Physiological responses to saline irrigation in two summer mungbean vigna radiata (l ) wilczek genotypes

content in leaf of mungbean genotypes 3 Changes in dry matter g plant -1 content in root of mungbean genotypes 4 Changes in lipid peroxidation MDA content nmoles g -1 DW in leaf of 5 C

PHYSIOLOGICAL RESPONSES TO SALINE IRRIGATION IN TWO SUMMER MUNGBEAN

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

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 EXTERNAL EXAMINER

HEAD OF THE DEPARTMENT

DEAN, POST-GRADUATE STUDIES

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

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

TTC - 2,3,5- triphenyl tetrazolium chloride

4 Changes in relative water content (RWC %) in leaf of mungbean genotypes

5 Changes in relative water content (RWC %) in root of mungbean genotypes

6 Changes in relative stress injury (RSI %) in leaf of mungbean genotypes

7 Changes in relative stress injury (RSI %) in root of mungbean genotypes

-) content (mg g-1 DW) in leaf of mungbean

11 Changes in chloride (Cl

-) content (mg g-1 DW) in root of mungbean

12 Changes in sulphate (SO4

2-) content (mg g-1 DW) in leaf of mungbean

13 Changes in sulphate (SO4

2-) content (mg g-1 DW) in root of mungbean

14 Changes in in vitro pollen germination (%) and pollen tuber growth (µm) in

15 Changes in yield and yield attributes in two mungbean genotypes under

) content in leaf of mungbean genotypes

3 Changes in dry matter (g plant

-1

) content in root of mungbean genotypes

4 Changes in lipid peroxidation (MDA) content (nmoles g

-1

DW) in leaf of

5 Changes in lipid peroxidation (MDA) content (nmoles g

-1

DW) in root of

6 Changes in chlorophyll ‘a’ content (mg g

-1

DW) in leaf of mungbean

7 Changes in chlorophyll ‘b’ content (mg g

-1

DW) in leaf of mungbean

8 Changes in total chlorophyll content (mg g

-1

DW) in leaf of mungbean

9 Changes in chlorophyll stability index (%) in leaf of mungbean genotypes

10 Changes in quantum yield (Fv/Fm) in leaf of mungbean genotypes under

11 Changes in hydrogen peroxides (H2O2) content (moles g

-1

DW x 10-4) in

12 Changes in hydrogen peroxides (H2O2) content (moles g

-1

DW x 10-4) in

13 Changes in proline content (mg g

-1

DW) in leaf of mungbean genotypes

14 Changes in proline content (mg g

-1

DW) in root of mungbean genotypes

15 Changes in total soluble carbohydrates content (mg g

-1

DW) in leaf of

16 Changes in total soluble carbohydrates content (mg g

-1

DW) in root of

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

19 Changes in catalase (CAT) specific activity (Units mg protein) in leaves

20 Changes in catalase (CAT) specific activity (Units mg

-1

protein) in root of

21 Changes in peroxidase (POX) specific activity (Units mg

-1

protein) in leaf

22 Changes in peroxidise (POX) specific activity (Units mg

-1

protein) in root

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

32 Changes in ascorbate (AsA) content (µmoles g

-1

DW) in root of mungbean

33 Changes in pollen viability in two mungbean genotypes under saline

CHAPTER-I

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

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), phospholipid-hydroperoxide glutathione peroxidise (GPX) and ascorbate peroxidase (APX), and non-enzymatic 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,

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

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

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 halophytic 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,

non-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 N2 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:

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)

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 (Ψw) 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 Ψs 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)

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)

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,

Na2SO4 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

Swartz ecotypes that with the increase of salinity level (1.1-49.7 dS m ) 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

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

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 H2O2 and may trigger genetically programmed cell suicide (Farouk, 2011) H2O2 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 H2O2 (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 H2O2

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

salinity-induced oxidative stress, plants make use of a complex antioxidant system, which is

composed of low molecular mass antioxidants as well as ROS scavenging enzymes

(Blokhina et al., 2003; Mudgal et al., 2010)

The formation of ROS has been reported to increase under salinity stresses like

osmotic stress in Vigna aconitifolia (Kestwal et al., 2012), Cassia angustifolia seedling

(Agarwal and Pandey, 2004) and rice seedling (Dionisio-Sese and Tobita, 1998) Several studies have produced evidence roles for ROSs in mungbean plant under salinity

2.6.1 Superoxide dismutase (SOD)

SOD originally discovered by McCord and Fridovich (1969) react with superoxide radicals at almost diffusion-limited rates to produce hydrogen peroxide In general, plants contain a mitochondria matrix localized MnSOD and cytosolic Cu/ZnSOD present in the chloroplast stroma (Geetanjali and Neera, 2008) Comparing the mechanisms of antioxidant production in salt tolerant and salt sensitive plants, Dionisio-Sese and Tobita (1998) reported a decline in SOD activity in salt sensitive and increased in tolerant rice

varieties Similarly, results had been reported in wheat (Mandhania et al., 2006) and cotton leaves (Meloni et al., 2002) It was suggested that the ratio between superoxide dismutase

and H2O2 scavenging enzyme activities could be used as a working hypothesis for a

biochemical marker for salt tolerance in sorghum (Costa el al., 2005) In contrast, Kukreja

et al (2006) observed two-fold increase in SOD activity in chickpea roots under

short-term 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., 2010) Manivannan et al (2007) reported SOD activity was increased in leaves, stems and roots

with increased the NaCl stress and CaCl2 stress versus control in mungbean plant

2.6.2 Catalase (CAT)

CAT converts H2O2 to water and molecular oxygen In plant, CAT is found predominantly in peroxisomes and glyoxysomes where it functions chiefly to remove the

