt stress for different cultivars which have different level of salt tolerance. In case of salt-sensitive genotypes of soybean, catalase activity decreases with increasing salt concentration (Dogan et al. 2011) but tolerant genotypes show different response to salt stress. Increasing trend is observed in the catalase activity for salt-tolerant genotypes of soybean (Weisany et al. 2012). Root crops like potato also exhibit the same trend like soybean. In potato, catalase (CAT) activity

significantly

increases

with

the

increasing

salt

concentrations

(Daneshmand et al. 2009). In barley, a linear and significant increase in CAT activity is observed in response to increased salt concentration after exposure to salt stress (Reza et al. 2006). Pea plants are normally sensitive to salt stress. Catalase activity shows a declining trend with increasing the magnitude of NaCl stress in case of pea as well (Ozturk et al. 2012). In case of green bean, CAT activities increased with the increasing salt stress in both salt-sensitive and salttolerant varieties. CAT activities were higher in the salt-tolerant one than the saltsensitive cultivar (Yasar et al. 2008). 2.3.2 Peroxidase

Peroxidase (POX) enzyme also plays an important role for protecting plants from the damage of reactive oxygen species (ROS). In plants, a number of enzymes regulate H2O2 intracellular levels; peroxidase was considered to be the most important one among them (De Andr’e et al. 2006). Different studies show that salt stress influences the activity of peroxidase enzyme of plants when they are exposed to excess salinity problem. All most all the plants show similar response to salinity. Salt stress significantly increases the activity of antioxidant enzyme i.e. peroxidase (POX) in case of salt sensitive rice varieties but tolerant varieties exhibit the declining trends of activity of peroxidase enzyme activity with the increasing salt concentration (Nounjan et al. 2012). In case of soybean, peroxidase activity increases as a result of increasing salt concentration (Weisany et al. 2012). Both salt and drought stress significantly increase the peroxidase activity in potato (Daneshmand et al. 2009). Peroxidase activity also significantly increases in salt stressed pea seedlings (Ozturk et al. 2012). 12

2.3.3 Ascorbate peroxidase

Ascorbate peroxidase enzyme helps to protect plants from osmotic stress by neutralizing the reactive oxygen species (ROS). Over expression of the APX gene in plants has been reported to improve protection against oxidative stress (Wang et al. 1999). Salt stress increases the activity of antioxidant enzyme i.e. ascorbate peroxidase (APX) in rice (Nounjan et al. 2012). Ascorbate peroxidase activity also increases as a result of increasing salinity stress in soybean (Weisany et al. 2012). Another study on salt sensitive soybean cultivars reveals that salinity induced a significant decline in ascorbate peroxidase activities (Dogan et al. 2011). In case of Barley, there is a linear and significant increase in ascorbate peroxidase activity in response to increased salt concentration (Reza et al. 2006). Root crops like potato exhibit that there is a significant increase in ascorbate peroxidase activity with the increasing magnitude of NaCl concentration (Daneshmand et al. 2009). APX activities increased with increasing salt stress in both salt-sensitive and salttolerant varieties. APX activities were higher in the salt-tolerant one than the saltsensitive cultivar (Yasar et al. 2008). Ozturk et al. (2012) investigated the effects of long term salt stress on the changes in activities ascorbate peroxidase (APX) in leaves of salt sensitive pea cultivars in field conditions and the result revealed that ascorbate peroxidase activity significantly decreases in salt stressed seedlings. These enzymes (CAT, POD and APX) were also reported to be important in salt tolerance rice along with other crops like mulberry (Sudhakar et al. 2001), cotton (Meloni et al. 2003) and maize genotypes (DeNato et al. 2006). 2.4

Potassium (K) and sodium (Na) ratio

Salinity imposes both ionic toxicity and osmotic stress to plants, leading to nutrition disorder and oxidative stress (Hasegawa et al. 2000 and Zhu, 2003). Under saline condition the availability of Na+ is higher compared to the essential K+ in soil solution. The salt-sensitive and salt-tolerant plants show differential response in the aspect of K+/Na+ ratio in both root and shoot under salt stress. Salt stress disturbs cytoplasmic K+/Na+ homeostasis, causing an increase in Na+ 13

to K+ ratio in the cytosol (Zhu, 2003). Salt tolerance is directly associated with K contents because of its involvement in osmotic regulation and competition with Na. Plant salt tolerance requires not only adaptation to Na+ toxicity but also the acquisition of abundant K+ whose uptake by the plant cell is affected by high external Na+ concentrations (Zhang et al. 2010). The tolerant genotypes absorbed less Na+ and more K+, resulting in lower Na+: K+ ratio in the shoot and produced greater shoot biomass through faster growth that could further dilute the absorbed Na+. Contrastingly, the sensitive genotypes had higher Na+ and lower K+ contents, resulting in higher Na+/K+ ratio in the shoot (Nakhoda et al., 2012). If the rice plants are exposed to salt stress from tillering stage to maturity similar result is observed. That salinity increases Na +, decreases +

+

K+ and K /Na ratio in flag leaves of rice when imposed to salinity (Islam et al. 2009). There is a significant negative correlation between leaf Na+ and K+/Na+ ratio in different rice varieties differing in salt tolerance under salt stress (Haq et al. 2009). Salinity decreases K+ concentrations and increases Na+ concentrations both in grain and straw. The salt-sensitive cultivar has a higher concentration of Na+ and a lower concentration of K+ at increased salinity levels than salt-tolerant (Miah et al. 1992). Besides rice, when soybean plants are exposed to salinity K+/Na+ ratio decreases with increasing salinity (Dogan et al. 2011). Root crops like potato also show the decrease in K+/Na+ ratio under salt stress (Daneshmand et al. 2009). This decrease in K+/Na+ ratio under salt stress causes Na+ toxicity in plants and leads to cellular damage and deficiency in K in plant.

14

CHAPTER III

MATERIALS AND METHODS In this chapter a brief description on different materials used in the experiment and different methods followed for determining physiological and biochemical responses of salt-sensitive and salt-tolerant rice genotypes under salt stress have been presented under the following headings: 3.1 Pot experimentation A pot experiment was carried out in the net-house, Department of Soil Science, BAU, Mymensingh during the period of January, 2014 to March, 2014 to investigate the effects of salt stress on the growth as well as physiological and biochemical characteristics of salt-sensitive and salt-tolerant rice cultivars.

3.2 Description of the experimental site 3.2.1 Location The growth performance was carried out in the net-house of Department of Soil Science, BAU, Mymensingh. The experimental site is located at 24.75˚N latitude and 90.4˚E longitude and 18 meter above the sea level. The Agro-ecological Zone of the study area is Old Brahmaputra Floodplain (AEZ 9).

3.2.2 Soil The soil was collected at 15 cm depth from the Soil Science Field Laboratory, BAU, Mymensingh. The physico-chemical characteristics of initial soils samples have been shown in Appendix- I.

3.2.3 Climate The experimental area was under sub-tropical climate characterized by high temperature, high humidity and heavy rainfall with occasional gusty winds in kharif season (April-September) and less rainfall associated with moderately low temperature during the rabi season (October-March). Weather informations regarding temperature, relative humidity, rainfall and sunshine hours prevailed 15

in the experimental site during the whole period of study has been reported in Appendix- II. 3.3 Treatments and design of the experiment Four NaCl concentrations viz. control (No NaCl), 20 mM NaCl, 40 mM NaCl and 60 mM NaCl were used as treatments in the experiment. Four rice cultivars viz. BRRI dhan28 (salt-sensitive), BRRI dhan47 (salt-tolerant), Binadhan-8 (salttolerant) and Binadhan-10 (salt-tolerant) were treated with the four salt treatments. The experiment was laid out in a completely randomized design (CRD) with four replications. Total 64 (4×4×4) pots were used in this experiment. Equal size plastic pots (15 cm deep with 17 cm diameter at the top) having a surface area of 0.023 m2 at the opening ware used in the experiment. Considerable spacing was maintained among the pots for the ease of management practices. 3.4

Description of rice cultivars

3.4.1 BRRI dhan28 BRRI (Bangladesh Rice Research Institute) released BRRI dhan28 in 1994. It is a salt-sensitive high yielding rice variety. It is semi dwarf, early maturing rice variety. Plant height is about 90-95 cm. Its crop duration is almost 135-140 days and average yield is 7.5 tons per hectare. It is cultivated in boro season; widely grown in all over Bangladesh but grows well in non-saline soil.

