Effect of Salicylic acid, Lead and their interaction on growth and development of (Phaseolus vulgaris L.) plants

Table of Contents

Supervisor's Certification

Examination Committee Certification

Summary

List of Tables

List of Figures

List of Appendices

List of abbreviations

1 Introduction

2 Review of Literature
2.1 Effects of SA
2.1.1 Historical discovery of SA
2.1.2 Effect of SA on vegetative growth
2.1.3 Effect of SA on yield characteristics
2.1.4 Effect of SA on chemical characteristics
2.2 Effects of Pb
2.2.1 Effect of Pb on vegetative growth
2.2.2 Effect of Pb on yield characteristics
2.2.3 Effect of Pb on chemical characteristics
2.3 Interaction effects of SA and Pb

3 Materials and methods
3.1 Location
3.2 Preparation of the soil and pots
3.3 Preparation of SA solutions
3.4 Preparation of Pb solutions
3.5 Description of glass house experiments
3.5.1 Experiment (1): Effect of different concentrations of SA on growth and development of bean plant
3.5.2 Experiment (2): Effect of SA applied by different methods on growth and development of bean plant
3.5.3 Experiment (3): Effect of different concentrations of Pb on growth and development of bean plant
3.5.4 Experiment(4):Interaction effect of different concentrations of SA and Pb on growth and development of bean plant
3.6 Statistical analysis
3.7 Experimental parameters
3.7.1 Vegetative growth parameters
3.7.1.1 Plant height (cm)
3.7.1.2 Number of branches.plant-[1]
3.7.1.3 Number of leaves.plant-[1]
3.7.1.4 Leaf area (cm[2]).plant-[1]
3.7.1.5 Dry weight of shoot system (g.plant-[1])
3.7.2 Yield components
3.7.2.1 Number of pods.plant-[1]
3.7.2.2 Number of seeds.pod-[1]
3.7.2.3 Dry weight of 100 seeds (g)
3.7.3 Chemical analysis of leaves and seeds
3.7.3.1 Chlorophyll content (mg.g-[1] fresh weight)
3.7.3.2 Water content (g.plant-[1])
3.7.3.3 Total protein (%)
3.7.3.4 Proline determination ( µg.g-[1] fresh weight)
3.7.3.5 Total phenol determination (µg.g-[1] fresh weight)
3.7.3.6 Total carbohydrates (%)
3.7.4 Mineral nutrient contents in dry weight
3.7.4.1 Total nitrogen ( mg.g-[1] )
3.7.4.2 Total phosphorus (mg.g-[1])
3.7.4.3 Total potassium (K+) and sodium (Na+) (mg.g-[1])
3.7.4.4 Total calcium, magnesium (mg.g-[1]), zinc ,manganese and iron contents (µg.g-[1])
3.7.4.5 Total Pb contents in plant and soil (µg.g-[1])
3.7.5 Preparation of stomatal slide