H2O2 form during the photorespiration (Geetanjali and Neera, 2008) and also during salt

stress and other abiotic stress condition (Willekens et al., 1995) Increase in CAT activity

upon salinization has been observed in Ipomoea pes-capraesweet (Venkateshan and

Chellappan, 1999), sugarbeet (Bor et al., 2003), cowpea (Cavalcanti et al., 2007) and chickpea (Sheokand et al., 2008) Contrary to the above reports, diminished catalase activity has been reported in salt stressed plants of Phaselous mungo (Dash and Panda, 2001), Catharanthus roseus (Jaleel et al., 2007) and K virginica (Guo et al., 2009)

Similarly, a significant decrease in CAT activity was also observed in leaves of both

salt-tolerant and salt-sensitive varieties of pea (Corpus et al., 1993)

Changes in CAT activity from mungbeans under salinity stress have been reported

in different tissues by Manivannan et al (2007), root and shoot by Panda (2001), leaves and root by Chakrabarti and Mukherji (2003 b), seedling by Hossain et al (2011) Saha et

al (2010) revealed that CAT activity increased in roots by 64% but slight decreased in

leaves in mung bean under different salinity levels Zaffar et al (2007) showed that CAT

activity increased by 2.4 fold at concentration 200mM at 20 DAS in tolerance mungbean

genotypes Yasar et al (2008) observed higher CAT activity in leaves of salt tolerant

genotype than sensitive under 50mM and 100mM NaCl treatment Shabina and Khan (2004) reported differential effect on the activity of CAT in different cultivars was

exposed to salinity levels Sumithra et al (2006) reported that greater increased (4.7 fold)

in CAT activity of tolerant as compared to control cultivar where as sensitive exhibited only 2 fold increased at 300 mM salt concentration

2.6.3 Peroxidase (POX)

POX localized in almost all compartments of the plant cell, it plays role in stability the level of H2O2 Peroxidases, besides their main function in H2O2 elimination, can also catalyse O2 and H2O2 formation by a complex reaction in which NADH is oxidized using trace amounts of H2O2 (Blokhina et al., 2003) Peroxidases are also involved in biosynthesis of cell wall lignifications and suberization (Passardi et al., 2004) Experimental evidence shows that salinity causes increases in POX activity in Cassia

angustifolia (Agarwal and Pandey, 2004), Brassia napus (Dolatabadion et al., 2008) and

chickpea plants (Sheokand et al., 2008) However, decrease in POX activity with increase

in NaCl concentration has been observed in Calamus tenuis (Khan and Patra, 2007) and

wheat (Farouk, 2011)

Chakrabarti and Mukherji (2003 a, b) reported that application of NaCl in mung

bean caused increased peroxidase activity in leaf and root Zaffar et al (2007) recorded

POX activity increased 2.4 and 2.8 fold under 200 mM NaCl at 20 and 40 DAS in mung

bean plant, respectively Arulbalachandran et al (2009) revealed peroxidase activity was

increased in shoot and root with increasing salt concentrations in cowpea, black gram and green gram compared to respective control and the POX activity was more in root than in shoot However, POX activities were highly significant decreasing in mung bean due to increasing NaCl concentration observed by Mohammed (2007)

2.6.4 Ascorbate peroxidase (APX)

Ascorbate peroxidase (APX) uses ascorbate as its reducing substrate It is involved

in scavenging of H2O2 in water and AsA-GSH cycle and utilizes AsA as the electron donor

(Ahmad et al., 2008) APX seems to play a more important role in scavenging ROS than

other antioxidative enzymes since ascorbate, in addition to reacting with H2O2 may react

with superoxide anion (O2), singlet oxygen ( O2) and hydroxyl radical (OH) (Shigeoka et

al., 2002) Hernandez et al (1999) reported rapid increased in APX activity in response to

90 mM and 110 mM NaCl concentrations in Pisum sativum leaves Sheokand et al (2008)

also reported an increase in APX activity under salt stress in chickpea

Salinity induced increase APX activity in both tolerant and sensitive plants were

reported in green bean (Yasar et al., 2008), cowpea (Maia et al., 2010), Pistacia vera (Abbaspour, 2012) Hernandez et al., 2000 reported that tolerant Pisum sativum response

to long term NaCl treatment increased ascorbate peroxidase 3 fold However, from NaCl sensitive plant no changes in the specific activity of APX Similar observe were report by

Amor et al (2006) in Cakile maritime Decreased APX activity has been reported in two tolerance mutants legume leaves Clitoria ternatea and Lathyrus sativus under salt stress

(Talukdar, 2011) Decreased APX activity in leaves and remain unchanged in root were

recorded by Fusun and Mehmet (2007) Kukreja et al (2006) reported APX specific

activity increased by 9 and 99% at 2.5 and 10 dS m-1 salinity levels in chickpea roots