3.4.2 Binadhan-8 Binadhan-8 is a salt tolerant high yielding rice variety released from BINA (Bangladesh Institute of Nuclear Agriculture) in 2010. It is semi dwarf, early maturing and medium bold grain rice variety. Plant height is about 105 cm. Binadhan-8 requires 130-135 days to mature. Under salt stress, average grain yield is 5.5 t/ha. It can tolerate 8-10 dS/m salinity at mature stage. It can be grown in aus, aman and boro season. This variety is most suitable in saline areas of Bangladesh and also other non saline areas. Binadhan-8 is becoming popular in the saline belt of Bangladesh due to its salt tolerance and better yield. 16

3.4.3 Binadhan-10 Binadhan-10 was released by BINA in 2012. It is a salt tolerant high yielding rice variety. Disease incidence and pest attacks are very low. Plant height is about 110 cm. It can tolerate up to 10-12 dS/m salinity. Plant duration is around 127-132 days and average grain yield is 6.0 t/ha which is higher than other salt tolerant varieties. The variety is suitable to be grown in both aman and boro seasons.

3.4.4 BRRI dhan47 BRRI dhan47 was released by BRRI (Bangladesh Rice Research Institute) in 2006. It is a salt-tolerant variety. Plant height is about 105 cm. The crop duration is almost 152 days and average yield is 6.0 t/ha. It is cultivated in boro season and suitable to be grown in coastal areas of Bangladesh. It can tolerate 8 dS/m salinity at seedling stage and 4 dS/m salinity at mature stage. 3.5

Crop cultivation

3.5.1 Pot preparation Soils were collected from the Soil Science Field Laboratory, BAU. The soil was air dried and ground using a wooden hammer. Plant debris and other unwanted materials were removed from the soil. The plastic pots were filled with 8 kg soil so that enough space was kept to maintain flooded condition.

3.5.2 Fertilizer application Full doses of chemical fertilizers viz. triple super phosphate (0.8 g/pot), muriate of potash (1.0 g/pot), gypsum (1.2 g/pot) and zinc oxide (0.1 g/pot) were added to soils during pot preparation. Urea fertilizer was applied in two split doses; first dose (0.3 g pot-1) was applied after 14 days (22 January, 2014) of transplanting and second dose (0.3 g pot-1) was applied at active tillering stage (18 February, 2014) .

3.5.3 Transplanting Thirty-days-old seedlings of all the rice varieties were collected from the Soil Science Field Laboratory, Bangladesh Agricultural University (BAU) and 17

Bangladesh Institute of Nuclear Agriculture (BINA), Mymensingh. Then three seedlings of each variety were transplanted in each pot on 08 January, 2014.

3.5.4 Salinity development The pure salt (NaCl) was used for developing salinity. For control treatment, no NaCl was added to soils. For 20 mM, 40 mM and 60 mM NaCl treatments, 15.24 g, 30.47 g and 45.71 g of NaCl were added to the pots, respectively at active tillering stage (24 February 2014). These amounts of NaCl were dissolved in 1000 ml of water and then the solutions were poured uniformly into the pots according to the treatments.

3.6 Intercultural operations 3.6.1 Weeding Weeds were uprooted by hand as and when necessary.

3.6.2 Irrigation Normal tape water was used as irrigation. Measured water was added to keep water level up to the top of the pot, avoiding anaerobic condition and maintaining proper imposition of salinity levels.

3.6.3 Plant protection measures No plant protection measures were performed since plants were not infested with any insects, pests or diseases.

3.6.4 General observation of the experimental pots Observations were regularly made. All the stages of plants and plant’s response as per treatments were observed carefully.

3.7 Harvesting The crops were harvested at maximum tillering stage. The whole plant with roots was carefully uprooted from the soil so that the root systems of the plants remain unaffected. The crop was harvested on 12 March 2014 at 62 DAT (days after

18

transplanting) for all the rice cultivars used in the experiment. The harvested crop of each pot was separately collected and properly tagged.

3.8 Analysis of post-harvest soil samples The changes in chemical properties of post-harvest soil including pH, electrical conductivity (EC), exchangeable sodium percentage (ESP) and organic matter content under different salt stress conditions for different rice cultivars were determined following the standard method. The physico-chemical characteristics of post-harvest soil samples for different treatments have been shown in Appendix- III.

3.9 Collection of growth related data 3.9.1 Plant height (cm) Plant height was taken before harvest as the length between the bases of the plant to the tip.

3.9.2 Shoot fresh weight (g) Immediately after harvesting, the shoot samples were separated from the root and the fresh shoot weight was taken carefully.

3.9.3 Shoot dry weight (g) Shoot dry weight was taken after the samples were sun-dried properly.

3.9.4 Root length (cm) Root length was taken after harvesting since it was determined by measuring the length between the bases of the plant to the root tip.

3.9.5 Root fresh weight (g) Root fresh weight was taken immediately after harvesting. After separating the root samples from the shoot root fresh weight was taken using an electric balance.

19

3.9.6 Root dry weight (g) After sun-drying, root dry weight was taken with the help of an electric balance.

3.9.7 Number of tillers per hill Number of tillers per hill was counted carefully for each of the plant before harvesting.

3.10

Biochemical analysis of rice cultivars

3.10.1 Chlorophyll contents Chlorophyll content was measured at maximum tillering stage according to method of Porra et al. (1989). An aliquot amount of fresh green leaf of rice was suspended in 10 ml of 80% acetone, mixed well and kept at room temperature in the dark for 7 days. The supernatant was collected after centrifugation at 5000 rpm for 15 min and the absorbance was recorded at 645 nm and 663 nm in a spectrophotometer to estimate total chlorophyll content in the green leaves (including chlorophyll a and chlorophyll b). The chlorophyll a and b contents were calculated using the following equations: Chlorophyll a (µg/ml) = 12.25(A663) - 2.55(A645) Chlorophyll b (µg/ml) = 20.31(A645) - 4.91(A663)

3.10.2 Intracellular proline contents Proline content was measured at maximum tillering stage according to the method of Bates et al. (1973). An aliquot amount of fresh green leaf of rice was homogenized in 10 ml of 3% sulfosalicylic acid and the homogenate was centrifuged at 5000 rpm for 15 min. Two milliliters of the supernatant were reacted with 2 ml of acid ninhydrin (1.25 g ninhydrin dissolved in 30 ml of glacial acetic acid and 20 ml of 6 M phosphoric acid) and 2 ml of glacial acetic acid for 1 hour at 100°C and the reaction was then terminated in an ice bath. The colored reaction mixture was extracted with 4 ml of toluene and the absorbance was recorded at 520 nm.

20

3.11

Preparation of enzyme extract

An aliquot amount of fresh green leaf was homogenized with 5 mL of 50 mM Tris–HCl buffer (pH 8.0) for CAT, and 50 mM KH2PO4 buffer (pH 7.0) for POX and APX. The homogenate was centrifuged at 5000 rpm for 20 min and the supernatant was then used as enzyme extract for antioxidant enzymes (catalase, peroxidase and ascorbate peroxidase) assay.

3.12

Analysis of activity of antioxidant enzymes

The activity of antioxidant enzymes was measured from green leaves at maximum tillering stage of rice cultivars.

3.12.1 Catalase Catalase (CAT) activity was determined by the method of Aebi (1984). The reaction mixture consisted of 50 mM Tris–HCl buffer (pH 8.0), 0.25 mM EDTA, 20 mM H2O2 and 25 μl of sample solution. The reaction was started by the addition of H2O2. The activity was calculated from the decrease in absorbance at 240 nm for 2 min when the extinction coefficient was 40 M-1 cm-1.

3.12.2 Peroxidase Peroxidase (POX) activity was determined according to Nakano and Asada (1981). The reaction buffer solution contained 50 mM KH2PO4 buffer (pH 7.0), 0.1 mM EDTA, 0.1 mM H2O2, and 10 mM guaiacol. The reaction was started by the addition of the sample solution to the reaction buffer solution. The activity was calculated from change in absorbance at 470 nm for 30 sec where an extinction coefficient is 26.6 mM-1 cm-1.