4 Results
4.1 Experiment (1): Effect of different concentrations of SA on growth and development of bean plant
4.1.1 Vegetative growth characteristics
4.1.1.1 Plant height (cm)
4.1.1.2 Number of leaves .plant-[1]
4.1.1.3 Number of branches .plant-[1]
4.1.1.4 Dry weight of shoot system (g.plant-[1])
4.1.1.5 Leaf area (cm[2]).plant-[1]
4.1.2 Yield characteristics
4.1.2.1 Number of pods .plant-[1]
4.1.2.2 Number of seeds .pod -[1]
4.1.2.3 Dry weight of 100 seeds (g)
4.1.3 Chemical characteristics of seeds and leaves
4.1.3.1 Biochemical contents
4.1.3.1.1 Chlorophyll contents of leaves (mg.g-[1]fresh weight )
4.1.3.1.2 Total protein contents of leaves (%)
4.1.3.1.3 Total carbohydrate content of seeds (%)
4.1.3.2 Mineral nutrient contents of leaves
4.1.3.2.1 Total nitrogen (mg.g-[1])
4.1.3.2.2 Total phosphorus (mg.g-[1])
4.1.3.2.3 Total potassium (mg.g-[1])
4.1.3.2.4 Total calcium (mg.g-[1])
4.1.3.2.5 Total sodium (mg.g-[1])
4.1.3.2.6 Total magnesium (mg.g-[1])
4.1.3.2.7 Total manganese (µg.g-[1])
4.2 Experiment(2): Effect of different methods of SA application on growth and development of bean plant
4.2.1 Vegetative growth characteristics
4.2.1.1 Plant height (cm)
4.2.1.2 Number of leaves .plant-[1]
4.2.1.3 Number of branches .plant-[1]
4.2.1.4 Dry weight of shoot system (g.plant-[1])
4.2.2 Yield characteristics
4.2.2.1 Number of pods. plant -[1]
4.2.2.2 Number of seeds .plant-[1]
4.2.2.3 Dry weight of 100 seeds (g)
4.2.3 Chemical characteristics of seeds and leaves
4.2.3.1 Biochemical contents
4.2.3.1.1 Chlorophyll contents of leaves (mg.g-[1]fresh weight)
4.2.3.1.2 Total protein contents of leaves (%)
4.2.3.1.3 Total carbohydrate of seeds (%)
4.2.3.2 Mineral nutrient contents of leaves
4.2.3.2.1 Total nitrogen(mg.g-[1])
4.2.3.2.2 Total phosphorus(mg.g-[1])
4.2.3.2.3 Total potassium (mg.g-[1])
4.2.3.2.4 Total calcium(mg.g-[1])
4.2.3.2.5 Total sodium (mg.g-[1])
4.2.3.2.6 Total magnesium(mg.g-[1])
4.2.3.2.7 Total manganese (µg.g-[1])
4.3 Experiment (3): Effect of different concentrations of Pb on growth and development of bean plant
4.3.1 Vegetative growth characteristics
4.3.1.1 Plant height (cm)
4.3.1.2 Number of leaves .plant -[1]
4.3.1.3 Number of branches .plant-[1]
4.3.1.4 Dry weight of shoot system (g.plant[1])
4.3.2 Yield characteristics
4.3.2.1 Number of pods.plant-[1]
4.3.2.2 Number of seeds.pod-[1]
4.3.2.3 Dry weight of 100 seeds(g)
4.3.3 Chemical characteristics of leaves and seeds
4.3.3.1 Biochemical contents
4.3.3.1.1 Chlorophyll contents of leaves (mg.g-[1] fresh weight)
4.3.3.1.2 Water contents of shoot system (g.plant-[1])
4.3.3.1.3 Total protein contents of leaves (%)
4.3.3.1.4 Total carbohydrate contents of seeds (%)
4.3.3.1.5 Proline contents of leaves (µg.g-[1] fresh weight)
4.3.3.1.6 Total phenol content of leaves ( µg.g-[1] fresh weight)
4.3.3.2 Mineral nutrient contents of leaves
4.3.3.2.1 Total nitrogen (mg.g-[1])
4.3.3.2.2 Total phosphorus (mg.g-[1])
4.3.3.2.3 Total potassium and sodium (mg.g-[1])
4.3.3.2.4 Total calcium (mg.g-[1])
4.3.3.2.5 Total magnesium (mg.g-[1]) and manganese (µg.g-[1])
4.3.3.2.6 Total iron and zinc (µg.g-[1])
4.3.3.2.7 Total lead content (µg.g-[1])
4.3.4 Stomatal characteristics
4.3.4.1 Number of closed stomata .unit of area -[1]
4.3.4.2 Total number of stomata .unit of area -[1]
4.4 Experiment (4): Interaction effects of SA and Pb on growth and development of bean plant
4.4.1 Vegetative growth characteristics
4.4.1.1 Plant height (cm)
4.4.1.2 Number of leaves .plant-[1]
4.4.1.3 Number of branches .plant-[1]
4.4.1.4 Dry weight of shoot system (g.plant-[1])
4.4.2 Yield characteristics
4.4.2.1 Number of pods.plant-[1]
4.4.2.2 Number of seeds .pod-[1]
4.4.2.3 Dry weight of 100 seeds (g)
4.4.3 Chemical characteristics of seeds and leaves
4.4.3.1 Biochemical contents
4.4.3.1.1 Chlorophyll contents of leaves (mg.g-[1]fresh weight)
4.4.3.1.2 Water content of shoot system (g.plant-[1])
4.4.3.1.3 Total protein content of leaves (%)
4.4.3.1.4 Total carbohydrate content of seeds (%)
4.4.3.1.5 Proline content of leaves (µg.g-[1] fresh weight)
4.4.3.1.6 Total phenol contents of leaves (µg.g-[1] fresh weight)
4.4.3.2 Mineral nutrient contents of leaves
4.4.3.2.1 Total nitrogen (mg.g-[1])
4.4.3.2.2 Total phosphorus (mg.g-[1])
4.4.3.2.3 Total potassium (mg.g-[1])
4.4.3.2.4 Total calcium (mg.g-[1])
4.4.3.2.4 Total sodium (mg.g-[1])
4.4.3.2.5 Total magnesium (mg.g-[1])
4.4.3.2.6 Total manganese (µg.g-[1])
4.4.3.2.7 Total zinc (µg.g-[1])
4.4.3.2.8 Total iron (µg.g-[1])
4.4.3.2.9 Total lead content (µg.g-[1])

5 Discussion

6 Conclusions and recommendations

7 References

8 Appendices

Summary in Arabic

Summary in Kurdish

Supervisor’s Certification

I certify that this thesis was prepared under my supervision at the Department of Biology, College of Science, University of Salahaddin-Erbil and hereby recommend it to be accepted in partial fulfillment of the requirements for the Master Degree of Science in Biology.

Signature:

Supervisor

Prof. Dr. Karim Salih Abdul

Date: / 9 / 2013

Chairman’s Certification

In view of the available recommendation I forward this thesis for debate by the examining committee.

Signature:

Dr. Yassen A.R. Goran

Head of Biology Department

Date: / 9 / 2013

Examination Committee Certification

We (the Examination Committee) certify that we have read this thesis and as the examining committee examined the student (Halala Rahman Qadir) in its contents and what is related to it and our opinion it meets the standing of a thesis for the degree of Master of Science in Biology.

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Dedication

To

- My dear parents with my respect

- My brothers and sisters

- My friends with best regards

Halala

Acknowledgement

First, I thank Allah for his blessing that help me to perform this work, blessing and peace upon Prophet Mohammad (Allah’s peace and pray be upon him).

I would like to express my special thanks of gratitude of my supervisor (Prof. Dr. Karim Salih Abdul) for his patients in guiding me throughout the duration of the project and for his perseverance in proofreading my manuscript.

