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) INTRODUCTION 1-4 REVIEW OF LITERATURE 5-19 MATERIAL AND METHODS 20-36 RESULTS 37-72 DISCUSSION 73-83 SUMMARY AND CONCLUSIONS 84-86 BIBLIOGRAPHY i-xiii LIST OF TABLES S No Title Page No Changes in water potential (-MPa) in leaf of mungbean genotypes under saline irrigation 39 Changes in osmotic potential (-MPa) in leaf of mungbean genotypes under saline irrigation 40 Changes in osmotic potential (-MPa) in root of mungbean genotypes under saline irrigation 41 Changes in relative water content (RWC %) in leaf of mungbean genotypes under saline irrigation 41 Changes in relative water content (RWC %) in root of mungbean genotypes under saline irrigation 42 Changes in relative stress injury (RSI %) in leaf of mungbean genotypes under saline irrigation 43 Changes in relative stress injury (RSI %) in root of mungbean genotypes under saline irrigation 44 Changes in Na+/K+ ratio in leaf of mungbean genotypes under saline irrigation 50 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 Changes in plant height (cm) in mungbean genotypes under saline irrigation 37 Changes in dry matter (g plant-1) content in leaf of mungbean genotypes under saline irrigation 38 Changes in dry matter (g plant-1) content in root of mungbean genotypes under saline irrigation 38 Changes in lipid peroxidation (MDA) content (nmoles g-1 DW) in leaf of mungbean genotypes under saline irrigation 45 Changes in lipid peroxidation (MDA) content (nmoles g-1 DW) in root of mungbean genotypes under saline irrigation 45 Changes in chlorophyll ‘a’ content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation 46 Changes in chlorophyll ‘b’ content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation 47 Changes in total chlorophyll content (mg g-1 DW) in leaf of mungbean genotypes under saline irrigation 47 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 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Wilczek] genotypes Name of the degree holder : Duong Hoang Son Title of the degree : Doctor of Philosophy Admission No : 2010BS100D Name and address of Major advisor : Dr Neeraj Kumar Scientist of Plant Physiology Department of Botany & Plant Physiology, College of Basic Sciences & Humanities CCS Haryana Agricultural University, Hisar-125004, Haryana, (India) Degree awarding University/Institute : CCS HAU, Hisar-125004 (Haryana) India Year of award of degree : 2013 Major subject : Plant Physiology Total number of pages in the thesis : 86 + xiii Number of words in the abstract : Approximate 590 Key words: Salinity tolerance; Mungbean; Growth; Physiology; Seed yield; SDS-PAGE The effects of saline irrigation on morpho-physiological traits, antioxidant defense mechanism and protein profile (SDS-PAGE) in two summer mungbean (Vigna radiata L.) genotypes viz MH 421 and SML 668 differing in their sensitivity to salinity was investigated under natural conditions of screen house At the flowering stage (30-35 DAS), plants were exposed to single saline irrigation (Cl-dominated) of levels 2.5, 5.0 and 7.5 dS m-1 Sampling was done after and 14 days of treatment (DAT) The control plants were irrigated with distilled water The water potential (Ψw) of leaf and osmotic potential (Ψs) of leaf and root decreases in both the genotypes MH 421 showed more negative values Ψw of leaf i.e from 0.20 to -0.46 MPa as compared to -0.18 to -0.40 MPa in SML 668, respectively The Ψs of leaf decreased from -1.36 to -1.89 MPa in MH 421 and -1.36 MPa to -1.80 MPa in SML 668 with increase in salinity level from control to 7.5 dS m-1 at DAT A similar trend of change in the osmotic potential (Ψs) of root was observed The proline content of leaf increased significantly from 11.4 to 144.5 % and 15.7 to 219.4 % and the total soluble carbohydrate (TSC) content from 16.6 to 79.8 % and 22.5 to 81.9 % in SML 668 and MH 421, respectively, with increasing level of saline irrigation from control to 7.5 dS m-1 A marked increase in hydrogen peroxide H2O2, lipid peroxidation (MDA content) and relative stress injury (RSI %) was noticed in leaf and root which was much higher in SML 668 than MH 421 A similar trend was