Tài liệu Physiological responses to saline irrigation in two summer mungbean [vigna radiata (l.) wilczek] genotypes

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P H Y S I O L O G I C A L RE S P O N S E S T O S A L I N E I R R I G A T I O N I N T W O S UM M E R M U N G B E A N [ V I G N A R A D I A T A ( L . ) W I L C Z E K ] G E N O T YP E S By DUONG HOANG SON (2010BS100D) Thesis submitted to the Chaudhary Charan Singh Haryana Agricultural University in partial fulfillment of the requirements for the degree of Doctor of Philosophy In Plant Physiology DEPARTMENT OF BOTANY AND PLANT PHYSIOLOGY COLLEGE OF BASIC SCIENCES AND HUMANITIES CCS HARYANA AGRICULTURAL UNIVERSITY HISAR – 125 004 2013 CERTIFICATE–I This is to certify that this thesis entitled, “Physiological responses to saline irrigation in two summer mungbean [Vigna radiata (L.) Wilczek] genotypes” submitted for the degree of Doctor of Philosophy in the subject of Plant Physiology to the CCS Haryana Agricultural University, Hisar, is a bonafide research work carried out by Mr. Duong Hoang Son under my supervision and guidance and that no part of this thesis has been submitted for any other degree. The assistance and help received during the course of investigation have been fully acknowledged. (Dr. Neeraj Kumar) Major Advisor Scientist of Plant Physiology Department of Botany and Plant Physiology College of Basic Sciences and Humanities CCS Haryana Agricultural University Hisar-125 004 (Haryana) India CERTIFICATE–II This is to certify that this thesis entitled “Physiological responses to saline irrigation in two summer mungbean [Vigna radiata (L.) Wilczek] genotypes”, submitted by Mr. Duong Hoang Son to the CCS Haryana Agricultural University, Hisar, in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the subject of Plant Physiology, has been approved by the Student’s Advisory Committee, after an oral examination on the same, in collaboration with an external examiner. MAJOR ADVISOR HEAD OF THE DEPARTMENT DEAN, POST-GRADUATE STUDIES EXTERNAL EXAMINER ACKNOWLEDGEMENTS All above, I want to express my deep sense of indebtedness to my family, especially my parent, my wife Quynh Mai and my sons Nhat Ha and Nhat Minh for their love, understanding and great companion. I have no words to express my deep sense of gratitude and indebtedness to my advisor, Dr. Neeraj Kumar, Scientist, Department of Botany and Plant Physiology, for his great kindness, constant encouragement and precious time to me in all aspects from the first day I came to India as well as during my study and investigation. I owe deep and fervent thanks to Dr. A.S. Nadwal, Additional Director Research - Directorate of Researh, who spent a lot of time reading and discussing my thesis. It gives me immense pleasure to record my sincere gratitude towards the learned members of my advisory committee: Dr. S.K. Sharma, Sr. Scientist (Soil Science), Dr. Ramesh Hasija, Sr. Scientist (Statistic) and Dr. Satish Kumar, Associate Dean PGS, for their intellectual enlightenment, sympathetic interest and pertinent suggestions throughout the pursuit of this study. This study could not be completed without their kind help and support. It is my profound privilege to express my heartiest thanks to Dr. (Mrs.) Sunita Sheokand, Sr. Scientist, Dr. K.D. Sharma, Scientist, Dr. Rajiv Angrish, Sr. Scientist and Dr. H.R. Dhingra, Professor of the Department of Botany and Plant Physiology for their timely help and willing cooperation. Distinctive words of thanks are due to Dr. (Mrs.) Rupa Dhawan, former Head, Dr. J.K. Sandooja, Head, Department of Botany and Plant Physiology, for providing the necessary facilities and cordial help whenever required. I am thanking to all my friends for their help during my study, cheerful company and the research fellows and seniors especially Dr. Anita Kumari and Dr. Gunjan Geera for her guidance in the analysis of antioxidant enzymes, protein profile and informative discussions during the writing-up of this thesis, and Mr. Suraj Bhan and Mr. Raghubir Signh for technical assistance. I am thankful to Dr. Le Van Banh, Director, and Dr. Cao Van Phung, Head of Soil Science department, CuuLong Delta Rice Research Institute, Vietnam, for providing me opportunity to study in India Financial help provided by Vietnam International Education Development (VIED), Ministry of Education and Training (MOET) in the form of 322 project fellowship is duly acknowledged. (Duong Hoang Son) Date: Hisar ABBREVIATIONS APS - Ammonium per sulphate APX - Ascorbate peroxidase AsA - Ascorbate ATP - Adenosine tri phosphate CAT - Catalase CD - Critical difference CRD - Complete randomized design CSI - Chlorophyll stability index DAT - Days after treatment DMSO - Dimethyl sulphoxide DW - Dry weight EDTA - Ethylene diamine tetra acetic acid FW - Fresh weight G - Genotype G×V - Genotype × variety GR - Glutathione reductase GPX - Glutathione peroxidase GST - Glutathione S-transferase RWC - Relative water content MPa - Mega Pascal MW - Molecular weight NPQ - Non-photo chemical quenching OD - Optical density PS1 - Photosystem I PSII - Photosystem II POX - Peroxidase PVP - Polyvinyl pyrolidone ROS - Reactive oxygen species SDS-PAGE - Sodium dodecyl sulphate- Polyacrylamide gel electrophoresis SOD - Superoxide dismutase TTC - 2,3,5- triphenyl tetrazolium chloride w - Water potential s - Osmotic potential CONTENTS CHAPTERS TITLE PAGE(S) 1 INTRODUCTION 1-4 2 REVIEW OF LITERATURE 5-19 3 MATERIAL AND METHODS 20-36 4 RESULTS 37-72 5 DISCUSSION 73-83 6 SUMMARY AND CONCLUSIONS 84-86 BIBLIOGRAPHY i-xiii LIST OF TABLES S. No. Title Page No. 1 Changes in water potential (-MPa) in leaf of mungbean genotypes under saline irrigation. 39 2 Changes in osmotic potential (-MPa) in leaf of mungbean genotypes under saline irrigation. 40 3 Changes in osmotic potential (-MPa) in root of mungbean genotypes under saline irrigation. 41 4 Changes in relative water content (RWC %) in leaf of mungbean genotypes under saline irrigation. 41 5 Changes in relative water content (RWC %) in root of mungbean genotypes under saline irrigation. 42 6 Changes in relative stress injury (RSI %) in leaf of mungbean genotypes under saline irrigation. 43 7 Changes in relative stress injury (RSI %) in root of mungbean genotypes under saline irrigation. 44 8 Changes in Na+/K+ ratio in leaf of mungbean genotypes under saline irrigation. 50 9 Changes in Na+/K+ ratio in root of mungbean genotypes under saline irrigation. 50 10 Changes in chloride (Cl-) content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation. 51 11 Changes in chloride (Cl-) content (mg g-1 DW) in root of mungbean genotypes under saline irrigation. 52 12 Changes in sulphate (SO42-) content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation. 53 13 Changes in sulphate (SO42-) content (mg g-1 DW) in root of mungbean genotypes under saline irrigation. 53 14 Changes in in vitro pollen germination (%) and pollen tuber growth (µm) in two mungbean genotypes under saline irrigation. 69 15 Changes in yield and yield attributes in two mungbean genotypes under saline irrigation. 71 16 Changes in ECe of soil under saline irrigation. 72 LIST OF FIGURES S. No. Title 1 Changes in plant height (cm) in mungbean genotypes under saline irrigation. 37 2 Changes in dry matter (g plant-1) content in leaf of mungbean genotypes under saline irrigation. 38 3 Changes in dry matter (g plant-1) content in root of mungbean genotypes under saline irrigation. 38 4 Changes in lipid peroxidation (MDA) content (nmoles g-1 DW) in leaf of mungbean genotypes under saline irrigation. 45 5 Changes in lipid peroxidation (MDA) content (nmoles g-1 DW) in root of mungbean genotypes under saline irrigation. 45 6 Changes in chlorophyll ‘a’ content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation. 46 7 Changes in chlorophyll ‘b’ content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation. 47 8 Changes in total chlorophyll content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation. 47 9 Changes in chlorophyll stability index (%) in leaf of mungbean genotypes under saline irrigation. 48 10 Changes in quantum yield (Fv/Fm) in leaf of mungbean genotypes under saline irrigation. 48 11 Changes in hydrogen peroxides (H2O2) content (moles g-1 DW x 10-4) in leaf of mungbean genotypes under saline irrigation. 54 12 Changes in hydrogen peroxides (H2O2) content (moles g-1 DW x 10-4) in root of mungbean genotypes under saline irrigation. 55 13 Changes in proline content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation. 55 14 Changes in proline content (mg g-1 DW) in root of mungbean genotypes under saline irrigation. 56 15 Changes in total soluble carbohydrates content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation. 57 16 Changes in total soluble carbohydrates content (mg g-1 DW) in root of mungbean genotypes under saline irrigation. 57 17 Changes in superoxide dismutase (SOD) specific activity (Units mg-1 protein) in leaf of mungbean genotypes under saline irrigation. 58 18 Changes in superoxide dismutase (SOD) specific activity (Units mg-1 protein) in root of mungbean genotypes under saline irrigation. 59 Page No. 19 Changes in catalase (CAT) specific activity (Units mg-1 protein) in leaves of mung bean genotypes under saline irrigation. 60 20 Changes in catalase (CAT) specific activity (Units mg-1 protein) in root of mungbean genotypes under saline irrigation. 60 21 Changes in peroxidase (POX) specific activity (Units mg-1 protein) in leaf of mungbean genotypes under saline irrigation. 61 22 Changes in peroxidise (POX) specific activity (Units mg-1 protein) in root of mungbean genotypes under saline irrigation. 61 23 Changes in ascorbate peroxidase (APX) specific activity (Units mg-1 protein) in leaf of mungbean genotypes under saline irrigation. 