I would like to express my sincere gratitude to presidency of Salahaddin University. I would also like to thank Dean of College of Science (Dr. Nadhum J. Ismaeil) and Head of Biology department, (Dr. Yassen A.R. Goran).

My grateful thanks to (Dr. Akram O. Ismail) for his help during data analysis of the study, and to (Dr. Abdulghani Sarmami) for his scientific suggestions.

My special thanks to (Dr. Muhammad Q. Khurshid, Dr. Mohammad Salim, Mr. Badir Qadir, Trifa Dhahir, Tara Muhammad).

Special thanks are due to Mr. Sarkawt for his help in information about SPSS program.

I would like to express my greatest gratitude to the people who have helped and supported me throughout my project especially my friends (Guan, Faeroz, Balqes, Karwan, Nehayat, Beriwan).

I also thank my parents and family for their support. Without them, this would not be possible.

Finally, I thank all for their tremendous contribution and support both morally and financially towards the completion of this project.

Halala

Summary

This study consisted of four experiments conducted in the greenhouse of Biology Department in the College of Science- University of Salahaddin- Erbil, during 18/4/ 2012 to 28/7/2012. The first experiment consisted of foliar spray of different concentrations of Salicylic acid (0, 50, 100, 200 and 400ppm), the second experiment included the application of different methods of Salicylic acid (250ppm) (presoaking seeds, foliar spray, injection, and soil application). The third experiment consisted of different concentrations of Lead (0, 5, 10, 15 and 20ppm) as soil irrigation, and the fourth experiment consisted of interaction application of Salicylic acid and Lead on vegetative growth, yield and chemical components of common bean. The results obtained were analyzed statistically using Complete Randomized Design (C.R.D) for the first three experiments and Factorial Complete Randomized Design (Factorial C.R.D) for the fourth experiment, with four replications for each treatment. Comparison of means were carried out by using Duncan’s Multiple Range Test at the probability of (0.05) for vegetative and yield parameters and (0.01) for the chemical constituents.

The main results are summarized in follows:

Effects of Salicylic acid

Effect of different concentrations of foliar spray of Salicylic acid and it’s application at different methods (presoaking of seeds, foliar spray, injection, and soil application), in the first and second experiment, due to significant increases of vegetative growth characteristics like plant height, number of leaves.plant-[1], number of branches.plant-[1], dry weight of shoot system, leaf area.plant-[1], and yield component such as dry weight of 100 seeds. The chemical constituents were also increased, such as chlorophyll a content of leaves, total potassium, sodium, magnesium and manganese contents of leaves as compared with control plants.

Effects of Lead

Soil irrigation with different concentrations of lead in experiment three, significantly decreased the vegetative growth characteristics such as plant height, number of leaves.plant-[1], number of branches.plant-[1], dry weight of shoot system. The chemical components such as chlorophyll a, b and total chlorophyll contents, total potassium, calcium and manganese content of leaves were also decreased, while there were significant increases in proline, total lead contents of leaves and the number of closed stomata while decreased total number of stomata. unit of area-[1] in upper epidermis as compared with control.

Interaction effects of Salicylic acid and Lead

It was found that SA significantly decreased the negative effects of Pb on the vegetative growth characteristics such as plant height, number of leaves.plant-[1], number of branches.plant-[1], dry weight of shoot system, and yield components such as number of seeds.pod-[1], dry weight of 100 seeds. The negative effects of Pb on chemical components such as chlorophyll a and total chlorophyll contents, total protein content of leaves and total sodium by the effect of salicylic acid.