2.6.4 Glutathione S-transferase (GST)

GSTs are a family of multifunctional enzymes that play important roles in oxidative stress resistance (Joseph and Jini, 2011) These dimeric enzymes catalyze the conjugation of GSH to avariety of electrophylic, hydropobic, and often toxic substrates,

thereby reducing their toxicity (Hossain and Fujita, 2010) Roxas et al (2000) study the

effect of NaCl concentration at 100 mM on GST of tobacco Results revealed that wild type showed approximately 2.5 fold lower GST activity as compare 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 (2006) 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 conditions, and most marked in roots GST increased by 95% was reported at 10 dS m-1 salinity level in chickpea roots (Kukreja et al., 2005) GST activity was reported to increase in salinity stress in mungbean (Hossain et al., 2011)

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 of ROS GPX decomposes peroxides to water or alcohol while simultaneously oxidizing glutathione (GSH) GPX competes with catalase for H2O2 as a substrate and is the major

source of protection against low levels of oxidative stress (Ahmad et al., 2008; Hossain and Fujita, 2010)

GPX activity was reported to decrease upon salinization of rice (Lee et al., 2001)

However, GPX activity was reported increased in leaves of tomato plant grown with 100

mM NaCl (He and Zhu, 2008) GPX expression can be considered as a molecular maker for oxidative stress in plant Similarly, GPX activity was also reported to increase in

salinity stress in tomato (Mittova et al., 2003 a, b) and mungbean (Hossain et al., 2011)

2.6.7 Glutathione reductase (GR)

GR catalyses the rate limiting last step of AsA-GSH pathway (Ahmad et al., 2008)

GR catalases the NADPH dependent reaction of disulphide bond of GSSG and is thus important in providing protection against oxidative damage in plants by maintaining the

reduced form of glutathione (Foyer et al., 1991) GR activity has been reported to increase

in B juncea seedlings with increase in salinity level (Verma and Mishra, 2005) Similarly,

Abbaspour (2012) reported an increased GR activity with increase in NaCl concentration

in Pistacia vera Salt tolerance in the leaves of Calamus tenuis has been correlated with elevated GR activity (Khan and Patra, 2007) Yasar et al (2008) reported in salt-tolerant

cultivar of green bean GR activity is greater than the salt-sensitive cultivar which indicates that the tolerant plants exhibit a more active ascorbate-glutathione cycle than the non-tolerant cultivar

The increase in GR activity has also been observed in salt tolerant varieties of rice

(Dionisio-Sese and Tobita, 1998), pea (Hernandez et al., 2000), in Brassica juncea (Verma and Mishra, 2005) and wheat (Sairam et al., 2002) than their respective sensitive varieties

Decreased GR activity was observed in leaves but increased in root of maize as compared

to control under salt stress (Neto et al., 2005) Salinity stress enhanced increase GR activity has been demonstrated in leaves of mungbean (Nafees et al., 2010) and in mungbean seedling (Hossain et al., 2011) Sumithra et al (2006) reported that increased

GR activity with increased salt stress levels in mungbean cultivars Pusa Bold showed over 6.9 fold more GR activity than control at 300 mM NaCl while CO-4 showed only 4.6 fold under same condition

2.6.8 Ascorbate (AsA)

AsA is the most abundant antioxidant and serves as a major contributor to the cellular redox state and protects plants against oxidative damage resulting from aerobic metabolism and a range of biotic and abiotic stresses (Smirnoff, 2000) It is substrate of cAPX and the corresponding organellar iso-forms, which are critical components of AsA-GSH cycle for H2O2 detoxification (Nakano and Asada, 1981) Salt stress caused a decreased in total AsA in tomato (He and Zhu, 2008) and wheat (Farouk, 2011)

Hernandez et al (1999; 2000) reported NaCl concentration at 70 mM decreased AsA in

both NaCl tolerant and NaCl sensitive pea cultivars The decline in ascorbate content was

also observed to be linear with increasing salinity level in chickpea (Kukreja et al., 2006)

The higher ascorbate content was observed in the salt-tolerant cultivars as compared to salt

sensitive cultivars of Oryza sativa under salt stress (Vaidyanathan et al., 2003) Amor et

al (2006) associated high salt-tolerance in Cakile maritima, a coastal halophyte with high

ascorbate AsA content was reported to be increased in salinity stress in mungbean

(Hossain et al., 2011) Salinity stress severely increased the ascorbate content in leaf of

Vigna radiata reported by Maia et al (2010)

2.7 Ionic composition

Detrimental effect of salt may be due the toxicity of specific ion, elevation of osmotic pressure or the increase in alkalinity which may restrict the availability of water or

influence cellular physiology and metabolic pathway (Mudgal et al., 2010) Salinity of soil

and water is caused by the presence of excessive amounts of salts Most commonly, high

Na+ and Cl- cause the salt stress High salt (NaCl) uptake competes with the uptake of other nutrient ions, especially K+, leading to K+ deficiency (Parida and Das, 2005) When the chloride content in the root zone exceeds a threshold value, it adversely affected the availability of S to plants Therefore, sulphur requirement of crops is increased considerably under Cl- dominated saline conditions (Sharma and Khajanchi, 2009)

In recent year, a lot of studies has been done on mungbean regarding with respect

to ionic composition under saline conditions (Sabina and Khan, 2004; Zaffar et al., 2007;