62 24 Changes in ascorbate peroxidase (APX) specific activity (Units mg-1 protein) in root of mungbean genotypes under saline irrigation. 63 25 Changes in glutathione reductase (GR) specific activity (Units mg-1 protein) in leaf of mungbean genotypes under saline irrigation 63 26 Changes in glutathione reductase (GR) specific activity (Units mg-1 protein) in root of mungbean genotypes under saline irrigation. 64 27 Changes in glutathione S transferase (GST) specific activity (Units mg-1 protein) in leaf of mungbean genotypes under saline irrigation. 65 28 Changes in glutathione S transferase (GST) specific activity (Units mg-1 protein) in root of mungbean genotypes under saline irrigation. 65 29 Changes in glutathione peroxidase (GPX) specific activity (Units mg-1 protein) in leaf of mungbean genotypes under saline irrigation. 66 30 Changes in glutathione peroxidase (GPX) specific activity (Units mg-1 protein) in root of mungbean genotypes under saline irrigation. 66 31 Changes in ascorbate (AsA) content (µmoles g-1 DW) in leaf of mungbean genotypes under saline irrigation 67 32 Changes in ascorbate (AsA) content (µmoles g-1 DW) in root of mungbean genotypes under saline irrigation 68 33 Changes in pollen viability in two mungbean genotypes under saline irrigation 68 CHAPTER-I INTRODUCTION Environmental stresses are the most important constraints limiting crop productivity. Among these salinity either of soil or water is a serious problem for agriculture all over the world (Majid et al., 2011). Salinity limited the growth and development of plant by altering their morphological, physiological, biochemical attributes and production in most of the arid and semi arid regions of the world (Mudgal et al., 2010; Kandil et al., 2012). There are different causes of the development of soil salinity. The major forms are viz. (i) natural or primary salinity and (ii) secondary or human-induced salinity. Primary salinity is occurred due to the long-term natural accumulation of salts in the soil or surface water. Secondary salinity occurs due to anthropogenic activities that disrupt the hydrologic balance of the soil between water applied (irrigation or rainfall) and water used by crops (Geetanjali and Neera, 2008). Salinity created due to high salt concentration in the soil solution is two-fold. First, many of salt ions are toxic to plant cells and second, high salt represents a water deficit or osmotic stress. Specific ion toxicity is usually associated with excessive intake of sodium, chloride or other ions and causes disrupt plant potassium and calcium nutrition (Zhu, 2007). The deleterious effect of the saline irrigation on plant involve osmotic stress, ion toxicity and mineral deficiency (Ashraf and Harris, 2004) and reduction in growth and alterations in several physiological processes including N2- fixation (Nandwal et al., 2000 a, b; Kukreja et al., 2005). Water potential and osmotic potential become more negative whereas turgor pressure increases with increasing salinity (Mudgal et al., 2010). Osmotic adjustment involves either inorganic ions or low molecules weight organic solutes. These play a crucial role in higher plants grown under saline conditions. The compatible osmolytes generally found in higher plants are low molecular weight sugars, organic acids, polyols and nitrogen containing compounds such as amino acids, amides, proteins and quaternary ammonium compounds (Dionisio-Sese and Tobita, 1998; Ashraf and Harris, 2004; Mudgal et al., 2010; Sabina and Mehar, 2011). Several physiological and biochemical processes like pigment content and photosynthesis, carbohydrate metabolism, protein synthesis, energy and lipid metabolism are affected by salinity. Salt stress disturbs intracellular ion homeostasis in plants, which leads to damage in maintaining cell turgor, enzyme activities, membrane dysfunction, attenuation of 1 metabolic activity and other secondary effects that cause growth inhibition and ultimately lead to cell death (Hasegawa et al., 2000; Saha et al., 2010; Zhu, 2007). A common consequence of salt stress is that they result, at some stage of exposure, in an increased production of reactive oxygen spices (ROS) (Ahmad et al., 2008; Kukerja et al., 2005). ROS production such as superoxide anion (O-2), hydrogen peroxide (H2O2) and hydroxyl radicals (OH-) caused oxidative damage (Zhu, 2001; Parida and Das, 2005; Kukreja et al., 2005). These ROS are highly reactive and in the absence of any protective mechanism caused cellular damage through oxidation of lipids, proteins and DNA injury (Mohammed, 2007). To control the level of ROS and to protect plant cells have to cope constantly with the damages produced by the ROS, and as a protective system they have evolved a complex series of enzymatic [superoxide dismutase (SOD), catalase (CAT) and peroxidases (POX)], detoxifying lipid peroxidation (LP) products [glutathione S-transferases (GST), phospholipidhydroperoxide glutathione peroxidise (GPX) and ascorbate peroxidase (APX), and nonenzymatic antioxidants [ascorbate (AsA), glutathione (GSH), phenolic compounds and tocopherols]. In addition, a whole array of enzymes is needed for the regeneration of the active forms of the antioxidants [monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR)], which are responsible for scavenging excessively accumulated ROS in plants under stress conditions (Sairam et al., 2002; Blokhina et al., 2003; Kukreja et al., 2010; Saha et al., 2010; Hossain et al., 2011). Salinity interfered with the nodule initiation in cowpea and mungbean and also caused a reduction in number, weight as well as nitrogen fixing efficiency of nodules (Balasubramnian and Sinha, 1976). In chickpea, nodules were observed in inoculated plants grown at 6 dS m-1 but nitrogen fixation was completely inhibited. Findings indicated that symbiosis is more salt sensitive than both rhizobium and host plant (Mudgal et al., 2010). Salinity adversely affects both qualitative and quantitative features of male functions, i.e. number of pollen produced, their viability, germination and tube growth, thereby reducing the relative male fitness to less than half of the control ovule (Abdulla et al., 1978; Dhingra and Varghese, 1985, 1986). Ottaviano et al. (1975) demonstrated that decline in male fitness character may result in reduction in both the number and quality of offspring. Salinization though did not affect ovule production but the number of pollen deposited on the stigmatic surface during pollination and formation of pods and healthy seeds decrease substantially with increase in salinity level (Dhingra and Sharma, 1992). In arid and semi-arid regions, salinity (both soil and water) is one of the major factors responsible for deterioration of soil and making it unfit for agriculture (Ashraf and Harris, 2 2004). 20 % of world’s cultivated land and 50 % of all irrigated lands are affected by salinity (Zhu, 2001). Furthermore, more than half of all ground water is naturally saline particularly in arid and semi arid regions of the world (Yeo, 1999). In India, salt affected soil is about 6.73 mha (2.96 mha saline and 3.77 mha sodic), whereas in Haryana 49.16 th-ha of land is affected by salinity (Ali, 2009). Ions containing soil salinity are Na+, Cl-, Ca+ and Mg++. High salinity, most commonly mediated by NaCl, is one of major abiotic stresses globally (Parida and Das, 2005). The problem of salinity is being further aggravated because of use of poor quality water for irrigation and poor drainage. In arid and semi-arid regions, insufficient precipitation results in extensive reliance of irrigation and a considerable proportion of underground water in most of these regions is of poor quality, however, the productivity of this crop is not optimal under such conditions. Mungbean [Vigna radiata (L.) Wilczek] is a short duration (70-80 days), warm season legume crop of this region. Approximately, an area of 2.5 million ha in world has been used for its cultivation from which 0.8 million tons of seeds are produced per annum (Ahmad et al., 2011). India is largest producer and consumer of mungbean and it alone accounts for about 65% of the world acreage and 54% of the world production of this crop (Singh and Singh, 2011). Recently, In India mungbean is grown in an area of 3.77 m ha with production of 1.52 m tones. However, it productivity is only 406 kg ha-1 (AICRP on MULLaRP, 2009). In Haryana, the approximate values are 21 thousand ha, 5.0 thousand tones and 260 kg ha-1, respectively. For developing country like India, mungbean is a main protein source for the vegetarian diet. It is the best in nutritional value, having 62-65% carbohydrate, 25-28% protein, 3.5-4.5% ash and 1-1.5% fat (Navneet et al., 2011). Mungbean is also characterized by its ability to improve the physiological, chemical and biological properties of soil. It can also increase the soil fertility through biological nitrogen fixation from atmosphere. The green plant and hay are utilized as fodder. So, it may be considered as an inevitable component of sustainable agricultural (Hussain et al., 2008). Mungbean may also be sown as an inter crop or as a green manure or cover crop. In order to overcome these problems, genotypes which are resistant to salinity are to be identify. Selection and breeding programmed to increase salt tolerance will be more successful if selection is based directly on the physiological mechanism (s) or character (s) confirming tolerance (Sairam et al., 2002; Kumar et al., 2008). Despite its great economic importance little work has been done on genotypic variations for salt tolerance. It would therefore, be important to identify the morpho-physiological and biochemical traits for salinity resistance in this crop. Salt tolerant mungbean crop may be an alternative for 3 increasing production in these saline soils. In view of these facts, the present investigation was planned with the following objectives: 1. To study the morpho-physiological traits and antioxidant defense mechanism in mungbean under saline conditions. 