List of Tables

1 Some physical and chemical properties of the soil used in the experiments.

2 Some physical and chemical properties of SA

3 Effect of different concentrations of SA on plant height at different stages of growth

4 Effect of different concentrations of SA on number of leaves at different stages of growth

5 Effect of different concentrations of SA on number of branches at different stages of growth

6 Effect of different concentrations of SA on chlorophyll content of leaves (mg.g-[1] fresh weight)

7 Effect of different concentrations of SA on some mineral nutrient content of leaves

8 Effect of SA applied at different methods on plant height at different stages of growth

9 Effect of SA applied at different methods on number of leaves at different stages of growth

10 Effect of SA applied at different methods on number of branches at different stages of growth

11 Effect of SA applied at different methods on chlorophyll content of leaves (mg.g- [1]fresh weight)

12 Effect of SA applied at different methods on some mineral nutrient content of leaves

13 Effect of different concentrations of Pb on plant height at different stages of growth

14 Effect of different concentrations of Pb on number of leaves at different stages of growth

15 Effect of different concentrations of Pb on number of branches at different stages of growth

16 Effect of different concentrations of Pb on chlorophyll content of leaves (mg.g-[1] fresh weight)

17 Effect of different concentrations of Pb on some biochemical contents of leaves and seeds

18 Effect of different concentrations of Pb on some mineral nutrient content of leaves

19 Effect of Pb concentrations on the number of stomata in upper epidermis

20 Interaction effects of SA and Pb on plant height at different stages of growth

21 Interaction effects of SA and Pb on number of leaves at different stages of growth

22 Interaction effects of SA and Pb on number of branches at different stages of growth

23 Interaction effects of SA and Pn on yield characteristics

24 Interaction effects of SA and Pb on chlorophyll content of leaves (mg.g-[1]fresh weight)

25 Interaction effects of SA and Pb on some biochemical content of leaves and seeds

26 Interaction effects of SA and Pb on some mineral nutrient content of leaves

List of figures

1 Effect of different concentrations of SA on dry weight of shoot system

2 Effect of different concentrations of SA on leaf area

3 Effect of different concentrations of SA on number of pods.plant-[1]

4 Effect of different concentrations of SA on number of seeds.pod-[1]

5 Effect of different concentrations of SA on dry weight of 100 seeds.

6 Effect of different concentration of SA on total protein content of leaves

7 Effect of different concentration of SA on carbohydrate content of seeds

8 Effect of SA applied at different methods on dry weight of shoot system

9 Effect of SA applied at different methods on number of pods.plant-[1]

10 Effect of SA applied at different methods on number of seeds.pod-[1]

11 Effect of SA applied at different methods on dry weight of 100 seeds

12 Effect of SA applied at different methods on leaf protein content

13 Effect of SA applied at different methods on seed carbohydrate content

14 Effect of different concentrations of Pb on dry weight of shoot system

15 Effect of different concentrations of Pb on number of pods.plant-[1]

16 Effect of different concentrations of Pb on number of seeds.pods-[1].

17 Effect of different concentrations of Pb on dry weight of 100 seeds.

18 Effect of different concentrations of Pb on water content of shoot system

19 Upper epidermis from the new leaf of bean plant after 30 days from Pb application. 40X of a compound microscope was taken with VGA camera

20 Interaction effects of SA and Pb on dry weight of shoot system

21 Interaction effects of SA and Pb on water content of shoot system.

List of Appendices

1 ANOVA for the effect of different concentrations of SA on plant height after 45 days from application (Exp. 1)

2 ANOVA for the effect of different concentrations of SA on dry weight of shoot system (g.palnt-[1]) (Exp.1)

3 ANOVA for the effect of different concentrations of SA on chlorophyll a content (mg.g-[1] fresh weight) (Exp. 1)

4 ANOVA for the effect of SA application at different methods on phosphorus content (mg.g-[1]) (Exp. 2).

5 ANOVA for the effect of SA application at different methods on protein content (%) (Exp. 2).

6 ANOVA for the effect of different concentrations of Pb on proline content (µg.g-[1]) (Exp. 3)

7 ANOVA for the effect of different concentrations of Pb on water content of shoot system (g. plant -[1]) (Exp. 3)

8 ANOVA for the effect of different concentrations of Pb on total Calcium content (mg.g-[1]) (Exp. 3)

9 AVOVA for the interaction effects of SA and Pb on total protein content of le a ves (%)

List of Abbreviations

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Chapter One Introduction

1. Introduction

The Common Bean (Phaseolus vulgaris L.) is a herbaceous annual plant species, belongs to fabaceae family domesticated independently in ancient Mesoamerica and now grown worldwide both for dry beans and as green bean. Among major food legumes the Common Bean is the third most important worldwide, superseded only by Soybean [ Glycine max (L.) Merr.], and Peanut (Arachis hypogaea L.) (Zeka, 2007). The common bean grown for their tender and green pods, or seeds as an important source of protein (20–25%) and complex carbohydrates (50–60%) (Martiniz et al., 2011), dietary fiber, minerals such as iron, zinc, calcium, and phosphorus and vitamins (Carvalho et al., 2012).

Phenolics are compounds possessing one or more aromatic rings with one or more hydroxyl groups (Michalak, 2006). Salicylic acid (SA) is an endogenous growth regulator of phenolic nature, which participates in the regulation of physiological processes in plants. SA has been found to play a key role in the regulation of plant growth, development, and interaction with other factors and in the responses to environmental stresses. Furthermore, it’s role is evident in seed germination, plant growth, fruit yield, glycolysis, flowering, ion uptake and transport, photosynthetic rate, stomatal conductance and transpiration …etc (Sadeghipour and Aghaei, 2012 A, B). It is implicated in hardening response to a biotic stressors and mediates some positive acclimination response to a biotic stress, such as heavy metals, herbicides, low temperature, salinity (Mohsenzadeh et al., 2011), and osmotic stress. Along these stresses, toxic heavy metal stress is an emerging and more effective stress for major crops (Bhardwaj et al., 2009).

Heavy metals are defined as that group of elements that have specific weights higher than about 5g/cm[3]. Such as number of them (Co, Fe, Mn, Mo, Ni, Zn, Cu) are essential micronutrients and are required for normal growth and take part in redox reactions, electron transfers and other important metabolic processes in plants. Metals which are considered nonessential (Pb, Cd, Cr, Hg etc.) are potentially highly toxic for plants (Michalak, 2006). Excessive concentrations of trace elements (Cd, Co, Cr, Hg, Mn, Ni, Pb and Zn) are toxic and lead to growth inhibition, decrease in biomass and death of the plant (Zenk, 1996). Heavy metals inhibit physiological processes such as respiration, photosynthesis, cell elongation, plant-water relationship, N-metabolism and mineral nutrition (Zornoza et al., 2002).