3.12.3 Ascorbate peroxidase Ascorbate peroxidase (APX) activity was measured following the method of Nakano and Asada (1981). The reaction buffer solution contained 50 mM KH2PO4 buffer (pH 7.0), 0.1 mM EDTA, 0.1 mM H2O2, and 0.5 mM ascorbate. The reaction was started by adding the sample solution to the reaction buffer solution. The

21

activity was calculated from the change in absorbance at 290 nm for 1 min when the extinction coefficient was 2.8 mM-1 cm-1.

3.13

Potassium (K) and sodium (Na) ratio

3.13.1 Preparation of plant samples The K and Na contents in shoot and root samples were measured according to the standard method. Shoot and root samples were dried in an oven at about 65°C for 48 hours and then ground in a grinding machine to pass through a 20 mesh sieve. The ground plant materials (shoot and root) were stored in small paper bags and placed in desiccators.

3.13.2 Digestion of plant samples Grinding plant samples of 0.5 g (shoot and root separately) were transferred into 100 ml digestion vessel. 10 ml of di-acid mixture (HNO3: HClO4= 2:1) was added into the vessel. After leaving for a while the flasks were heated at a temperature slowly raised to 200°C. Heating was stopped when the dense white fume of HClO4 occurred. After cooling, the contents were taken into a 50 ml volumetric flask and the volume was made with distilled water. The digests were used for the determination of K and Na.

3.13.3 Determination of potassium (K) Two ml of digested samples for shoot and root were taken and both were diluted 50 ml volumetric flask individually to make desired concentration. Potassium was determined from the extracted solution by using flame photometer. This method was proposed by Brown and Lilleland (1946).

3.13.4 Determination of sodium (Na) Two milliliters of digested samples for both shoot and root were taken and both were diluted 50 ml volumetric flask individually to make desired concentration. Na was determined from the extracted solution by using flame photometer. This method was proposed by Brown and Lilleland (1946).

22

3.14

Statistical analysis

Data were analyzed statistically to examine the treatment effects using statistical package MStatC. The mean differences were adjudged by Duncan’s Multiple Range Test (DMRT) (Gomez and Gomez, 1984) and ranking was indicated by letters.

23

CHAPTER IV RESULTS This experiment was carried out to determine the effects of salt stress on morphological viz. root and shoot growth of rice plants as well as biochemical responses viz. antioxidant enzyme activities in imparting tolerance to NaCl oxidative stress, chlorophyll, proline and K+ and Na+ contents of leaves and roots of rice varieties exhibiting differences in salinity tolerance. Results obtained on different morphological and biochemical parameters of salt-sensitive (BRRI dhan28) and salt-tolerant (BRRI dhan47, Binadhan-8 and Binadhan-10) rice cultivars have been presented in this chapter. 4.1 Effects of salt stress on physiological parameters of rice cultivars 4.1.1 Plant height Soil salinity caused a significant decrease in plant height of all the rice cultivars (salt-sensitive and salt-tolerant) used in the experiment (Table 4.1 and Appendix IV). The lowest salt stress used in this experiment (20 mM NaCl) significantly decreased plant height in both salt-sensitive and salt-tolerant rice cultivars except in Binadhan-10. At the highest salts stress (60 mM NaCl), the tallest plants (58.5 cm) were obtained in a salt-tolerant rice variety (BRRI dhan47) and the shortest plants (45.0 cm) were obtained in a salt-sensitive variety (BRRI dhan28). Table 4.1: Effect of salinity on plant height of different rice cultivars Treatments

Plant height (cm) BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Control

62.5a

66.5a

65.5a

64.5a

20 mM NaCl 40 mM NaCl

60.0b 51.0c

63b 60c

61b 55.5c

63.5a 54b

60 mM NaCl

45.0d

58.5c

52d

49c

SE ()

0.613

0.454

0.496

0.361

CV (%)

4.87

4.27

3.47

5.10

24

It was found that at 20 mM NaCl treatment the percent decrease in plant height was the lowest in Binadhan-10 and the highest in Binadhan-8 (Figure 4.1). At higher salinity levels (40 mM and 60 mM NaCl), the percent decrease in plant height was higher in salt-sensitive (BRRI dhan28) cultivar compared to salt tolerant cultivars. Among the salt-tolerant rice cultivars, the percent decrease in plant height was higher in Binadhan-10 and lower in BRRI dhan47 at higher salinity levels.

30

Percent decrease in Plant height

25 20 BRRI dhan28

15

BRRI dhan47

10

Binadhan-8 5

Binadhan-10

0 20 mM NaCl

40 mM NaCl 60 mM NaCl

Treatments

Figure 4.1: Effect of salinity on percent decrease in plant height of different rice cultivars 4.1.2 Root length Root length of both salt-sensitive and salt-tolerant rice cultivars was drastically reduced with the increasing salinity levels (Table 4.2 and Appendix IV). It was observed that at 20 mM NaCl stress condition, root length was significantly decreased in all the rice cultivars compared to control condition. There was no significant difference in root length at 40 mM and 60 mM NaCl treatments in salt tolerant rice cultivars (Binadhan-10 and BRRI dhan47).

25

Table 4.2: Effect of salinity on root length of different rice cultivars Treatments

Root length (cm) BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

SE ()

27.5a 17.5b 14.5c 12.5d 0.448

25.5a 21b 15.5c 14c 0.500

26.5a 18.5b 16.5c 13d 0.541

27a 22.5b 18c 16c 0.617

CV (%)

4.31

4.53

5.04

5.19

Control 20 mM NaCl 40 mM NaCl 60 mM NaCl

At 20 mM NaCl treatment, the percent decrease in root length was the highest in BRRI dhan28 and the lowest in Binadhan-10. At 40 mM and 60 mM NaCl treatments, the percent decrease in root length was the highest in BRRI dhan28 and the lowest in Binadhan-10. Among the salt-tolerant rice cultivars percent decrease in root length was the lowest in Binadhan-10 at all the NaCl stresses. At 20 mM and 60 mM NaCl stresses, percent decrease in root length was the highest in Binadhan-8 compared to BRRI dhan47. At 40 mM NaCl treatment, decrease in root length was higher in BRRI dhan47 than Binadhan-8 (Figure 4.2). 60

Percent decrease in Root length

50 40 30

BRRI dhan28 BRRI dhan47

20

Binadhan-8

Binadhan-10

10 0 20 mM NaCl 40 mM NaCl 60 mM NaCl

Treatments

Figure 4.2: Effect of salinity on percent decrease in root length of different rice cultivars 26

4.1.3 Shoot fresh weight Salt stress caused a significant decrease in shoot fresh weight of all the rice varieties used in the experiment (Table 4.3). At 20 mM NaCl stress, shoot fresh weight significantly decreased in all the rice cultivars irrespective of salt tolerance. At 40 mM and 60 mM NaCl treatments, both the salt-sensitive (BRRI dhan28) and salt-tolerant (BRRI dhan47, Binadhan-8 and Binadhan-10) rice genotypes exhibited significant reduction in shoot fresh weight. It was observed that reduction of shoot fresh weight due to salt stress was the highest in saltsensitive rice cultivar compared to salt-tolerant rice cultivars. At 60 mM NaCl treatment, the highest shoot fresh weight was obtained in Binadhan-10 followed by BRRI dhan47 and Binadhan-8 exhibited the lowest shoot fresh weight among the salt-tolerant rice varieties. Table 4.3: Effect of salinity on shoot fresh weight of different rice cultivars Treatments

Shoot fresh weight (g) BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Control

43.12a

32.56a

30.29a

28.78a

20 mM NaCl 40 mM NaCl

29.84b 16.72c

26.57b 18.20c

23.78b 16.16c

25.42b 23.36c

60 mM NaCl

6.32d

14.22d

12.43d

15.45d

SE ()

0.634

0.637

0.165

0.219

CV (%)

4.27

4.82

4.47

4.72

It was observed that percent reduction in shoot fresh weight was always higher in the salt-sensitive rice cultivar compared to the salt-tolerant rice cultivars. At all NaCl stresses, salt-sensitive rice cultivar (BRRI dhan28) exhibited the highest percent reduction in shoot fresh weight among all the rice cultivars used in the experiment. Among the salt-tolerant rice cultivars, Binadhan-10 exhibited lower reduction percentage in shoot fresh weight compared to BRRI dhan47 and Binadhan-8 under salt stress. Percent reduction in shoot fresh weight was the highest in Binadhan-8 followed by BRRI dhan47 when exposed to salt stresses (20 mM, 40 mM and 60 mM NaCl) among the salt-tolerant rice cultivars (Figure 4.3).