2. To study the protein profile (SDS-PAGE) of leaves and roots in mungbean under saline conditions. 4 CHAPTER-II REVIEW OF LITERATURE The sustainability of irrigated agriculture in many arid and semiarid areas of the world is at risk because of a combination of several interrelated factors, including lack of fresh water and drainage, the presence of high water tables, and salinization of soil and groundwater resources (Geetanjali and Neera, 2008). Among them; salinity is a major abiotic stress in plant agriculture worldwide restricting many plant physiological and biochemical processes such as photosynthesis, protein synthesis, energy and lipid metabolisms (Parida and Das, 2005). Seed germination, seedling growth and vigour, vegetative growth, flowering and fruit set are adversely affected by high salt concentration, ultimately causing diminished economic yield and also quality of produce (Hasegawa et al., 2000; Sairam and Tyagi, 2004). Plants are classified as glycophytes and/or halophytes according to their capacity to grow on high salt medium. Unfortunately, the major crops are almost universally nonhalophytic. For example, bean yield is inhibited almost entirely at 50 mol m-3 (Sairam and Tyagi, 2004; Mass and Grieve, 1987). The deleterious effects of salinity on plant growth are associated with low osmotic potential of soil solution creating water stress in plant. Secondly, they cause severe ion toxicity. Finally, the interactions of salts with mineral nutrition may result in nutrient imbalances and deficiencies. The consequence of all these can ultimately lead to plant death as a result of growth arrest and molecular damage (Zhu, 2001; Jain et al., 2003; Sairam and Tyagi, 2004; Parvaiz and Satyawati, 2008). Plants develop defense strategies against salt stress based on (i) selective accumulation or exclusion of ions, (ii) control of ion uptake by roots and transport into leaves, (iii) compartmentalization of ions at cellular and whole plant levels, (iv) synthesis of compatible sulutes, (v) change in photosynthetic pathway, (vi) alteration in membrane structure, (vii) induction of antioxidant enzymes and induction of plant hormone (Parida and Das, 2005). Legumes are key component of sustainable agriculture and can offer many economic and environmental benefits because of their input of fixed N 2 improving the physical and chemical properties of the soil and help to reintroduce agriculture to these lands (Rolfe and Gresshoff, 1988; Geetanjali and Neera, 2008). Voluminous literature is available on the effect of salt stress on the physiological and biochemical aspects of growth and development. Relevant available literature on salinity has been reviewed as follow: 5 2.1 Growth parameters Morphological, the most typical symptom of salinity is stunted plant growth. Suppression of growth occurs in all plant, but their tolerance levels and rates of growth reduction at lethal concentrations of salt vary widely among different plant species (Parida and Das, 2005). Salt tolerance is usually assessed as the percent biomass production in saline versus control conditions over a prolonged period of time (Munns, 2002). In mungbean plant, genotypes revealed remarkable differences at all the growth stages for the symptoms. Ionic injury was evident in form of tip burning, chlorosis and necrotic spot on leaflet of both young and old ages. Results also showed that number and area of green leaves were more affected by salinity than the total shoot dry weight in mungbean plants (Wahid et al., 2004). The shoot root ratio of mungbean increase at high salt levels (Ashraf and Rasul, 1988). Salt stress by NaCl at 100, 200 and 300 mM cause marked decreases in root and shoot lengths, number of lateral roots and leaves, total leaf area plant -1, fresh and dry weight of shoot and roots as well as percentage of water content in mungbean plant (Sumithra et al., 2006). Effect of NaCl at 100 and 150 mM on mungbean seedling caused drastic effects on roots compared to shoots. Accompanying reductions in length, number of root hairs and branches, roots become stout, brittle and brown in color (Saha et al., 2010). Mohamed and Kramany (2005) studied effect of saline water (2000 and 4000 ppm) for irrigation on four varieties of mungbean resulted decreased dry weight of leaves and stems plant-1; leaves area and depression in dry matter accumulation in both ages 35 and 50 days after treatment. Salt stress was observed more effectively at vegetative, flowering and seed filling stages rather than seed development stage in mung bean genotypes. Delayed maturity due to salt stress pushes the plant also be desiccation stress causing shriveled seeds (Ahmed, 2009). 