Sabina and Mehar, 2011) K+ concentration was similar in different plant parts but Na+ was high in shoot and root at pre-flowering and flowering stages of growth of mungbean grown

at various salinity levels (Hafeez et al., 1988) Nandwal et al (2000 b) revealed that

salinity nutrient solution significantly increased Na+/K+ ratio in the leaves and roots as well as in nodules Similar, NaCl induced salinity resulted in sharp increase in Na+, Cl- and decrease in K+ accumulation in the root, stem and leaf on mungbean seedling (Parveen et

al., 2004) However, Zayed and Zeid (1998) reported that NaCl applied in mungbean

seedling resulted Na+ accumulation in shoot while reduction in root, K+ content decreased

in both shoot and root, Ca2+ content accumulation in shoot but remained unchanged in root, Cl- accumulation high in root and slightly in shoot and Sulfur content decreased markedly with increasing the salt concentration

Increased saline irrigation on mungbean caused increase in Na+, Cl-, SO4

2-

and decrease in K+, accumulation in leaves (Pooja and Sharma, 2010) Similar, Na+, Cl-increased and decreased in K+, Ca2+ and Mg2+ accumulation with increased salinity levels

in shoot and root (Mohammed, 2007) Wahid et al (2004) reported that all the mungbean

genotypes displayed the accumulation of Na+, Cl- in leaf as the salt levels progressed Old

+

and Ca content under increased salt levels in all stages Tolerant genotypes of mungbean had lower Na+/K+ ratio in shoots and roots than the susceptible genotypes (Sumithra et al.,

2006)

2.8 SDS-PAGE

Plants exposed to the biotic stress of high soil salinity by modify their metabolism

to cope with environment change A metabolic change common to most if not all plants is the accumulation of low molecular weight organic solutes under salt stress (Zhu, 2007) The possibility is that in 3-D structure is governed by hydrophobic/hydrophilic, ionic interaction and interaction between side chains of constituent amino acids Thus free amino acid could interfere with these side chain bandings and introduce conformational changes in the enzyme protein and thus affect their activity (Rai, 2002) So, salt-induced proteins have been identified in plants species and have been classified into two distinct groups: salt stress proteins which is accumulate only due to salt stress, and stress associated proteins which also accumulate with other biotic stress (Ashraf and Harris, 2004)

Mittova et al (2003 b) reported that salt induced accumulation -ECS isoprotein

was identified by SDS-PAGE in salt tolerant but it absent in sensitive tomato cultivar In onion, two new bands 14 kDa and 54 kDa polypeptides were identify in responsed to salt

stress (El-baky et al., 2003)

Maria et al (2000) studied salt modulation of vaculolar H+-PPase and V-ATPase

protein in Vigna unguiculata hypocotyls Maria revealed that the relative amount of

protein of both subunits varied according to changes of enzyme activities in all condition Sheoran and Garg (1979) reported two new isoenzyme (0.15 and 0.42 kDa) in cotyledons, three new isoenzyme (0.05, 0.49 and 0.62 kDa) in embryo axis, three new isoenzyme (0.34, 0.38 and 0.65 kDa) appeared and isoenzyme (0.38 kDa) disappeared as compared to control under different salt compositions were identify by SDS-PAGE in mungbean The salt treatment is characterized by specific band at molecular weight 39.5 kDa in mungbean (Mohammed, 2007)

2.9 Pollen viability, pollen germination and pollen tube growth

Very little information regarding the effects of salinity on the development of the reproductive parts of flower plants is available The failure of seed set and consequent decreased yield may be due to adverse effect of saline irrigation on pollen viability (Humaira and Rafiq, 2006) Salinity delayed flowering, decreased pollen production, inhibited tube elongation was observed in chickpea (Dhingra and Varghese, 1993) Similar, some evidence adverse effect of saline for decline in quantitative production of

pollen, their viability, germination and tube growth has been reported in maize (Dhingra and Varghese, 1985) and pea (Dhingra and Sharma, 1992)

Khan and Khatoon (1997) reported that high NaCl salinity induces sterility in C

roseus pollens but may not have direct effect at low NaCl level; instead pollens are rather

activated by low level of NaCl However, Hameda and Ralph (1992) revealed that pollen viability and germination were reduced response to salinity levels in maize In rice, pollen viability was reduced at both panicle and booting stage under saline condition (Khatun and Flowers, 1995) Gupta and Murty (1978) compared pollen germination and tube length in four different mungbean cultivars namely T-44, K-851, UG-152 and UG-157 UG-152 and K-851 yielded optimum pollen germination on a medium containing 20 % sucrose, UG-

157 at 30 % and T-44 at 35 % sucrose Maximum tube length (270 µm) was achieved in

cv T-44 on a medium containing 35% sucrose

2.10 Yield and its attributes

The effect of salt on tissue and organ development is reflected in altered patterns

of plant growth and development (Volkmar et al., 1998) Salinity stress causes changed in

various physiological processes and particularly increase in Na+ and Cl- ions seedling

under salinity leads to decrease in yield attributes (Pandey et al., 2010) Dry matter and

grain yield almost regulatory decreased with increased of EC and SAR of the irrigation water and the saturation extract of the soil and soil ESP (Gandhi and Paliwal, 1975) Various studies regarding the effect of saline over crops yield and yield component has

been reported in wheat (Farooq and Azam, 2006), cotton (Soomro et al., 2001), mungbean (Shabina and Khan, 2004; Khan et al., 2010, Pooja and Sharma, 2010)