Lead (Pb), is one of the heaviest non-essential metals released into the natural environment from a range of anthropogenic activities (Ekmekçi et al., 2009). Upon release, it gets accumulated in the soil and causes toxicity to plants and animals (Kaur et al., 2010). Significant increases in the Pb content of cultivated soils have been observed near urban and industrial areas where it tends to accumulate in the surface ground layer (Hussain et al., 2006). Pb contamination in soils not only aroused the changes of soil microorganism and it’s activities and resulted in soil fertility deterioration, but also directly affected the change of physiological indices and, furthermore, resulted in yield decline (Majer et al., 2002). Pb is taken up by plants mainly through the root system and partly in minor amounts through the leaves and trichomes. Pb toxicity in plant causes such problems as reduction in growth and production levels, yellowing of young leaves, reduction in absorption of essential elements such as iron and reduction in the rate of photosynthesis. The Pb contamination in the plant environment is known to affect the seed germination, seedling growth, photosynthesis, respiration, nitrate assimilation and other processes (Singh et al., 2003).

This study was carried out to investigate the effect of each of SA and Pb on growth and development of common bean plant under normal conditions, further more the ability of SA to alleviating of Pb toxicity was studied through the interaction effects of different concentrations of each of them.

Chapter Two Review of Literature

2. Review of Literature

The review of literature includes the results of studies reported regarding the effects of SA and Pb on vegetative growth, yield characteristics and chemical components. Then the interaction studies of both factors were reviewed regarding the same topics.

2.1 Effects of SA

2.1.1 Historical discovery of SA

The name of salicylic acid (SA) is derived from the word Salix , the scientific name of the willow tree. Both American Indians and the ancient Greeks knew that the leaves and bark of willow trees could be used as a painkiller and antipyretic. Salicylic acid have been known to possess medicinal properties since the 5th century B.C., when Hippocrates prescribed salicylate-rich willow leaf and bark for pain relief during childbirth (Hayat and Ahmed, 2007).

Salicylates from plant sources have been used in medicines since antiquity. In 1828 in Munich it was isolated for the first time as small amount of salicin; the glucoside of salicyl alcohol, from willow bar. Ten years later Raffaele Piria named it SA, from the Latin word Salix for willow tree. The first commercial production of synthetic SA began in Germany in 1874 (Popova et al., 1997).

Aspirin, a close analog of salicylic acid, was introduced by the Bayer Company in 1898 and rapidly became one of the most popular pharmaceutical preparations in the world. During the 19th century many compounds belonging to the group of salicylates were isolated from a variety of plants (Weissman, 1991).

The first indication for a physiological effect of SA was the discovery of flower-inducing action and bud formation in tobacco cell cultures. The stimulatory effect of SA on flowering was latter demonstrated in other plant species and this was ground for suggesting that SA functions as an endogenous regulator of flowering (Popova et al., 1997).

2.1.2 Effect of SA on vegetative growth

Gutierrez-Coronado et al., (1998 ) showed that application of SA on soybean significantly increased the growth of shoots, roots, root length, fresh and dry weight of root, SA increased root growth up to 100% were measured in the field. Shakirova et al., (2000) found that foliar application of SA caused significant increases in seedling growth and yield of spring wheat. Khan et al., (2003) observed that foliar application of SA (10-[5],10-[3]mol.L-[1]) on soybean, has not affected plant height, root length, root dry mass, while leaf area, shoot dry mass increased by treatments with 10-[5] mol.L-[1] SA as compared with control plants. Sandoval-Yepiz (2004) studied the effect of SA (10-[6], 10-[8], 10-[10]M) on Tagetes erecta , it was found that SA increased the number of leaves formed, and leaf area had values over 10% of that of the control. Similar values were recorded for the diameter of the rosette plant beside the floral characters; the biomass of the shoot was significantly affected by the application of low concentration of SA. Naz (2006) demonstrated that the application of different concentrations of SA (50, 100, 200, 300, 400, 500, 600 ppm) on (Arachis hypogaea L.) increased plant height, number of branches, leaf area, leaf dry weight, stem dry weight, total dry weight, absolute growth rate, crop growth rate, leaf area index, leaf area ratio, leaf area duration increased with increased concentrations of SA. Hegazi and El-shraiy (2007) indicated that the spray application of SA (10-[2], 10-[3]M) on common bean (Phaseolus vulgaris L.) generally had a positive effect on vegetative growth parameters (plant height, leaves number, shoots and roots fresh and dry weight) as compared with controls. In their study Chandra et al., (2007) indicated that SA (0.02%) affected on four genotypes of cowpea (Vigna unguiculata L.) significantly increased leaf length, dry weight of plant, while plant height was not affected as compared to control. El-shraiy and Hegazi (2009) demonstrated in their study on pea plant (Pisum sativum L.) ASA significantly increased plant height, number of leaves, and plant fresh and dry weight as compared with control plants. Orabi et al., ( 2012) reported that the response of cucumber (Cucumber sativus L.) to different concentrations of SA (2 and 4 mM), led to significant increases in plant height, number of leaves, fresh and dry weights of leaves or stems and leaf area.plant-[1], as compared to the corresponding controls. Aftab et al., (2010) studied the effect of SA (0.25, 0.50, 1.0 mM) on growth and photosynthesis on (Artemisia annua L.); they showed that SA significantly enhanced the growth represented by shoot height and shoot weight as compared to the control plants. Devi et al., (2011) mentioned that foliar spray of SA (50ppm) led to increase of plant height, number of branches, dry weight.plant-[1], and leaf area index of soybean plant. Khandaker et al., (2011) noted the response of red amaranthus (Amaranthus tricolor L.) plants to foliar application of SA (10-[3],10-[4],10-[5]M), showed that SA increased plant height, stem length, leaf number, leaf size, root length as compared to untreated plants. Farahabakhsh and Saiid ( 2011) studied the effect of different concentrations of SA (100 and 200 ppm) on maize (Zea mays L.) in green house conditions, they observed that SA caused significant increases in shoot dry weight, stem length, number of leaves, leaf area. Sadeghipour and Aghaei (2012A) indicated that plant height, leaf area index was significantly increased by presoaking seeds of SA (0.5 mM) in (Phaseolus vulgaris L.) Farouk et al., (2012) showed that SA (50, 100 mg.l-[1]) especially 100mg.l-[1] as presoaking seed and shoot spraying increased plant height, fresh and dry weight of shoot, leaf number per plant, leaf area per plant as compared to controls in pea plants. SA treatment resulted in increasing the plant height, number of branches, and plant dry weight in (Pisum sativum L.). Ali and Mahmoud (2013) showed the effect of different concentrations of SA (0, 50, 100, 150 ppm) on mungbean (Vigna radiata L.) by foliar spray caused significant increase in vegetative growth parameters such as plant height and number of branches as compared with control plants.