27

90

% reduction in shoot FW

80 70 60 50 BRRI dhan28

40

BRRI dhan47 30

Binadhan-8

20

Binadhan-10

10

0 20 mM NaCl 40 mM NaCl 60 mM NaCl

Treatments Figure 4.3: Effect of salinity on percent reduction in shoot fresh weight of different rice cultivars 4.1.4 Shoot dry weight With the increase in salinity levels, shoot dry weight decreased in both saltsensitive and salt-tolerant rice genotypes (Table 4.4). At 20 mM NaCl stress condition, there was a significant reduction in shoot dry weight of all the rice cultivars in this experiment except Binadhan-10. But at 40mM and 60 mM NaCl treatments, shoot dry weight decreased significantly in all varieties including Binadhan-10. It was observed that reduction of shoot dry weight due to salt stress was the highest in BRRI dhan28 (salt-sensitive) compared to salt-tolerant rice varieties. The highest shoot dry weight at 40 mM NaCl treatments was found in BRRI dhan47 and at 60 mM NaCl condition Binadhan-10 exhibited the highest shoot DW among the salt-tolerant rice cultivars.

28

Table 4.4: Effect of salinity on shoot dry weight of different rice cultivars Treatments Control 20 mM NaCl 40 mM NaCl 60 mM NaCl SE () CV (%)

Shoot dry weight (g) BRRI dhan28 10.71a 7.07b 4.32c 1.43d 0.329 9.15

BRRI dhan47 7.57a 6.73b 4.53c 2.86d 0.154 4.92

Binadhan-8 7.43a 4.83b 3.79c 2.51d 0.165 6.18

Binadhan-10 5.67a 5.53a 4.20b 2.96c 0.132 4.86

It was also observed that percent shoot dry weight reduction was the highest in salt-sensitive (BRRI dhan28) one and compared to salt-tolerant rice cultivars at all levels of salinity. At 60 mM NaCl treatment, among the salt-tolerant rice varieties BRRI dhan47 and Binadhan-8 exhibited higher reduction percentage in shoot dry weight than Binadhan-10. Binadhan-8 showed the highest reduction percentage in shoot dry weight compared to other salt-tolerant genotypes but the reduction was lower than the salt-sensitive one. At 20 mM and 40 mM NaCl treatment, the reduction in shoot dry weight was also the highest in Binadhan-8 and the lowest in Binadhan-10 among the salt-tolerant rice genotypes (Figure 4.4).

% reduction in shoot DW

90 80 70

60 50

BRRI dhan28

40

BRRI dhan47

30

Binadhan-8

20

Binadhan-10

10 0 20 mM NaCl 40 mM NaCl 60 mM NaCl

Treatments Figure 4.4: Effect of salinity on percent reduction in shoot dry weight of different rice cultivars 29

4.1.5 Root fresh weight Salt stress caused a drastic decrease in root FW in both salt-sensitive and salttolerant rice cultivars (Table 4.5). At 20 mM NaCl stress, root FW significantly decreased in all the rice cultivars irrespective of salt-tolerance. At 40 mM and 60 mM NaCl treatments, the rice genotypes exhibited significant reduction in root FW. It was observed that reduction of root fresh weight due to salt stress was the highest in BRRI dhan28 compared to salt tolerant rice varieties. Among the salttolerant rice genotypes, at the highest salinity level (60 mM NaCl) the highest root FW was obtained in Binadhan-10 following Binadhan-8 when BRRI dhan47 exhibited the lowest root FW among the salt-tolerant rice varieties. But at lower salinity levels BRRI dhan47 maintained higher root FW compared to others. Table 4.5: Effect of salinity on root fresh weight of different rice cultivars Treatments

Root fresh weight (g) BRRI dhan28 BRRI dhan47

Binadhan-8

Binadhan-10

Control

19.58a

19.05a

18.32a

17.09a

20 mM NaCl 40 mM NaCl

16.63b 11.76c

17.58b 13.57c

12.46b 10.86c

15.40b 10.21c

60 mM NaCl

4.45d

6.29d

6.82d

7.28d

SE ()

0.361

0.288

0.412

0.290

CV (%)

4.78

3.54

7.13

3.87

Like other parameters, salt-tolerant rice genotypes exhibited the lowest percent reduction in root fresh weight from control and the highest percent reduction was observed in salt-sensitive one. The percent reduction in root fresh weight was the highest in BRRI dhan28 and the lowest was in Binadhan-10 when the plants were treated with 60 mM NaCl. When the rice plants differing in salt tolerance were exposed to 40 mM NaCl treatments, percent reduction in root FW was the highest in BRRI dhan28 and the lowest in BRRI dhan47. Among the salttolerant rice cultivars, Binadhan-10 exhibited lower percent reduction in root FW compared to Binadhan-8 under salt stress. At 20 mM NaCl treatment, the reduction in root FW was the highest in Binadhan-8 and the lowest was in BRRI dhan47 (Figure 4.5). 30

% reduction in root FW

80 70 60 50 40

BRRI dhan28

30

BRRI dhan47

20

Binadhan-8

Binadhan-10

10 0 20 mM NaCl 40 mM NaCl 60 mM NaCl

Treatments Figure 4.5: Effect of salinity on percent reduction in root fresh weight of different rice cultivars 4.1.6 Root dry weight Salinity caused a reduction in root dry weight in both salt-sensitive and salttolerant rice varieties (Table 4.6). At 20 mM NaCl stress, there was a significant reduction in root dry weight in all the rice varieties in this experiment irrespective of salt tolerance. The lowest root dry weight was found in salttolerant rice (BRRI dhan47) cultivar compared to other rice cultivars at higher salinity level. The highest root dry weight was obtained in Binadhan-10 at 60 mM NaCl treatment while the lowest was in BRRI dhan47 among the salt-tolerant rice cultivars. Table 4.6: Effect of salinity on root dry weight of different rice cultivars Treatments

Root dry weight (g) BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Control

7.49a

5.64a

6.39a

5.92a

20 mM NaCl 40 mM NaCl 60 mM NaCl SE () CV (%)

4.46b 2.61c 1.73d 0.113

5.08b 2.59c 1.54d 0.106

4.62b 3.12b 1.78c 0.107

4.88b 3.22c 1.91d 0.136

4.84

4.97

4.60

6.35

31

It was found that, percent reduction in root dry weight was the highest in saltsensitive rice cultivar. At 20 mM NaCl treatment, BRRI dhan47 exhibited the lowest reduction percentage in root dry weight whereas Binadhan-8 showed the highest reduction percentage in root dry weight compared to other salt-tolerant cultivars. When the plants were exposed to 40 mM and 60 mM NaCl treatments, the lowest percent reduction in root dry weight was found in Binadhan-10 and the highest was in BRRI dhan47 among the salt-tolerant rice cultivars (Figure 4.6).

% reduction root DW

80 70 60 50 40

BRRI dhan28

30

BRRI dhan47

20

Binadhan-8 Binadhan-10

10 0

20 mM NaCl 40 mM NaCl 60 mM NaCl

Treatments Figure 4.6: Effect of salinity on percent reduction in root dry weight of different rice cultivars 4.1.7 Number of tillers per hill Salt stress caused a drastic decrease in number of tillers per hill in both the saltsensitive and salt-tolerant rice cultivars (Table 4.7). The number of tillers per hill decreased significantly with the increasing salinity levels. Among the salttolerant rice cultivars, at 20 mM NaCl treatment, number of tillers per hill in Binadhan-10 and BRRI dhan47 was significantly decreased whereas in Binadhan8 the decrease was not significant. The salt-sensitive variety BRRI dhan28 showed significant difference in number of tillers per hill at 20 mM NaCl treatment. The 32

highest number of tillers per hill was obtained in Binadhan-10 among the four test cultivars irrespective of salinity levels. Table 4.7: Effect of salinity on number of tillers per hill of different rice cultivars Treatments Control 20 mM NaCl 40 mM NaCl 60 mM NaCl SE () CV (%)

Number of tillers per hill (no.) BRRI dhan28 13a 12b 8c 6d 0.338 5.71

BRRI dhan47 13a 12b 9c 8d 0.270 4.46

Binadhan-8 15a 14a 10c 8d 0.336 4.99

Binadhan-10 16a 15b 12c 11d 0.264 3.73

It was observed that at 20 mM NaCl salt stress, the percent decrease in number of tillers per hill was less than 10% in both the salt sensitive and salt tolerant rice cultivars. At 40 mM and 60 mM NaCl treatments, the decrease was the highest in salt-sensitive (BRRI dhan28) cultivar. Among the salt-tolerant cultivars, the percent decrease in number of tillers per hill was the highest in Binadhan-8 and the lowest was in Binadhan-10 (Figure 4.7).