Yield and yield component decreased with increased salinity levels The reduction

in productivity of mungbean cultivars reach up to 50% under salt stress (Mohamed and El

Kramany, 2005) Similar, Wahid et al (2004) reported that four mungbean genotypes

exhibited sensitive to salinity at all stages and showed yield reduces ranged from 39% to 51% Increasing levels of salinity applied to mungbeans resulted in reduced number of pods plant-1, number of seeds pod-1, weight of pod-1, 1000-seed weight, seed yield-1 (Kabir

et al., 2004; Wahid et al., 2004) Soil salinity is reported to cause a decline in seed yield of

mungbean and this reduction was ascribed to different factors such as reduction in branches plant-1, number of pods, number of seeds pod-1 and seed plant-1 (Mohamed and El Kramany, 2005)

Garg et al (1996) studied the effect of different saline irrigation (NaCl, Na2SO4

and NaHCO3) at two level 5 and 10 dS m-1 on yield in cluster bean, mungbean and moth bean Increased salts concentration progressively decreased pods per plant and seed yield

Among three salts, NaHCO3 was most detrimental; Na2SO4 was more detrimental than NaCl for yield of these legumes This study has revealed the higher sensitive of mungbean and moth bean than cluster bean Mensah and Ihenyen (2009) also reported that salt tolerance of different mung bean cultivars were differential reduction in growth and yield when grown in salt affect soil

2.11 ECe of soil

Mung bean shows suppressed growth event in marginally saline stress areas and most cultivars exhibit a salt tolerance threshold to < 2 dS m-1 (Minhas et al., 1990) Most

of mungbean cultivars are salt sensitive, their seed yield threshold at ECe less than 1.8 dS

m-1 and relative crop yield slope obtained at 20% (Francois and Maas, 1999) Maliwal and Paliwal (1982) determined that critical level of salinity for mung bean germination from 12

to 18 mmhos/cm Irrigation saline water treatment resulted increase ECe concentration in

individual soil salinity cultivar (Aragues et al., 2010) Increased saline irrigation levels

caused increased ECe, SAR, Na, Ca, Mg, Cl in soil leads to reduced crop growth and development (Picchioni and Miyamoto, 1990)

Differential ECe levels and soil texture due to salt water caused increased SAR and ESP of the soil (Gandhi and Paliwal, 1975) Mitchell and Shennan (1991) studied the effects of irrigation and deficit irrigation with saline drainage water on processing tomato yield and quality in two years It was observed that in first year ECe values increased with the time in the salinity treatment from first flower and leads to double after 4 weeks before harvest as compared to control at all soil depths A similar result was obtained in second year However, ECe increased with more depths of soil and extent of days irrigate as same saline irrigation concentrate Similar, Soil pH, ECe, ESP, SAR were observed increasing

with increasing saline irrigation concentration and days interval (Soomro et al., 2001)

CHAPTER-I I

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 screen house under natural condition in the Department of Botany and Plant Physiology, CCS Haryana Agricultural University, Hisar, 125004, Haryana

3.1 Experimental Material

3.1.1 Raising of crop

The crop was raised in earthen pots filled with 5 kg of dune sand Some of the physico-chemical characteristics of the experimental dune sand were as follows: (i) Mechanical analysis = sand (92.2%), silt (3.8%), clay (4.2%) (ii) Texture = sand (iii) Saturation capacity = 25%, (iv) pHc = 8.72, (v) ECe = 0.95 dSm-1 at 25°C, (vi) Available nutrients (mg kg-1) = N (10.3), P (2.5), K (18.0), (vii) Taxonomic class = Typic torrispamments

Before sowing, the seeds were surface sterilized with 0.1 % HgCl2 for two minutes

and washed with distilled water twice Then seeds were inoculated with culture (Rhizobium

leguminosarum, S-24) obtained from the Department of Microbiology, CCS Haryana

Agricultural University, Hisar Nitrogen free nutrient solution (Wilson and Reisenauer, 1963)

was given at required time intervals with the following compositions:

3.1.2 Irrigation

Whenever needed, the pots were irrigated to field capacity with distilled water Before salinity treatments, whenever needed, the pots were irrigated from the surface only However, after the salinity treatments, they were irrigated 50 % from the surface and 50 % (of the total) from the subsurface (through a slightly inclined embedded plastic feeder tube having a pad of glass wool at its lower end) In this way uniformly required ECe was

maintained throughout the dune sand

3.1 3 Treatments

The dune sand was irrigated with artificially prepared saline water for chloride (Cl-) dominated salinity (Cl: SO4 ratio 7:3) by using mixture of different salts like NaCl, MgCl2, MgSO4 and CaCl2 where Na: Ca + Mg in the ratio of 1:1 and Ca: Mg in the ratio of 1:3 on milliequivalent basis dissolved in distilled water

Amount of salt used for preparing chloride dominated saline water

by Duncan’s Multiple Range Test at P<0.05 Vertical bars in figures indicate value of standard error (SE ±) mean

(i) Plant height: The plant height was measured from the surface of sand to the apex of

the plant i.e main shoot length using meter scale and expressed in cm plant-1

(ii) Dry matter of roots and shoots plant -1: The separated plant roots and shoots were

oven-dried at 60ºC for 48h and finally at 80ºC for 24 h or till a constant dry weight was obtained It was recorded in g plant-1

3.2.2 Plant water status

3.2.2.1 Water potential (Ψ w )

Water potential of third leaf was measured with the help of pressure chamber (Model