From the studies mentioned above, it could be concluded that SA has different effects on plant height, number of leaves, number of branches, leaf area, etc… depending on plant species, concentrations and developmental stages.

2.1.3 Effect of SA on yield characteristics

Exogenous application of SA to different crops has been shown to elicit yield and yield components. An increase in number of pods and yield has been found in (Vigna radiata L.) (Singh and Kaur, 1980). Sujatha (2001) showed that application of SA at 100 ppm concentration increased number of pods plant-[1], number of seeds pod-[1], seed weight plant-[1] and seed yield ha-[1] of mungbean (Vinga radiata L.). Naz (2006) observed that foliar application of SA had different effects on ground nut (Arachis hypogaea L.), it has increased pod number.plant-[1], pod dry weight, pod yield as compared to the control plants. Murtaza et al., (2007) found that number of pods, number of seeds, weight of 100 seeds, biological yield, and green pod yield increased with SA application at 10-[5],10-[4]M by three different modes (seed treatment, seed treatment plus foliar spray and foliar spray) in pea plant. Hegazi and El-Shraiy (2007) studied the response of common bean (Phaseolus vulgaris L.) to different concentrations of SA (10-[2], 10-[3]M) under green house conditions; they showed that the application of SA at 10-[3]M was the best treatment for yield parameters (pod number, pod fresh and dry weight) as compared with control plants. El-shraiy and Hegazi (2009) in their study indicated to the effect of ASA on Pea (Pisum sativum L.) and found that ASA enhanced significant increase in 1000seed weight, pod parameters (length of pod, pods number per plant, seed number per pod, pods fresh and dry weights) as comparing with controls. Khan et al., (2010) showed that application of SA (0.1, 0.5, 1.0 mM) on mungbean (Vigna radiata L.), increased pod length, pod number, seed number, seed yield in comparison to the control. Devi et al., (2011) studied the response of soybean to SA, the data showed that foliar spraying of SA increased number of pods.plant-[1], seeds. pod-[1], dry weight of 100 seeds, seed yield, straw yield and harvest index as compared to the untreated plants. Hashmi et al., (2012) studied the exogenous application of SA on Fennel (Foeniculum vulgare Mill), the plants were sprayed three times at doses (10-[5], 10[4], 10-[3] M), which significantly increased seed yield and yield attributes, number of fruits, 1000 seed weight. SA applied at 10-[4]M resulted in the highest seed yield, as compared with control. Sadeghipour and Aghaei ( 2012B) studied the response of common bean (Phaseolus vulgaris L.) to seed soaking of SA (0.25, 0.5, 0.75, 1 mM), the data showed that SA increased number of pods plant-[1], number of seeds pod-[1], 100 seed weight and seed yield. In addition, foliar sprays of SA in pea increased pod length, pod diameter, pod weight, seed weight.fresh pod-[1], 1000seed weight, number of fresh seeds.pod-[1], green pod yield, number of fresh seed. (El-Hak et al., 2012). Ali and Muhmoud (2013) showed that foliar spray of SA (50, 100, 150 ppm ), significantly increased the number of pods plant-[1], number of seeds pod-[1], 1000 seeds weight, seed weight plant-[1] and seed yield ha-[1] as compared with control, the superiority in this respect to the high SA concentration (150ppm) which gives the highest value for these traits .

It could be concluded from this review that SA positively affected yield characteristics in most cases such as number of pods per plant, number of seeds per pod, dry weight of seeds and seed yield.