Percent decrease in Number of tillers per hill

60 50 40 30

BRRI dhan28 BRRI dhan47

20

Binadhan-8 Binadhan-10

10 0 20 mM NaCl 40 mM NaCl 60 mM NaCl

Treatments Figure 4.7: Effect of salinity on percent decrease in number of tillers per hill of different rice cultivars 33

4.2 Effects of salinity on biochemical attributes of rice cultivars 4.2.1 Chlorophyll contents Chlorophyll is one of the most important pigment components of a plant. Photosynthesis is the important process of the plant for food production which occurs in green part of the plant in the presence of chlorophyll. So chlorophyll content in plant is the indicator of the food production. Chlorophyll content may vary with varying salt concentration and eventually affecting the plant growth and development. Chlorophyll-a and chlorophyll-b contents in rice leaf exhibited differential responses to salt stress. It was found that there was an increase in chlorophyll-a content in salt-tolerant rice genotypes with the increasing salt concentration. The salt-sensitive rice genotype (BRRI dhan28) showed a decrease in chlorophyll-a content at and above 40 mM NaCl stress. For the salt-tolerant rice varieties, chlorophyll-a content increased up to 40 mM NaCl stress but decreased at 60 mM NaCl treatment. Binadhan-10 exhibited the highest chlorophyll-a content among the rice genotypes under the highest salinity level (Table 4.8). Table 4.8: Effect of salinity on chlorophyll-a content in different rice cultivars Treatments

Chlorophyll-a content (µg/ml) BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Control

9.06a

7.75b

8.35b

8.17b

20 mM NaCl

9.12a

8.03b

8.41b

8.21b

40 mM NaCl

8.22b

8.84a

8.67a

8.44a

60 mM NaCl

7.21c

7.34c

6.88c

8.38a

SE ()

0.109

0.084

0.069

0.025

CV (%)

2.18

1.84

1.48

0.51

Both the salt-sensitive and salt-tolerant rice genotypes showed the same trend in chlorophyll-b content under salt stress. Chlorophyll-b content was decreased in all the rice genotypes with the increasing salt concentrations (Table 4.9). At 20 34

mM NaCl stress, chlorophyll-b content decreased but this decrease was not significant which was true for all the varieties. When the plants were exposed to higher salinity level, significant reduction was occurred in both salt-sensitive and salt-tolerant rice genotypes. Salt stress caused a minimum damage in chlorophyll-b content in Binadhan-10 at different NaCl stresses compared to other test cultivars. Table 4.9: Effect of salinity on chlorophyll-b content in different rice cultivars Treatments

Chlorophyll-b content (µg/ml) BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Control

10.39a

10.97a

10.32a

9.97a

20 mM NaCl

10.04a

9.95a

9.91a

9.80a

40 mM NaCl

8.44b

7.23b

8.53b

9.39b

60 mM NaCl

4.76c

5.37c

3.91c

9.36b

SE ()

0.115

0.281

0.156

0.114

CV (%)

2.37

6.03

3.32

2.06

There was a significant reduction in total chlorophyll content both in saltsensitive and salt-tolerant rice cultivars with the increasing salinity levels (Figure 4.8). Salt stress decreased total chlorophyll content in all the rice varieties but the reduction in total chlorophyll content was not significant in Binadhan-10 at all salinity levels (0-60 mM NaCl). There was no significant decrease in total chlorophyll content in all other rice cultivars when they were exposed to 20 mM NaCl stress condition irrespective of salt tolerance. Significant reduction was found in both salt-sensitive (BRRI dhan28) and salt-tolerant (BRRI dhan47 and Binadhan-8) rice cultivars at 40 mM and 60 Mm NaCl stress conditions. It was found that Binadhan-10 exhibited the highest total chlorophyll content under 60 mM NaCl stress condition while Binadhan-8 exhibited the lowest amount of total chlorophyll content. These results are in agreement with the higher sensitivity of chlorophyll to NaCl observed in sensitive plants as compared with tolerant plants.

35

22 20

Control

20 mM NaCl

40 mM NaCl

60 mM NaCl

Total chl content ((µg/ml)

18 16 14 12 10 8

a

a

b c

a

a

b c

a

a

b

c

a

a

a a

6 4 2 0 BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Figure 4.8: Effect of salinity on total chlorophyll content of different rice cultivars 4.2.2 Intracellular proline contents Salt stress causes an accumulation of proline in plant cell which may help plants to avoid cellular damages. At 20 mM NaCl stress condition, there was a significant increase in intracellular proline content in all the salt-tolerant rice cultivars BRRI dhan47, Binadhan-8 and Binadhan-10 as well as in salt-sensitive (BRRI dhan28) rice cultivars. When the plants were exposed to 40 mM NaCl stress condition, there was a significant increase in intercellular proline content in both salt-sensitive and salt-tolerant rice genotypes. At 60 mM NaCl stress condition, the highest intercellular proline content was obtained in Binadhan-10 and the lowest intercellular proline content was found in the salt-sensitive one (BRRI dhan28) compared to other rice varieties. The salt-sensitive rice genotype BRRI dhan28 exhibited the significant reduction in intercellular proline content 36

whereas three salt-tolerant genotypes showed the significant increase in intercellular proline content at 60 mM NaCl stress condition. 12

Control 20 mM NaCl 40 mM NaCl 60 mM NaCl

Proline content (mM)

10

8

6

c

b a d

c

c

b a a

c b a

d

c b a

4

2

0 BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Figure 4.9: Effect of salt stress on intercellular proline content in four rice cultivars differing in salt tolerance With the increase in salt concentration, intercellular proline content was significantly increased in all salt-tolerant rice varieties (BRRI dhan47, Binadhan-8 and Binadhan-10) while salt-sensitive one (BRRI dhan28) resulted in an increase in intercellular proline content at lower level of salinity (20 mM NaCl and 40 mM NaCl treatment) and caused a significant decrease in proline content at higher salinity level (60 mM NaCl). Therefore, it is clear that Binadhan-10 maintained the higher intercellular proline content at all salt stress conditions compared to other rice cultivars (Figure 4.9).

37

4.3 Effects of salinity on antioxidant enzymes activity in rice An increase in activity of antioxidant enzymes might help in protecting plants against salt stress. To investigate the changes in antioxidant defense systems in rice under salt stress condition, activities of antioxidant enzymes catalase (CAT), peroxidase (POX) and ascorbate peroxidase (APX) were measured. 4.3.1. Catalase (CAT) The salt-sensitive and salt-tolerant rice genotypes exhibited significant differences in CAT activity under NaCl stress condition (Figure 4.10). There was a significant increase in CAT activity with the increasing salinity level in the salttolerant rice genotypes. There was no significant difference in CAT activity in Binadhan-10 at 40 mM and 60 mM NaCl stress conditions. In case of salt-sensitive BRRI dhan28, CAT activity decreased significantly with the increasing salt concentration. At control condition, the highest CAT activity was obtained in salt-sensitive (BRRI dhan28) rice cultivars while Binadhan-10 exhibited the lowest CAT activity compared to other rice cultivars. 7

Control 20 mM NaCl

CAT (mmol/min/g FW)

6

40 mM NaCl 60 mM NaCl

5

4

3

2

a

a b c

d

c b a

d

c b a

b

b a a

1

0 BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Figure 4.10: Effect of salinity on catalase (CAT) activity in different rice cultivars 38

4.3.2. Peroxidase (POX) There was a remarkable difference in POX activity in both salt-sensitive and salttolerant rice genotypes under salt stress condition. Peroxidase (POX) activity significantly decreased in both salt-tolerant (BRRI dhan47, Binadhan-8 and Binadhan-10) and salt-sensitive rice genotype (BRRI dhan28) with increasing salinity level (Figure 4.11). At 40 mM NaCl stress, POX activity increased in Binadhan-8 but this increase was not significant. At control condition, the highest POX activity was obtained in salt-tolerant (BRRI dhan47) rice cultivar and Binadhan-10 exhibited the lowest POX activity compared to other rice cultivars. At 60 mM NaCl stress condition, POX activity was the highest in BRRI dhan47 while the lowest POX activity was found in the salt-sensitive (BRRI dhan28) rice cultivar.