3005, Soil Moisture Equipment Corporation, Santa Barbara, CA, USA), between 8 AM to 10

AM The plants were gently removed from the sand The third fully expanded trifoliate leaf from the top was cut from the plant with the help of sharp edged knife and sealed in the pressure chamber one by one with the cut end protruding outside and the pressure was developed till the sap just appeared at end That pressure (-) bars was recorded as water potential of the respective tissue which was exposure as (-MPa) by using the conversion factor -l0 bar = -1MPa

3.2.2.2 Osmotic potential (Ψ s )

The osmotic potential of root and third leaf from the top of plant were taken to measure for Ψs The airtight syringes were used to keep the leaves and roots samples in deep freezer at -150C The sap from these leaf tissues was extracted on filter paper discs and Ψs

measured using Vapor Pressure Osmometer (Model 5100-B, Wescor Inc Logan, Utah, USA) The osmometer was calibrated by using Osmolality Reference Standards of Sodium Chloride (Wescor mc, USA) The readings of osmometer, thus, obtained in miliosmoles kg-1 were converted to molality and finally to (-) bars with the help of calibration curve which was expressed in (-) MPa by using the following conversion factor

40 Osmol = -1 bar -l0 bar = -1MPa

3.2.2.3 Relative water content (RWC %)

The plants were sampled and third leaf from the top were detached between 9 AM to

10 AM, quickly sealed in humified polythene bags, and transported to the laboratory on ice Sand was removed with the help of a soft brush Then the leaves and roots were separated and weighed immediately to take their fresh weight Then the leaves and roots were kept in petri dishes full of distilled water separately for 3 h After that the leaves and roots (fully turgid) were weighed again and then kept in oven at 65°C for 72 h till a constant dry weight These three weights were used to calculate RWC (%) of leaves and roots according to the formula given by Weatherley (1950)

RWC (%) = [Fresh weight – Dry weight / Turgid weight – Dry weight] x 100

3.2.3 Membrane injury

3.2.3.1 Relative stress injury (RSI) (%)

Membrane injury index was measured as percent proportion of ion leakage in to the external aqueous medium to the total ion concentration of the stressed tissue as measured by the EC of the external medium (Sullivan and Ross, 1979)

Procedure

The third fully expanded leaf of uniform size was taken in test tubes containing 10 ml

of deionised water in two sets One set was kept at 25 °C for 30 minutes and another set at

100 °C for 15 minutes in boiling water bath and their respective electrical conductivity [EC1 and EC2] were measured by conductivity meter RSI % was calculated with formula:

RSI (%) = [1- (EC1 / EC2)] × 100

3.2.3.2 Lipid peroxidation

The level of lipid peroxidation was measured in terms of malondialdehyde (MDA) present in the tissues (leaf and root) MDA is a product of lipid peroxidation and was measured by thiobarbituric acid (TBA) reaction with minor modifications of the method of Heath and Packer (1968)

Reagents

(i) 0.1 % Trichloroacetic acid (TCA)

(ii) 20 % TCA containing 0.5 % thiobarbituric acid (TBA)

Extraction

Three hundred mg of leaf and root were homogenized separately with 5 ml of 0.1 % TCA The homogenate was centrifuged at 8000 x g for 15 min The supernatant was then directly used for the assay

Procedure

One ml of the supernatant was taken and precipitated by 4 ml of 20 % TCA containing TBA The mixture was heated in a water bath shaker at 95°C for 30 minutes and quickly cooled in an ice-bath After centrifugation at 8000 x g for 10 minutes the absorbance

of the reaction mixture was read at 532 nm and the value for non-specific absorption at 600

nm was subtracted The concentration of MDA was calculated using its extinction coefficient

of 155 mM-1cm-1 and the MDA content was expressed as nmoles g-1 DW

3.2.3.3 Chlorophyll content

Chlorophyll content was estimated according to the method of Hiscox and Israelstam

(1979) using dimethyl sulfoxide (DMSO)

Procedure

Third leaf from the top of plant was detached and weighed and was kept into a test tube containing 5 ml of DMSO The test tube was then placed into oven at 60ºC for 2 h to facilitate the extraction of pigment After 2 h and attaining the room temperature the absorption was read at 645 and 665 nm on a computer added spectrophotometer DMSO was used as blank Calculations for different pigments were made according to Welburn (1994)

Chl ‘a’ (mg/g-1) = 12.19 A665 – 3.45 A645

Chl ‘b’ (mg/g-1) = 21.99 A645 – 3.32 A665

Total chlorophyll = Chl ‘a’ + Chl ‘b’

Quantity of all these pigments was calculated in mg g-1 tissue DW

3.2.3.4 Chlorophyll stability index

Chlorophyll stability index (CSI) was estimated by method of Koleyoreas (1958) Normal and heated leaf were extracted in 80% acetone and absorbance was recorded at 652

nm for estimation of CSI by following formula:

CSI = [Absorbance of saline treated leaf at 652 nm / Absorbance of normal leaf at

652 nm] x 100

3.2.3.5 Chlorophyll fluorescence / quantum yield of PS II

Photochemical efficiency / quantum yield of PS II of third leaf from the top of plant was determined with intact plants in the field with an OS-30P Chlorophyll Fluorometer (Opti-Science, Inc., Hudson, USA) Initial (F0) and maximum (Fm) fluorescence were recorded and variable fluorescence (Fv), derived by subtracting Fo from Fm Quantum yield of PS II / photochemical efficiency which is Fv/Fm ratios were calculated