2.2 Plant water relation Dissolved solutes in the root zone create a low osmotic potential that lower the soil water potential. The general water balance of plant is thus affected, because the shoot needs to have an even lower potential to maintain a “down hill” gradient of water potential between the soil and leaves (Taiz and Zeiger, 1998). Plants subjected with salt stress revealed that halophytes accumulate salts whereas glycophytes tend to exclude the salts. Halophytes have evolved mechanisms to accumulate ions in order to lower cell osmotic potential. This osmotic adjustment is necessary because the plant have to continue to extract water from the salty solution to meet transpiration demands of their leaves (Zhu, 2007). 6 2.2.1 Water potential (Ψ w ) Decrease in plant water potential under salt accumulation, must immediately be offset by decrease in osmotic potential, through increase solute content for turgor potential to be maintain (Mudgal et al., 2010). However, the water potential (Ψw) increased markedly due to application of K at both control and salt treatment (Kabir et al., 2004). Salinity induced reduction in leaf (Ψ w) of number of plant spices like mungbean (Nandwal et al., 2000 a, b) and pea (Hernandez et al., 1999). 2.2.2 Osmotic potential (Ψ s ) Plants may maintain water uptake from saline soil by a process known as osmotic adjustment (Sheldon et al., 2004). However, osmotic adjustment might be an adaption for plants surviving under salt stress conditions but may also reduce growth due to ion toxicity, ion deficiency and/or other physiological process (Volkmar et al., 1998). Effect of NaCl and PEG stress on mung bean plant showed that the contribution of inorganic solutes was high in saline stress and organic solute decreased in both treatments (Saffan, 2008). Zayed and Zeid (1998) revealed that osmotic potential of mungbean seedlings under water stress induced by PEG were affected much more than under salinity. The values of Ψ s of leaves, roots and nodules became more negative with increasing salt stress in mungbean genotypes i.e. K-851 and a mutant. However, values were more negative in mutant than in K-851 (Nandwal et al., 2000 a, b). 2.2.3 Relative water content (RWC %) Responses of two green gram (P. aureus) cultivars differing in salt stress suggested possible different behaviors of cultivars differing in salt tolerance with respect to plant fresh and dry weight, water content (Misra and Dwivedi, 2004). Zayed and Zeid (1998) revealed that salinity stress to decreased the osmotic potential in mung bean seedlings growth medium induced reduce water content, the reduction was 10% as compared to control. Water contents of 86-88% should be optimum for mobilization of reverses from the cotyledons to the embryo axis and the attainment of this level was delay with increased salinity in mungbean seedling (Promila and Kumar, 2000). Kabir et al. (2004) reported that salinity decreased relative water content and water retention capacity, while increased water saturation deficit and water uptake capacity in mungbean plant. Relative water content in roots and shoots were declined upon salinization in mungbean plants (Sumithra et al., 2006). Similarly, a significant decrease in RWC of leaves, roots and nodules was observed at vegetative and flowering stages, when single saline irrigation was given in mungbean genotypes (Nandwal et al., 2000 a, b). 7 2.3 Membrane injury 2.3.1 Membrane stability ROSs are generating through oxidative stress and involved in the injury mechanism due to salt stress. ROS can function to product peroxidants of membrane lipid, protein and nucleic acid (Katsuhara et al., 2005). Liang et al. (2003) proposed that accumulation of H2O2 lead to lipid peroxidation, causing membrane damage and leakage of various micro, macromolecules and electrolytes out of the cell. Cell membrane stability is technique that has often been used for screening against salinity tolerance in various crops due to malfunctioning of the cellular membranes by increasing their permeability to ions and electrolytes (Farooq and Azam, 2006). There are different ionic mechanisms involved in the perception of the ionic and osmotic components of salt stress (Shabala, 2000). Increased of electrolyte leakage with increasing of saline stress has been reported in wheat leaf senescence (Farouk, 2011) and in wheat young leaf (Farooq and Azam, 2006). Similar results has been observed in green gram (Panda, 2001), barley (Li, 2008), chickpea plants (Kukreja et al., 2006; Sheokand et al., 2008). Dionisio Sese and Tobita (1998) studied the fourth cultivars of rice Oryza sativa subjected to different level of salt stress. The amount of electrolyte leakage from the leaves were observed gradual increasing in all cultivars with increasing salt levels while remain unchanged in salt tolerant rice. Similar results have been observed in Clitoria ternatea and Lathtrus sativus leaf (Talukdar, 2011). However, Maia et al. (2010) demonstrated that the tolerant cultivar of cowpea i.e. Pitiuba, and the susceptible cv. Perola maintained stable electrolyte leakage similarly in both cultivar. Cavalcanti et al. (2004) reported that leaf membrane damage was observed increased with long time applied of salt stress. 