60 Control 20 mM NaCl

POX (μmol/min/g FW)

50

40 mM NaCl 60 mM NaCl

40

30

20

10

a

b c c

a

b c d

a b b c

a

BRRI dhan47

Binadhan-8

Binadhan-10

b c d

0 BRRI dhan28

Figure 4.11: Effect of salinity on peroxidase (POX) activity in different rice cultivars 39

4.3.3. Ascorbate Peroxidase (APX) Salt stress caused a significant increase in ascorbate peroxidase (APX) activity in all the salt-tolerant (BRRI dhan47, Binadhan-8 and Binadhan-10) rice genotypes but salt-sensitive (BRRI dhan28) cultivar exhibited a significant decrease in APX activity with the increasing salinity level. At control condition, the highest APX activity was found in Binadhan-8 and Binadhan-10 exhibited the lowest APX activity compared to other cultivars. At 60 mM NaCl stress condition, APX activity was the highest both in BRRI dhan-8 and Binadhan-10. On the other hand, BRRI dhan28 exhibited the lowest APX activity at 60 mM NaCl stress condition (Figure 4.12).

18

Control

APX (μmol/min/g FW)

16

20 mM NaCl 40 mM NaCl

14

60 mM NaCl 12 10 8 6

4

a b c

d

c

c b a

d c

b

a

d

c b a

2 0 BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Figure 4.12: Effect of salinity on ascorbate peroxidase (APX) activity in different rice cultivars

40

4.4 Potassium (K) and sodium (Na) ratio Shoot and root samples were analysed for the determination of K+/Na+ ratio in different rice genotypes under salt stress. 4.4.1 In Shoot The K+/Na+ ratio in rice shoot significantly decreased due to salt stress in both salt-sensitive and salt-tolerant rice varieties with the increase in salt concentration. At 20 mM NaCl treatment, this decrease in K +/Na+ ratio was not significant in Binadhan-10 and BRRI dhan47 but significant reduction was observed in Binadhan-8 and BRRI dhan28 (Figure 4.13). At lower salt concentrations (20 mM NaCl and 40 mM NaCl treatments) among the salttolerant varieties, the highest K+/Na+ ratio was observed in Binadhan-8 while Binadhan-10 exhibited higher K+/Na+ ratio compared to BRRI dhan47. At 60 mM NaCl stress condition, the K+/Na+ ratio in Binadhan-8 significantly reduced whereas Binadhan-10 exhibited the highest K+/Na+ ratio among the salt-tolerant varieties. 1.4

Control 20 mM NaCl

1.2

40 mM NaCl

Shoot K+/Na+ ratio

60 mM NaCl 1

0.8

0.6

0.4

0.2

a

b b c

a

a b c

a

b c d

a a b c

0 BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Figure 4.13: Effect of salinity on K+/ Na+ ratio in shoot of four different rice varieties 41

The percent decrease in shoot K+/Na+ ratio increased with the increasing salt stress compared to control (Figure 4.14). The highest percent decrease in shoot K+/Na+ ratio was observed in Binadhan-8 at the highest level of salinity (60 mM NaCl) and the lowest was in Binadhan-10 at different levels of salinity (0-60 mM NaCl).

Percent decrease in shoot K+/Na+ ratio

70 60 50 BRRI dhan28

40

BRRI dhan47 30 Binadhan-8 20

Binadhan-10

10 0

20 mM NaCl

40 mM NaCl

60 mM NaCl

Figure 4.14: Effect of salinity on percent decrease in K+/ Na+ ratio in shoot of four different rice varieties 4.4.2 In Root The K+/Na+ ratio in rice root significantly decreased due to salt stress in both the salt-sensitive

and salt-tolerant varieties (Figure 4.15). The salt-sensitive rice

variety BRRI dhan28 maintained lower K+/Na+ ratio compared to the salttolerant rice varieties. The K+/Na+ ratio was around 2-fold lower in 20 mM NaClstressed root than non-stressed root in both salt-sensitive (BRRI dhan28) and salttolerant rice genotypes (BRRI dhan47, Binadhan-8 and Binadhan-10). NaCl stress at 40 mM and 60mM caused a drastic decrease in K+/Na+ ratio in salt-tolerant rice varieties but the decrease in BRRI dhan28 was not significant. For all treatments, BRRI dhan47 exhibited the highest K+/Na+ ratio compared to other salt-tolerant rice varieties and Binadhan-8 exhibited lower K+/Na+ ratio than Binadhan-10. 42

1.4 Control 20 mM NaCl 40 mM NaCl 60 mM NaCl

1.2

Root K+/Na+ ratio

1 0.8 0.6 0.4 0.2

a b b b

a

b c c

a b b c

a

b bc c

0 BRRI dhan28

BRRI dhan47

Binadhan-8

Binadhan-10

Figure 4.15: Effect of salinity on K+/ Na+ ratio in root of four different rice varieties The percent decrease in root K+/Na+ ratio was also increased with the increasing salt stress compared to control (Figure 4.16). BRRI dhan47 showed the highest percent decrease in root K+/Na+ ratio among the rice cultivars at all salinity levels (0-60 mM NaCl). The salt-sensitive cultivar BRRI dhan28 showed lower level of reduction percentage in root K+/Na+ ratio compared to other cultivars when exposed to different levels of salinity. 70

Percent decrease in root K+/Na+ ratio

60 50

BRRI dhan28

40

BRRI dhan47 30

Binadhan-8 Binadhan-10

20 10 0 20 mM NaCl

40 mM NaCl

60 mM NaCl

Figure 4.16: Effect of salinity on percent decrease in K+/ Na+ ratio in root of four different rice varieties 43

CHAPTER V

DISCUSSION Salinity is a major problem for reduced rice yield in the coastal belt of Bangladesh. Reduced plant growth is a common phenomenon when grown under salt stress and usually expressed as stunted shoots. Plant height was significantly decreased with increasing salinity level in rice (Table 4.1 and Appendix 4.1). The percent decrease in plant height was higher in salt-sensitive one than the salt-tolerant rice cultivars (Figure 4.1). Islam et al. (2011) on hybrid rice and Miah et al. (1992) on two rice varieties also found that plant height decreased with increasing salinity level which was similar to our results. As roots are in direct contact with the surrounding solution having salinity, they are the first to encounter the saline medium and are potentially the first site of damage. The imposition of salinity had an inhibitory effect on the root growth of all the rice cultivars. The results from this study indicated a significant decline in root length as concentration of salinity increased (Table 4.2 and Appendix IV). Similar results have been found by other workers in rice as well as in other crops (Momayezi et al. 2010; Dadkhah et al. 2001). It was probably because salinity may affect final cell size as well as rate of cell production (Azaizeh et al. 1992) thereby producing shorter root. However, these results do not agree with those of Cramer and Nowak (1992), who reported that roots were less sensitive to salt stress. Shoot and root growth inhibition is a common response to salinity and one of the most important agricultural indices of salt stress tolerance as indicated by certain studies (Tuna et al. 2008 and Koca et al. 2007). The result of the present study demonstrated that shoot and root fresh weight were significantly decreased due to increasing levels of salinity (Table 4.3 and 4.5). The reduction in fresh weight was lower in salt-tolerant rice cultivars than the sensitive one (Figure 4.3 and 4.5). Similar observations were reported by Grieve and Fujiyama (1987) and Shannon and Grieve (1998) reported that shoot and root biomass of rice significantly decreased with increasing salt stress.