3.2.4 Biochemical studies

3.2.4.1 Proline content

Proline content was estimated by using the method of Bates et al (1973)

Reagents

(i) 3 % aqueous sulphosalicylic acid (w/v)

(ii) Acid ninhydrin (dissolving 1.25 g ninhydrin in 30 ml glacial acetic acid and 20 ml

Procedure

Two ml of supernatant was taken and 2.0 ml reagent acid ninhydrin and 2.0 ml acetic acid was added This mixture was then kept in boiling water bath for 1 h at 100 °C and thereafter reaction was terminated by keeping tubes in ice-bath Then 4.0 ml of toluene was added After vigorous shaking, the upper organic phase was taken after attainment of room temperature and absorbance was recorded at 520 nm by using toluene as blank

A standard curve was prepared by using graded concentration of proline in 3 % sulphosalicylic acid The proline content was expressed as mg g-1 DW

3.2.4.2 Total soluble carbohydrate (TSC)

Total soluble carbohydrates were determined with the method of Yemm and Willis (1954)

Extraction was done according to Barnett and Naylor (1966) Two hundred mg fresh samples of leaf and root were homogenized separately in 80% ethanol, refluxed for 15 minutes on a steam bath and centrifuged The residue was further refluxed with 40% ethanol Like this the extraction was repeated thrice The supernatant from different extraction was pooled and volume made to 5ml with 80% ethanol The extract so obtained was used for estimation of TSC

Reagents

Anthrone reagent: dissolving 0.4 g anthrone in 100 ml concentrated H2SO4

Procedure

Total soluble carbohydrates were estimated using anthrone reagent A 0.2 ml aliquot

of the extract was evaporated to dryness in a test tube in a boiling water bath on cooling the

residue left in the tube was dissolved in one ml of distilled water and mixed with 4.0 ml of the anthrone reagent Heat the mixture in a water bath for 10 minutes After cooling, absorbance was recorded at 620 nm using Spectrophotometer

Standard curve was prepared using graded concentration of glucose and the data were expressed as mg g-1 DW of the tissue

3.2.4.3 Hydrogen peroxide (H 2 O 2 )

H2O2 content of the leaves and roots was determined by a modified Patterson et al

(1984) method

Reagents

(i) 5 % trichloroacetic acid (TCA)

(ii) 100 mM potassium phosphate buffer (pH 8.4)

(iii) Colorimetric reagent: It was prepared by mixing 0.6 mM M4-(2-pyridyzalo) resorcinol and potassium titanium oxalate in 1:1 (v/v) ratio The mixture was kept on ice until use

Extraction

Three hundred mg of leaf and root were homogenized separately with 0.2 g of activated charcoal and 5 ml of 5 % TCA The homogenate was filtered through Whatman No.1 filter paper and centrifuged at 8000 x g for 10 min The supernatant was filtered through three layers of Whatman No.1 filter paper

Procedure

Two hundred µl of the extract was brought to 4 ml with potassium phosphate buffer The pH was adjusted to 8.4 with concentrated ammonia solution Then 2 ml of colorimetric reagent was added to the reaction mixture The absorbance was then read at 508 nm H2O2

was determined from the difference in absorbance between sample and blank The H2O2

content was calculated using its molar extinction coefficient of 3.6 x 10-4 moles and the H2O2

content was expressed as moles g-1 DW

3.2.5 Antioxidant defence system

leaves were washed with distilled water, dried with filter paper and macerated in a chilled pestle and mortar in presence of 3.0 ml of cold extraction buffer (potassium phosphate) containing 0.1 mM EDTA, 1% (w/v) PVP, 0.5% triton X-100 and 20% glycerol pH was adjusted to 7.8 The homogenate was centrifuged at 10,000 x g for 15 min at 4°C The supernatant was carefully decanted and used as the crude enzyme extract

H2O2 consumed during the reaction

3.2.5.2 Superoxide dismutase (SOD)

The specific activity of SOD was assayed by measuring its ability to inhibit the

photochemical reduction of NBT according to Beauchamp and Fridovich (1971)

w fluorescent lamps) The reaction was allowed to run for 20 minutes and the reaction was stopped by switching off the light The tubes were immediately covered with a black cloth The absorbance was recorded at 560 nm A non-irradiated reaction mixture, which did not develop color, served as control However, in the presence of SOD the reaction was inhibited and the amount of inhibition was used to quantify the enzyme

Log A560 was plotted as a function of volume of enzyme extract used in the reaction mixture From the resultant graph, volume of enzyme extract corresponding to 50 per cent inhibition of the photo-chemical reaction was obtained and considered as one enzyme unit

3.2.5.3 Glutathione reductase (GR)

The sample preparation and extraction buffer was same as used in CAT Specific activity was analysed by the method of Halliwell and Foyer (1978)

Procedure

Incubation mixture for enzyme assay consisted of 0.1 M phosphate buffer (pH 7.5), 5

mM oxidized glutathione (GSSG), 0.2 mM NADPH and 100 µl enzyme extract in a final volume of 1.5 ml Addition of GSSG, initiated the enzyme reaction The decrease in absorbance at 340 nm due to oxidation of NADPH was monitored The enzyme activity was calculated by using the extinction coefficient value of 6.2 mM-1 cm-1 for NADPH One unit of enzyme specific activity was equivalent to one nmol of NADPH oxidised during the reaction