2.1.4 Effect of SA on chemical characteristics

Khodary (2004) mentioned that application of SA (10-[2] M) on maize plant, led to significant increase in chlorophyll a, b, carotenoids, soluble sugars, polysaccharides and total carbohydrates as compared with control plants. Naz (2006) found that SA with different concentrations had affected on chlorophyll content, total phenol content, total sugar content, tannin content, polyphenol reductase activity of (Arachis hypogaea L.), these parameters increased with SA application as compared with controls. Chandra et al., (2007) observed the effect of SA on biochemical attribute in cow pea (Vigna unguiculata L.), they showed that SA significantly increased the peroxidase activity, protein content, total sugars as compared with control in both flowering stage and seed setting stage. El-shraiy and Hegazi (2009) studied the response of pea (Pisum sativum L.) to ASA (10, 20 ppm) and showed that ASA significantly increased total soluble protein, phenol, proline, total soluble sugar, total soluble carbohydrate, reduced sugar, non-reduced sugar as compared to control plants. Maity and Bera (2009) reported the effect of foliar application of SA on biochemical contents of green gram (Vigna radiata L.), once at pre-flowering stage and second at flowering stage of the crop, SA significantly increased chlorophyll a, b and total chlorophyll content, reducing and non reducing sugars, starch and soluble protein content in the leaves as compared to control plants. Unlu et al., (2009) reported that the application of SA (0.25, 0.50, 0.75, 1.0 mM) on cowpea (Vigna unguiculata L.walp), led to increased chlorophyll a, b and total chlorophyll, while 1.0mM decreased them. Shahba et al., (2010) studied the effect of SA (0.5, 1.0, 1.5 mM) on tomato (Lycopersicum esculentum Mill), the data showed that SA increased leaf sugar, root sugar and leaf protein at different SA concentrations. Khan et al., (2010) noticed that the application of SA (0.1,0.5,1.0 mM) on mungbean palnt (Vigna radiata L.) significantly decreased the content of Na+ and Cl-, and enhanced N, P, K and Ca content in comparison to the control plants. Fahad and Bano (2012) showed the response of maize (Zea mays L.) to foliar application of SA, the data showed that SA has no significant effect on chlorophyll a and carotenoids, protein and sugar contents however, chlorophyll b was significantly decreased as compared to the control plants. Hashmi et al., (2012) indicated that application of SA (10-[5], 10-[4], 10-[3]M) on fennel (Foeniculum vulgare L.), the results showed that SA at (10-[4]M) significantly increased chlorophyll a, b, total chlorophyll, carotenoid contents, nitrate reductase activity, carbonic anhydrase activity, leaf-N,-P, and-K contents. Sadeghipour and Aghaei (2012C) mentioned that the application of seed soaking of SA on (Phaseolus vulgaris L.), led to increased total chlorophyll contents as compared with control plants.

From the previous review it could be concluded that SA affect some chemical characteristics of the leaves and seeds, which differ by plant species, concentrations and developmental stages, for the studied plants.

2.2 Effects of Pb

Lead is one of the most toxic and frequently encountered metal. Lead continues to be used widely in many industrial processes and occurs as a contaminant in all environmental compartments (soils, water, the atmosphere, and living organisms). The effect of lead depends on the concentration, type of salts and plant species involved. Though effects are more pronounced at higher concentrations and durations, in some cases, lower concentrations might stimulate metabolic processes. This metal impairs plant growth, root elongation, seed germination, seedling development, transpiration, chlorophyll production, lamellar organization in the chloroplast, and cell division (Sharma and Dubey, 2005; Pourrut et al., 2011), photosynthesis, plant water status, mineral nutrition, and enzymatic activities (Patra et al., 2004).

2.2.1 Effect of Pb on vegetative growth

Gupta et al., (2006) noticed that treatments with 9, 10, 11 mg.l-[1] of Pb(NO3)2 caused significant reduction in plant height, fresh and dry weight in black gram. Mahmud and Salh (2007) studied the effect of Pb (1.00mM.kg-[1]) soil on vegetative growth of wheat, the data showed that Pb decreased shoot and root dry weight, leaf area, relative water content in leaf tissue, while increased shoot/root dry weight as compared to control plants. Piechalak et al., (2008) investigated the effects of Pb on bean plants ( Phaseolus vulgaris L. cv. Zlota Saxa), that exposed to Pb(NO3)2 (0.1, 0.5, 1mM) , the results showed an inhibition of root elongation, decrease in the number of root hairs, the production of fresh mass, and partial inhibition of growth of aboveground parts. Kibria et al., (2009) reported the effect of different levels of Pb (20, 40, 60, 80, 100mg.kg-[1]) on growth of (Amaranthus gangeticus L.) and (Amaranthus oleracea L.), they showed that dry weight of shoot and root of A. gangeticus and A. oleracea were significantly affected by Pb application. Kabir et al., (2010) studied the effect of Pb (5, 10, 15, 20, and 25 μmol.l-[1] ) on Thespesia populnea L., the data showed that there is significant decrease on seedling and root length, plant circumference and seedling dry weight, while Pb treatment at 10–25 μmol.l-[1] produced significant decrease on shoot length, number of leaves and leaf area as compared with controls. Ahmed et al., (2011) studied the response of (Zea mays L.) to different concentrations of Pb (0.01, 0.1, 1.0 mg.L-[1]), they observed that length and fresh and dry weight of shoot and root decreased by Pb and higher Pb levels which also decreased photosynthetic rate, water use efficiency, but increased transpiration rate as a result of increasing the stomatal conductance. Azad et al., ( 2011) studied the toxic effect of Pb on growth and some biochemical and Ionic parameters on Sunflower (Helianthus annuus L.) seedlings, the data showed that Pb treatment dramatically inhibited the accumulation of both shoot and root biomass and consequently, decreased their dry weight. Kadhim (2011) referred to the effect of Pb (2.5,5, and 10 mM ) on mung bean (Phaseolus aureus Roxb), the data showed that Pb significantly decreased the stem length, root length, leaf area, dry weight of stem, roots and leaves as compared with control plants. Sinhal et al., (2011) estimated that Pb at doses (19, 20, 21mg.l-[1]) caused reduction in plant height, fresh and dry weight of pigeon pea plant. Borah and Devi (2012) studied the response of (Pisum sativum L.) to different concentrations of Pb, and showed that there is decrease in plant height and the total number of leaflets in all treated plants as compared with controls. Aghaz et al., (2012) studied the response of dill (Anethum graveolens ) to different concentrations of Pb (300, 600µM) at green house conditions, the results showed that there was a significant effect of Pb stress on total dry weight, shoot dry weight and height, average relative growth rate, absolute growth rate, net assimilation growth rate, except leaf area duration, and relative growth rate which were not affected by Pb stress.