44

The data herein clearly demonstrated that the dry matter yield of root and shoot systems of the rice cultivars showed marked decrease as the salinity level was increased (Table 4.4 and 4.6). Reduced rates of new cell production may cause the inhibition of growth as reported by Shabala et al. (2000). Hakim et al. (2010) on twelve rice varieties found that dry shoot and root weight decreased significantly as the levels of salinity increased. Similar result was also observed by Talat et al. (2013) on two wheat cultivars. The reduction in dry weight accumulation could be attributed to increasing stiffness of the cell wall, due to altered cell wall structure induced by salinity as reported by Sweet et al. (1990). Salinity significantly decreased total number of tillers per hill both in saltsensitive and salt-tolerant rice cultivars (Table 4.7). The percent reduction in number of tillers per hill was higher in tolerant genotypes than the sensitive one (Figure 4.7). This result is also in agreement with those of Islam et al. (2011) and Ali et al. (2004). Zeng et al. (2000) also observed the reduction in tiller numbers per hill in rice and concluded that it could be the major cause of yield loss in rice under salt stress.In this study, when the rice genotypes were subjected to NaCl stress, all rice cultivars showed significant reductions in plant growth. Phenotypically Binadhan-10 genotype showed better growth than other salt tolerant varieties (Binadhan-8 and BRRI dhan47) under salt stress conditions (Appendix IV). The reduction in growth observed in the present investigation subjected to excess salinity is often associated with a decrease in rate of photosynthetic capacity due to lower level of chlorophyll content. Chlorophyll pigment reduction was observed with increasing salt concentration in all the genotypes, but the reduction was more pronounced in salt-sensitive genotype than salt-tolerant genotypes with an exception in Binadhan-8 (Figure 4.8). The concentrations of the pigments fractions (chlorophyll-a and chlorophyll-b) in the leaves of all rice cultivars are clearly demonstrated that the biosynthesis of photosynthetic pigments was affected by NaCl stress (Table 4.8 and 4.9). These results are also in agreement with those of Islam et al. (2007) and Abeer et al. (2013) in rice. Parida and Das (2005) suggested that decrease in chlorophyll content in response to salt 45

stress is a general phenomenon. Chen and Yu (2007) also observed a significant decrease in chlorophyll content at high NaCl level. Proline accumulates in larger amount than any other amino acids and regulates osmotic potential of the cell to avoid cellular damage under salt stress. Intercellular proline content increased significantly in the leaves of all the genotypes as the salt concentration increased (Figure 4.9).Among the salt-tolerant rice genotypes, Binadhan-10 accumulated 2.2 folds proline while the other genotypes Binadhan-8 and BRRI dhan47 accumulated 1.47 and 1.71 folds proline at highest level of salinity (60 mM NaCl) as compared to the control, respectively. The significant proline accumulation in lower amount was noted at 20 and 40 mM NaCl stress in the salt-sensitive genotype but proline content was decreased at 60 mM NaCl stress (Figure 4.9). Higher proline content in Binadhan-10 might be the one of the reason for higher salt tolerance when compared to other genotypes. Nounjan et al. (2012) on Thai aromatic rice (cv. KDML105; saltsensitive) and Summart et al. (2010) Thai jasmine rice also revealed that salt stress caused an increase in accumulation of intercellular proline content. Weisany et al. (2012) and Dogan et al. (2011) also reported that intracellular proline content was increased in soybean with the increasing salinity levels. In the present study, antioxidant enzyme activities changed significantly in response to the salinity stress. Catalase (CAT) showed a significant decrease in its activity under salt stress in salt-sensitive genotype but increased in all salttolerant rice genotypes (Figure 4.10). At given concentration of NaCl, the decrease in the activity of the enzyme was more pronounced in salt-sensitive (BRRI dhan28) genotype then salt-tolerant genotypes (BRRI dhan47, Binadhan-8 and Binadhan-10). Similarly, CAT inhibition by salt stress was also observed in rye, vigna and rice (Singha and Choudhuri 1990; Hertwig et al. 1992 and Nounjan et al. 2012). Dogan et al. (2011) also showed that salinity decreased CAT activity significantly in salt-sensitive soybean. Peroxidase (POX) showed a considerable decrease in its activity in response to the salt treatments in all the genotypes tested, irrespective of their tolerance levels (Figure 4.11). Ascorbate peroxidase (APX) activity showed increasing trend with the increasing salt concentration in 46

the medium in case of the salt-tolerant rice genotypes, but APX activity in saltsensitive genotype (BRRI dhan28) decreased with the increasing salinity as compared to the control (Figure 4.12). However, these results do not agree with those of Turan and Tripathy (2013) conducted an investigation on a salt-sensitive and relatively salt-tolerant rice cultivars and found that activities of CAT and APX increased in both cultivars in response to salt stress. Dogan et al. (2011) also found that salinity decreased APX activity significantly in salt-sensitive soybean. These results are consistent with observations of many researchers who also reported the APX activity coordinated with CAT and POX activities in rice as well as in cotton during salt stress (Vaidyanathan et al. 2003, Meloni et al. 2003 and Demirel and Turkan, 2005). CAT, POX and APX were the main enzymes involved in the detoxification of the deleterious oxygen species (Mittova et al. 2003). In the present study, significantly higher CAT, POX and APX activities were found in the salt-tolerant rice genotypes than the salt-sensitive genotype under increasing salinity stress suggesting that the higher antioxidant enzymes activity have a role in imparting tolerance to these genotypes against salt stress. Higher uptake of Na+ competes with the uptake of other nutrient ions, especially K+, and causes K+ deficiency which leading to lower K+/Na+ ratio in rice under salt stress. Elevated NaCl levels resulted in significant decreases in K+ in all the genotypes besides increased Na+. The K+/Na+ ratio was significantly higher in the salt-tolerant genotypes than the salt-sensitive genotype both in soot and root samples of rice (Figure 4.13 and 4.14). The trend of decrease in K+/Na+ ratio in shoot and root was not similar may be due to difference in timing of shoot and root sampling. Shoot sampling was done at 7 days after salinity intrusion and root sampling was done at 21 days after salinity intrusion. In the present study, the results suggested that Binadhan-10 showed higher salt-tolerance by keeping the optimal K+/Na+ ratio both in shoot and root (Figure 4.13 and 4.14). Islam et al. (2011), Miah et al. (1992) and Haq et al. (2009) found that K+/Na+ ratio decreased in rice with the increasing salinity level. Sakil (2015) also found that the reduction in K+/Na+ ratio was higher in salt-sensitive rice genotypes than the tolerant genotypes. 47

Chapter VI SUMMARY The pot experiment was carried out in the net-house of Department of Soil Science, Bangladesh Agricultural University, Mymensingh during the period of January, 2014 to March, 2014 to investigate the effects of salt stress on the growth as well as physiological and biochemical characteristics of salt-sensitive and salttolerant rice cultivars. The objectives of the study were to elucidate the physiological and biochemical characteristics of salt-sensitive and salt-tolerant rice genotypes in response to salt stress for screening suitable rice cultivar for cultivation in coastal areas. Four different rice cultivars (BRRI dhan28, BRRI dhan47, Binadhan-8 and Binadhan-10) differing in salt tolerance were exposed to 20 mM, 40 mM and 60 mM NaCl stress conditions. Rice plants showed differential responses to salt stress in physiological and biochemical aspects. Salt stress caused significant reduction in different growth parameters of rice as well as caused decrease in chlorophyll and intercellular proline content and disrupted the antioxidant defense system by modifying the enzymatic process. Plant height significantly decreased in both salt-sensitive and salt-tolerant rice genotypes when exposed to higher salinity level. The highest plant height was obtained in BRRI dhan47 and the lowest was in BRRI dhan28 compared to other varieties under salt stress. The percent decrease in plant height at higher salinity levels was the highest in Binadhan-10 and the lowest in BRRI dhan47 different rice cultivars. Under salt stress conditions, root length was significantly decreased in all the rice cultivars compared to control condition. Binadhan-10 exhibited the highest root length under different salinity level. The percent decrease from control in root length was the lowest in BRRI dhan47 at lower salinity level but at higher salinity level, the percent decrease in root length was the lowest in Binadhan-10. The percent decrease in root length was the highest in BRRI dhan28 under all salinity level. With the increase in salinity levels, shoot fresh and dry weight decreased in both salt-sensitive and salt-tolerant rice genotypes. The highest shoot fresh and dry weight was obtained in Binadhan-10 48