3.2.5.4 Ascorbate peroxidase (APX)

The enzyme specific activity was determined according to the method described by

Nakano and Asada (1981)

Procedure

The sample preparation and extraction buffer was same as used in CAT The composition of assay mixture was 50 mM phosphate buffer (pH 7.0), 0.5 mM sodium ascorbate, 1.0 mM H2O2 and 75 µl of enzyme extract in 1.5 ml final volume The reaction was initiated by the addition of H2O2 The decrease in absorbance due to oxidation of ascorbate at

290 nm was recorded spectrophotometrically for 2 min The enzyme activity was calculated

by using the extinction efficient value of 2.8 mM-1 cm-1 for ascorbate One unit of enzyme specific activity corresponded to one µmol of ascorbate oxidised during the reaction

mM-1 cm-1 for guaiacol One unit of enzyme specific activity was equivalent to µmol of H2O2

oxidised

3.2.5.6 Glutathione peroxidase (GPX)

The GPX specific activity was estimated according to the procedure described by

Hossain and Fujita (2010)

Extraction

Extraction conditions were standardized with respect to molarities and pH of buffer to achieve maximum extraction of enzyme in leaf All the steps of extraction were carried out at 0-4°C Five hundred mg of leaf and root from control and treated plants were excised The leaf were washed with distilled water, dried with filter paper and macerated in a chilled pestle and mortar in presence of 1.0 ml of cold extraction buffer (potassium phosphate) containing 0.1 mM KCl, 1 mM ascorbate, 5 mM β-mercaptoethanol and 10% glycerol pH was adjusted

to 7.0 The homogenate was centrifuged at 11,500 x g for 15 min at 4°C The supernatant was carefully decanted and used as the crude enzyme extract

3.2.5.8 Ascorbate (AsA)

Ascorbic acid content was determined with a modification of the procedure of

Takahama and Oniki (1992)

(3-4 min) DHA was reduced by 2 mM DTT and the ascorbate content calculated by using extinction coefficient 15 mM-1 cm-1

3.2.6 Ionic contents

3.2.6.1 Sodium (Na + ) and potassium (K + )

The sodium and potassium contents were determined from the oven dried (85°C) and ground material

Digestion

Fifty mg of dried and well ground plant material was digested in 3 ml of 9:1 mixture

of concentrated H2SO4 (91-100 %) and HClO4 (50 %) by heating gently on a hot plate till the solution became colourless The solution was cooled and the volume was made up to 25 ml with distilled water

Sodium and potassium contents were determined using Flame Photometer (Elico, India) and expressed in ppm values Prior to determination of sodium and potassium contents

of tissue digested, it was calibrated for operation over concentration of 100 and range

0-1000 ppm of sodium and potassium, respectively, using graded concentration of NaCl and KCl From ppm values, Na and K contents were converted to mg g-1 DW basis

Procedure

Fifty mg of the dry ground plant material was taken in a 100 ml glass beaker along with 50 ml of the supporting electrolyte It was continuously stirred for 15 minutes with the help of magnetic stirrer The coloured plant extract so obtained was decolourised by the addition of Darco-G-60 and filtered The acid extract was brought to pH 8.2 + 0.1 with the help of NaOH by using phenolphthalein as indicator till faint pink colour was obtained This solution was titrated with N/50 AgNO3 by adding 3-4 drops of K2Cr2O4 as indicator to reddish brown end point

Calculation

Chloride = [(ml of N/50 AgNO3 used for plant extract – Blank) x N x 1000] /

volume of plant extract taken

(ii) Barium chloride: Grinded BaCl2.2H2O crystals in a mortar, until they passed through

a 20 to 30 mesh sieve, but retained on a 60 mesh sieve

(iii) Standard SO42- solutions: Dissolved 0.1815 g of reagent grade K2SO4 in one liter of

distilled water This represents 100 mg/l stock solution of SO4

2- Transferred 12-.25, 2.50, 5.0, 7.5, 10.0, 12.5 and 15.0 of the 100 mg/l SO42-, stock solution in a series of

25 ml volumetric flasks to obtain 5, 10, 20, 30, 40, 50 and 60 mg/l SO4

2-, respectively

Procedure

Five ml aliquot of the digestion was transferred to a 25 ml volumetric flask To it 1 ml

of gum acacia solution was added to make the final volume 25 ml and shaken for one min Further 1.0 g of the sieved BaCl2 crystals were added and mixture shaken for 1 min Turbidity was measured after 25 to 30 min., after adding BaCl2 crystals, on spectrophotometer using a blue filter at a wavelength of 420 nm Simultaneously, a blank (without sample) was carried out Data were expressed as mg g-1 DW

(i) TTC solution: 0.5 % in 15 % sucrose

(ii) Sodium succinate crystals

Procedure

To the TTC solution taken in a test tube, few a crystal of sodium succinate were added A few drops of this solution was put in a clean microslide Small amount of pollen were suspended in the TTC drop and cover slip was applied It was incubated at 30-35ºC for

15 minutes in dark At the end of incubation period, preparation was scored for percentage of viable pollen grain under a light microscope Ten observations per replicate and three replicates per treatment were taken for this test Percentage viability was computed from this data