observed at 14 DAT Saline irrigation increased the Cl- and SO42- concentration in leaf by 2.8 and 3.1 fold in MH 421 and 3.0 and 3.6 fold in SML 668, respectively, at 7.5 dS m-1 Moreover, MH 421 had overall lower Na+/K+ ratio than SML 668 More negative values of Ψw of leaf, Ψs of leaf and root and better accumulation of osmotically active solutes, i.e proline, TSC and low Na+/K+ ratio in leaf and root of MH 421, helped in maintaining the higher RWC % of these organs than noticed in SML 668 ROS scavenging enzyme of leaf i.e SOD, CAT, POD, APX, GR, GPX and GST increased by 296.7, 158.5, 146.3, 159.1, 125.8, 95.9, 81.0 % in SML 668 and by 806.8, 180.3, 231.4, 174.9, 190.2, 112.6, 172.6 % in MH 421, respectively, upon increasing levels of saline irrigation from control to 7.5 dS m-1 after days of treatment (DAT) Despite the increase in the activity of these enzymes AsA content decreased by 52.5 and 41.3% Higher activities of antioxidant enzymes, lower accumulation H2O2, MDA and AsA content in MH 421 than in SML 668 indicated those enzymes play a key role in removal of ROS in MH 421 better than in SML 668, thus minimizing the cellular damage caused by ROS under saline irrigation Pollen viability (%), in vitro pollen germination and tube growth were also adversely affected with increasing salinity; the effect being most pronounced in SML 668 SDS-PAGE revealed more deletion than addition of polypeptide bands in SML 668 than MH 421 with increasing levels of saline irrigation in leaf and root at both the stages The yield parameters like number of pods plant-1, number of seeds plant-1, 100 seed weight and seed weight plant-1 decreased more in SML 668 with increasing salinity Hence, the mechanism of salt tolerance was better in MH 421 than in SML 668 as found from physiological traits studied MAJOR ADVISOR HEAD OF THE DEPARTMENT SIGNATURE OF STUDENT CURRICULUM VITAE Name Date of birth Place of birth Mother’s name Father’s name Permanent address : : : : : : Telephone E-mail Academic qualifications : : Degree University/ Board B.Sc University of Natural Sciences, Vietnam M.Sc Ph.D CCS HAU, Hisar, India CCS HAU, Hisar, India DUONG HOANG SON June 21, 1976 HaNoi (VietNam) Smt Hoang Thi Kim Quy Sh Duong Thanh Tu 27/10 LeBinh Street, NinhKieu Distric, CanTho City - VietNam 00847103-839451 dhoangsonvn@yahoo.com Year of passing 2001 %age of marks/OGPA 61.2 2007 2013 71.2 71.2 Co-curricular activities : - Medals/Honours received : - Subjects Biology (Major subject: Plant Physiology) Plant Physiology Plant Physiology (submitted) List of publications: (i) Research Papers - Published: 1 Son, D.H., Kumar, N., Nandwal, A.S., Kumar, S and Sharma, S.K 2013 Comparative physiology of two summer mungbean genotypes to salt stress International Journal of Biotechnology and Bioengineering Research (6): 603-608 (ii) Research paper presented and published in proceedings of Conference/Seminar: Son, D.H., Kumar, N., Nandwal, A.S., Bhasker, P., Kumar, S and Sharma, S.K 2013 Plant water status, osmolyte accumulation, membrane integrity and ionic distribution in two summer Vigna radiata L cultivars under saline irrigation Summary published in National Conference of Plant Physiology on: “Current trends in plant biology research”, pp 494-495, organised at Directorate of Groundnut Research, Junagadh, Gujarat, India, December 12-16, 2013 Kumar, N., Son, D.H., Nandwal, A.S., Devi, S., Yadav, R and Sharma, S.K 2013 Effect of saline irrigation on antioxidant responses in two summer mungbean (Vigna radiata L.) cultivars Summary published in National Conference of Plant Physiology on: “Current trends in plant biology research”, pp 599-600, organised at Directorate of Groundnut Research, Junagadh, Gujarat, India, December 12-16, 2013 Son, D.H., Kumar, N., Nandwal, A.S and Yadav, R 2012 Physiological responses to saline irrigation in two summer mungbean [Vigna radiata (L.) Wilczek] genotypes Abstract in National Seminar of Plant Physiology on: “Physiological and molecular approaches for development of climate resilient crops”, pp 84-85, organised at Directorate of Sorghum Research, Rajender Nagar, Hyderabad, India, December 12-14, 2012 