2.3.2 Lipid peroxidation An increased production of active and/or reactive oxygen species and an accumulation of lipid peroxidation products have been associated with a variety of salt stress (Rodriguez et al., 1999; Katsuhara et al., 2005). Oxidative damage to lipids was determined as lipid peroxidation by the formation of thiobarbaturic acid reactive substances (TABRS) in terms of amount of malondialdehyde (MDA) when plant subjected to salinity (El-baky et al., 2003; Mudgal et al., 2010). Recent investigations have shown increased MDA content with increasing salinity for Brassica juncea (Verma and Mishra, 2005), Cicer arietinum (Kukreja et al., 2005), Vigna unguiculata (Cavalcanti et al., 2007) and wheat (Farouk, 2011). 8 The accumulation of MDA was more in the salt susceptible than in the salt tolerant cultivars in rice (Dionisio-Sese and Tobita, 1998; Vaidayanathan et al., 2003). Changes of leaf Na + accumulation caused increase TBARS levels in cowpea (Cavalcanti et al., 2004). Kukreja et al. (2005) reported that increased H2O2 content of root with increasing salinization might be the cause for increased lipid peroxidation in chickpea. Saha et al. (2010) observed a higher increased lipid peroxidation in leaves than roots of V. radiata. 2.4 Chlorophyll content Compositions of the chloroplastic pigments have been reported to be altered under saline conditions and these changes depend upon the specific nature of ions contributing to the salinity, plant species and age of the plant (Levitt, 1980). Zaidi and Singh (1995) found inhibition in the total chlorophyll (Chl), Chl a:b rations with increase in the soil salinity. The total chlorophyll contents of leaves decrease in general under salt stress (Hernandez et al., 1999; Yasar et al., 2008). Garg et al. (1996) reported that NaCl at 10 dS m-1 decreased total chlorophyll in mungbean but did not recorded at 5 dS m-1. Another salt, Na 2SO4 and NaHCO3 not found any adverse effect on Chl content at 10 dS m-1. It has been suggested by Asharf and Rasul (1988) that all the Chl contents were reduced significantly at EC more than 6 dS m-1 in difference mungbean genotypes. In mungbean seedling, the total Chlorophyll and Chlorophyll a:b and carotenoid (Car) contents were greatly reduced under salt stress (Zayed and Zeid, 1998; Maity et al., 2000). Wahid et al. (2004) revealed that the chlorophyll and carotenoid contents were diminished under salinity in leaflets at young and old mungbean leave ages. Furthermore, Chl a:b ratio of young leaves of sensitive genotypes increased significantly while the tolerance ones tended to maintain a fairly values. Increased Chl a:b ratio was positively related to Na + and Cl-, this revealed an important role of photosynthetic pigment (mainly Chl b and Car) in the enhance salt tolerance of mungbean genotypes. 2.4.1 Chlorophyll fluorescence Part of the light energy absorbed by leaf chlorophyll pigments during photosynthesis is emitted as fluorescence. Chlorophyll fluorescence analysis is a powerful technique to provide a sensitive indicator of stress condition in plants (Maxwell and Johnson, 2000). A number of studies utilized chlorophyll fluorescence as parameters to examine factors limiting photosynthesis of salt effected plant (Maria et al., 2000; Saha et al., 2010) to compare salt treated and control plants (Song et al., 2001) or to differentiate between salt tolerant and sensitive genotypes (Suriyan and Chalermpol, 2010). The Fv/Fm ratio can be used to detect damage to photosymtem II and possible photo-inhibition (Ahmed et al., 2002). Lee et al. (2004) observed in Paspalum vaginatum 9 Swartz ecotypes that with the increase of salinity level (1.1-49.7 dS m-1) initial chlorophyll fluorescence (Fo) increased while maximum and variable (Fv/Fm) chlorophyll fluorescence ratio tended to decrease. Applied exogenous of sodium nitropursside (SNP) increased chlorophyll fluorescence and ultimately protects PS II activity under salt stress (Uchida et al., 2002). 