and the lowest value was observed in BRRI dhan28 under salt stress. The percent decrease in shoot fresh and dry weight was also lower in Binadhan-10 and BRRI dhan47 and higher reduction percentage was found in BRRI dhan28. Reduction in root fresh weight due to salt stress was the highest in BRRI dhan28 compared to salt tolerant rice varieties. Among the salt-tolerant rice genotypes, the highest root FW was obtained in Binadhan-10 and Binadhan-8 exhibited the lowest root fresh weight. The percent reduction in root fresh weight was the highest in BRRI dhan28 and the lowest was in Binadhan-10 when the plants were exposed to higher salinity levels. Root dry weight was also the highest in Binadhan-10 under salt stress and reduction percentage in root length was also lowest in Binadhan-10. Salt stress caused a drastic decrease in number of tillers per hill in both of the salt-sensitive and salt-tolerant rice cultivars compared to control. The highest number of tillers per hill was obtained in Binadhan-10 even plants were exposed to different level of NaCl stress. The percent decrease in number of tillers per hill was the highest in Binadhan-8 and the lowest was in Binadhan-10. There was reduction in chlorophyll content both in salt-sensitive and salt-tolerant rice cultivars. An increase in chlorophyll-a content was observed in salt-tolerant rice genotypes with the increasing salt concentration. The salt-sensitive rice genotype (BRRI dhan28) showed a decrease in chlorophyll-a content under salt stress. On the other hand, chlorophyll-b content was decreased in all the rice genotypes with the increasing salt concentration. Salt stress decreased total chlorophyll content in all the rice varieties but the decrease was not significant in Binadhan-10 and exhibited the highest total chlorophyll content under stress condition. Salt stress also caused an accumulation of proline in plant cell which may help plants to avoid cellular damages but proline accumulation was higher in salt-tolerant cultivars than the sensitive one. With the increase in salt concentration, intercellular proline content was significantly increased in all salttolerant rice varieties (BRRI dhan47, Binadhan-8 and Binadhan-10) while saltsensitive one (BRRI dhan28) resulted in an increase in intercellular proline 49

content at lower level of salinity and caused a significant decrease in proline content at higher salinity level. Antioxidant enzymes (CAT, POX and APX) showed differential responses to salt stress. Catalase (CAT) and ascorbate peroxidase (APX) activities were significantly increased in all the salt-tolerant rice genotypes but decreased with the increasing salinity level. On the other hand, Peroxidase (POX) activity was decreased in both salt-sensitive and salt-tolerant rice genotypes with the increasing salinity level. The K+/Na+ ratio in rice shoot and root significantly decreased due to salt stress in both salt-sensitive and salt-tolerant rice varieties with the increase in salt concentration. The highest percent decrease in shoot K+/Na+ ratio was observed in Binadhan-8 and the lowest was in Binadhan-10 at different levels of salinity. BRRI dhan47 exhibited the highest K+/Na+ ratio compared to other salt-tolerant rice varieties and Binadhan-8 exhibited lower K+/Na+ ratio than Binadhan-10. Binadhan-10 maintained the lowest reduction percent in root K+/Na+ ratio among the salt-tolerant rice cultivars.

50

CHAPTER VII

CONCLUSION Soil and/or water salinity is of increasing importance in agriculture. The main objective of this study was to investigate the physiological and biochemical responses of rice cultivars under salt stress as well as to provide plant breeders to use these responses as the selection criteria for salt tolerance. It is thus apparent from the present investigation that no single parameter could be suggested as sole indicator for salinity stress tolerance in rice. A combination of these characters can contribute to salt tolerance in rice. The results of this study showed that there were considerable differences between salt-sensitive rice varieties in response to salt stress. The significant differences in response to salt stress were closely related to differences in the activities of antioxidant enzymes. CAT and APX enzymes activities increased in salt-tolerant cultivars but decreased in salt-sensitive cultivars with the increasing salt stress. The salt-tolerant cultivars may protect them from oxidative damage by increasing CAT and APX activities under salt stress. These results suggest that salt-tolerant rice variety (Binadhan-10) may have better protection against oxidative damages by increasing the activity of antioxidant enzymes under salt stress. On the other hand, BRRI dhan47 also showed satisfactory level of salt tolerance but that was lower than Binadhan-10 in most of the physiological and biochemical characteristics. On the basis of our comparative analysis, the salt tolerant genotype Binadhan-10 successfully tolerated at higher salinity level (60 mM) by accumulating higher level of chlorophyll and proline, maintaining higher K+/Na+ ratio and increasing antioxidant enzyme (CAT and APX) activities than the other tolerant and sensitive genotypes. These findings about biochemical and physiological characteristics at the cellular level may serve as in vitro selection criteria for salt tolerance in rice. Therefore further research should be focused on intercellular and intracellular molecular mechanisms involved in salinity stress response for the determination of key pathways, controlling salinity tolerance in plants. 51

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APPENDICES APPENDIX I. Physicochemical characteristics of initial soil samples Characteristics

Values

Textural class pH (soil : water= 1:2.5) Cation exchange capacity (meq/100 g soil) EC (dS m-1)

Silt loam 6.15 13.6 0.20

Organic carbon (%) Organic matter (%) Exchangeable Na (meq/100 g soil) Total N (%) Available P (ppm)

1.09 1.90 0.383 0.11 12.8

Exchangeable K ( meq/100 g soil)

0.082

Available S (ppm)

11.50

APPENDIX II. Monthly record of air temperature, relative humidity, rainfall and sunshine hours during the period from January, 2014to April, 2014 Monthly air temperature (0c) Month

Maximum

Minimum

Mean

*Relative

Rainfall

Sunshine

humidity

(mm)

(hrs)

(%)

**

**

January

23.64

19.80

17.22

0.00

82.90

114.96

February

25.41

20.57

20.57

55.2

81.11

148.4

March

29.13

23.45

23.45

18.5

75.42

218.23

April

30.44

26.30

26.30

207.9

82.40

186.09

**=Monthly total *= Monthly average Source: Weather Yard, Department of Irrigation and Water Management, Bangladesh Agricultural University, Mymensingh. 66

APPENDIX III. Chemical characteristics of post-harvest soil Samples Rice Treatments pH EC Exchangeable OC OM %N cultivars (dS/m) Na (%) (%) (meq./100g soil) Control 6.88 0.141 0.277 0.995 1.720 0.15 Bina dhan-8

Bina dhan-10

BRRI dhan28

BRRI dhan47

20mM NaCl

6.64

0.984

0.623

0.861 1.489 0.16

40mM NaCl

6.68

1.938

1.212

0.889 1.555 0.15

60mM NaCl

6.48

3.98

1.403

1.032 1.785 0.14

Control

6.48

0.133

0.294

0.975 1.687 0.16

20mM NaCl

6.47

1.306

0.848

1.128 1.951 0.17

40mM NaCl

6.42

2.14

1.264

1.166 2.017 0.17

60mM NaCl

6.65

3.09

1.489

1.108 1.911 0.16

Control

6.83

0.126

0.294

0.995 1.720 0.17

20mM NaCl

6.46

1.572

0.831

1.090 1.886 0.15

40mM NaCl

6.46

2.35

1.177

1.032 1.785 0.15

60mM NaCl

6.43

3.43

1.524

1.090 1.886 0.15

Control

6.73

0.163

0.329

1.051 1.818 0.14

20mM NaCl

6.39

1.035

0.779

1.014 1.754 0.13

40mM NaCl

6.46

2.45

1.056

0.918 1.588 0.14

60mM NaCl

6.46

3.84

1.385

1.090 1.866 0.14

67

APPENDIX IV. Different photographs indicating the effect of salinity on shoot and root growth of rice cultivars used in the experiment

Control

20 mM NaCl

40 mM NaCl

60 mM NaCl

Photograph 1: Effect of salinity on the growth of a salt-sensitive variety (BRRI dhan28) under salt stress.

Control

20 mM NaCl

40 mM NaCl

60 mM NaCl

Photograph 2: Effect of salinity on the growth of a salt-tolerant variety (BRRI dhan47) under salt stress. 68

Control

Photograph 3:

20 mM NaCl

40 mM NaCl

60 mM NaCl

Effect of salinity on the growth of a salt-tolerant variety

(Binadhan-8) under salt stress

Control

Photograph 4:

20 mM NaCl

40 mM NaCl

60 mM NaCl

Effect of salinity on the growth of a salt-tolerant variety

(Binadhan-10) under salt stress 69

Control

20 mM NaCl

40 mM NaCl

60 mM NaCl

Photograph 5: Effect of salinity on root growth of a salt-sensitive variety (BRRI dhan28) under salt stress.

Control

20 mM NaCl

40 mM NaCl

60 mM NaCl

Photograph 6: Effect of salinity on root growth of a salt-sensitive variety (BRRI dhan47) under salt stress. 70

Control

20 mM NaCl

40 mM NaCl

60 mM NaCl

Photograph 7: Effect of salinity on root growth of a salt-tolerant variety (Binadhan-8) under salt stress.

Control

20 mM NaCl

40 mM NaCl

60 mM NaCl

Photograph 8: Effect of salinity on root growth of a salt-tolerant variety (Binadhan-10) under salt stress. 71

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