Son, D.H., Kumar, N., Nandwal, A.S., Devi, S., Kumar, S., Sharma, M.K., Sharma, S.K and Yadav, R 2014 Activities of antioxidant enzymes in two summer mungbean Vigna radiata (L.) cultivars in response to saline irrigation Abstract in National Seminar on: “Reorientation of Agricultural Research to Ensure National Food Security”, pp 32-33, organised at Chaudhary Charan Singh, Haryana Agricultural University, Hisar, India, January 6-7, 2014 UNDERTAKING OF COPYRIGHT I, Duong Hoang Son, Admission No 2010BS100D undertake that I give copyright to the CCS HAU, Hisar of my thesis entitled, “Physiological responses to saline irrigation in two summer mungbean [Vigna radiata (L.) Wilczek] genotypes” I also undertake that patent, if any, arising out of the research work conducted during the programme shall be filed by me only with due permission of the competent authority of CCS HAU, Hisar (Haryana) Signature of the Student [...]... marked in roots GST increased by 95% was reported at 10 dS m-1 salinity level in chickpea roots (Kukreja et al., 200 5) GST activity was reported to increase in salinity stress in mungbean (Hossain et al., 201 1) 2.6.6 Glutathione peroxidase (GPX) GPX is ubiquitously occurring enzymes in plant cells that involved in scavenging of H2O2 and sever to detoxify products of lipid peroxidation formed due to activity... thus important in providing protection against oxidative damage in plants by maintaining the reduced form of glutathione (Foyer et al., 199 1) GR activity has been reported to increase in B juncea seedlings with increase in salinity level (Verma and Mishra, 200 5) Similarly, Abbaspour (201 2) reported an increased GR activity with increase in NaCl concentration in Pistacia vera Salt tolerance in the leaves... 201 0) A rapid accumulation of proline under salt stress has been observed in mungbean crop (Singh et al., 199 4) Arora and Saradhi (200 2) 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 (200 8) observed... in addition to reacting with H 2O2 may react 13 with superoxide anion (O- 2), singlet oxygen (1O 2) and hydroxyl radical (OH -) (Shigeoka et al., 200 2) Hernandez et al (199 9) reported rapid increased in APX activity in response to 90 mM and 110 mM NaCl concentrations in Pisum sativum leaves Sheokand et al (200 8) also reported an increase in APX activity under salt stress in chickpea Salinity induced increase... sustainable agricultural (Hussain et al., 200 8) 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... et al., 200 1) or to differentiate between salt tolerant and sensitive genotypes (Suriyan and Chalermpol, 201 0) The Fv/Fm ratio can be used to detect damage to photosymtem II and possible photo-inhibition (Ahmed et al., 200 2) Lee et al (200 4) 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... observed increasing with increasing saline irrigation concentration and days interval (Soomro et al., 200 1) 19 CHAPTER-III MATERIAL AND METHODS The present investigations were conducted on two released genotypes of mungbean [Vigna radiata (L. ) Wilczek] namely MH 421 and SML 668 The seeds were procured from the Pulses Section, Department of Genetic and Plant Breeding and the experiment was conducted in the... mutant genotypes Similarly, in tomato, it has been detected that GST activity increased in leaves and roots of salt tolerant but unchanged in control tomato cultivars under 100m M NaCl (Mittova et al., 2003 b) Misra and Gupta (200 6) studied wild type and mutant of Canthranthus roseus subjected to salinity A higher GST activity was observed in leaf pairs (apical, middle, and basal) and roots under saline. .. 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., 200 9) Shabina and Mehar (201 1) subjected the seven varieties of mungbean to 50... al., 200 5) In contrast, Kukreja et al (200 6) observed two- fold increase in SOD activity in chickpea roots under shortterm salinization SOD activity increased under salt stress was observed in both leaves and roots of mungbean (Chakrabarti and Mukherji, 2003 a), mungbean seedling (Saha et al., 201 0) Manivannan et al (200 7) reported SOD activity was increased in leaves, stems and roots with increased