2.5 Biochemicals 2.4.1 Proline Accumulation of metabolites that acts as compatible solutes is one of the probable universal responses of plants to changes in the external osmotic potential. Metabolites with osmolyte function like simple sugars, sugar alcohols, complex sugar, and other quaternary amino acid are frequently observed in plant under unfavorable conditions (Jain et al., 2003; Bartels and Sunkar, 2005; Mudgal et al., 2010; Sabina and Mehar, 2011). Proline is probably the most wide distributed osmolytes found in plant and many other organisms, there is strong correlation between increased cellular proline levels and the capacity to survive the effects of high environmental salinity (Sairam and Tyagi, 2004; Bartels and Sunkar, 2005; Mudgal et al., 2010). A rapid accumulation of proline under salt stress has been observed in mungbean crop (Singh et al., 1994). Arora and Saradhi (2002) studied Vigna radiata were exposed to different concentrations of NaCl in light and dark. Proline accumulation in the shoots was higher in light than in dark, the increased in proline content upto 286% as compared to control in light under 200 mM NaCl. Saffan (2008) observed that the proline content increased in all plants (wheat, barley, mungbean and kidney bean) under effect of 200 mM NaCl. Similarly, proline content increased with increasing salt treatments in cowpea, black gram and green gram compared to control. Accumulation of proline was more in root compared to shoot (Arulbalachandran et al., 2009). Shabina and Mehar (2011) subjected the seven varieties of mungbean to 50 and 100 mM NaCl stress. Proline content significantly increased in stress plant over control of all the genotypes. However, the Punt mung exhibited higher adaptive potential under salinity stress as judged by accumulation of osmoprotectants when compared to other genotypes. Salt induced increase in proline concentration started shortly after the salt stress application. In agreement with the above, a better accumulation of proline in leaves, stems, roots and nodules under salt stress has been observed in various mungbean genotypes (Nandwal et al., 2000 a, b; Manivannan et al., 2007; Saha et al., 2010). 2.5.2 Total soluble carbohydrate Information, regarding the role of sugar in adaption of plants to salinity is, therefore, insufficient to conclude that they are universally associated with salt tolerance. 10 However, potential role for soluble sugar accumulation as an indicator for salt tolerance in breeding programs for some species (Asharf and Harris, 2004). Several physiological studies suggest that under stress conditions, carbohydrates accumulated to varying degree in different plant species (Geetanjali and Neera, 2008). Carbohydrates such as sugars and starch accumulated under salt stress. Their major functions are osmoprotection, osmotic adjustment, carbon storage and radical scavenging (Parida et al., 2002; Parida and Das, 2005). Mohammed (2007) reported that there is a highly significant decrease in reducing sugars and sucrose contents of mungbean plant with increasing salinity levels. Ashraf and Rasul (1988) reported that, increased salt concentration significantly reduced total carbohydrate in leaves, stem and roots of mungbean. Arulbalachandran et al. (2009) revealed that reducing sugar and starch content were increased in both shoot and root with increasing salt concentrations in cowpea, black gram and green gram as compared to their respective control. Accumulation of sugar and starch content were more in shoot rather than root in all three Vigna species. 2.5.3 Hydrogen peroxide (H 2 O 2 ) Salinity stress induced production of H 2O2 and may trigger genetically programmed cell suicide (Farouk, 2011). H 2O2 is widely generated in many biological systems and mediates various physiological and biochemical process in plant (Li and Xue, 2010). Salinity induced the generation of H 2O2 (Sairam and Tyagi, 2004). The chief toxicity of H2O2 are production of hydroxyl radicals and other destructive species such as lipid peroxides lead to damage vital cellular macromolecules (Vaidyanathan et al., 2003). Increased in H2O2 production under salinity has been reported in chickpea roots (Kukreja et al., 2005), tomato leaves (He and Zhu, 2008). A progressive increase in H 2O2 content with increasing the NaCl concentration was observed in Brassica juncea (Verma and Mishra, 2005). The higher H2O2 content was observed in the salt sensitive as compared salt tolerant cultivars of Oryza sativa under salt stress (Vaidyanathan et al., 2003). The H2O2 content increased under NaCl stress in mungbean (Nafees et al., 2010; Hossain et al., 2011; Neelam, 2013). Saha et al. (2010) revealed that endogenous H2O2 production increased with increasing salt stress in leaves and roots of mungbean. The maximum H2O2 content was observed in the salt-tolerant cultivars as compared to salt sensitive cultivars of mungbean under salt stress (Sumithra et al., 2006). 2.6 Antioxidant defence system (ADS) Reactive oxygen species (ROS) are produced in both unstressed and stressed cells. Plants have well developed defence systems against ROS, involving both limiting its formation as well as removal (Alscher et al., 2002). To overcome the effects of salinityinduced oxidative stress, plants make use of a complex antioxidant system, which is 11
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