From studies mentioned above, it could be concluded that Lead has different effects on vegetative growth which differ by plant species, concentrations and developmental stages.

2.2.2 Effect of Pb on yield characteristics

Hussain et al., (2006) studied the effect of Pb (20, 40 mg.L-[1]) on yield components of two Mush bean cultivars (Vigna mungo L.), they showed the decline in number of pods plant-[1], number of seeds .pod-[1], weight of 100seeds, seed yield.plant-[1] as compared to the untreated plants. Alzandi (2012) studied the response of pea (Pisum sativum L.) cultivars (little marvel, perfection and Alderman) to Pb (0,1mM) application to soil, the data showed that yield.pot-[1] and seed number.plant-[1], decreased with Pb while seed number pod-[1] was not affected, the percentage of seed weight were similar in both cultivars (little marvel and Alderman), while Perfection pea plant cultivar recorded the highest values as compared to controls. Fatoba et al., (2012) studied the toxic effects of Pb (0, 10, 20, 30 and 40ppm) on growth and productivity of (Arachis hypogaea L.) and (Glycine max L.), the data showed that Pb concentration in G. max at 20ppm decreased number of pod per plant and number of seeds per plant while significant decreases was recorded in A. hypogaea at 10ppm regarding number of seeds plant-[1].

From the previous review, it is obvious that Pb has negative effect on the number of pods plant-[1], number of seeds pod-[1], dry weight of 100 seeds, and seed yields in general.

2.2.3 Effect of Pb on chemical characteristics

Sengar and Pandey(1996) studied the response of (Pisum sativum L.) to Pb (0.01 to 1.0 mM), they showed that Pb decreased the chlorophyll contents of leaves. Parys et al ., (1998) observed the effect of Pb (1, 5 mmol.dm-[3]) on (Pisum sativum L.), the results showed that higher concentrations of Pb decreased photosynthetic and transpiration rate while increased respiration rate as compared with control. Seregin et al., (2004) studied the response of Zea mays to Pb, lead exposure decreased the concentration of divalent cations (Zn+[2], Mn+[2], Mg+[2], Ca+[2], and Fe+) in leaves of Z. mays . Zengin and Munzurogu (2005) demonstrated the effect of Pb concentration (1.5, 2.0, 2.5 mM) on Bean (Phaseolus vulgaris L.), they observed that Pb concentration caused significant reduction in total chlorophyll while increased retinol, ascorbic acid contents and amino acid proline as compared to the controls. Hussain et al., (2006) referred to the effect of Pb (20, 40 mg.l-[1]) on photosynthetic pigments on Vigna mungo L. The results showed that chlorophylls a, b and total chlorophyll were reduced significantly by Pb stress as compared with control plants. Mahmud and Salh (2007) obtained increase in leaf proline contents as a result of treatment with Pb (1.00 mM) on wheat plant. Ahmed et al., ( 2008) observed the effect of Pb concentarions (20, 50mg.L-[1]) on two mung bean [ Vigna radiata (L.) Wilczek] cultivars (Mung-1 & Mung-6), the results showed that Pb concentration caused significant inhibition of photosynthetic and transpiration rates and stomatal conductance as compared to the control plants. John et al., (2008) studied the response of (Lemna polyrizha L.) to different concentrations of Pb (10, 20, 30, 40 mg.l-[1]), the data showed decline in chlorophyll a and b, soluble protein contents, while proline increased as compared to the controls; lower concentration of Pb increased soluble sugar content however higher concentration decreased soluble sugar content. Bhardwaj et al., (2009) reported the response of (Phaseolus vulgaris L.) to different concentrations of Pb (2, 4, 6, 8 g.Kg-[1]), they showed that under the Pb stress the level of photosynthetic pigments decreased, chlorophyll was more effected as compared to carotenoids, soluble carbohydrate contents, soluble sugar, starch, soluble protein contents also decreased with increasing concentration of Pb, but total free amino acids increased as in comparison to control. Sharaf et al., (2009) studied the effect of Pb (100ppm) on biochemical contents of Bean and Lupin plants, the results showed that significant decreases in photosynthetic pigments, soluble carbohydrates, proteins and activities of certain enzymes of both broad bean and Lupin plants. Kibria et al., (2009) studied the effects of Pb on growth and nutrient uptake of Amaranthus ganeticus L. and Amaranthus oleracea L., the data showed that Pb application in soil significantly decreased N and P concentration in shoots as well as Ca, Zn and Mn in both shoots and roots of A. ganeticus. P, K and Fe in roots of A. ganeticus increased with increasing rates of Pb. The contents of P, Fe and Mn in shoots and Ca, Zn and Mn in roots of A. oleracea decreased with increased rates of Pb application. On the other hand, an increase of N, K and Zn concentration in shoots and K and Fe concentration in roots of A.oleracea were observed that Pb application in soil significantly increased Mg concentration in both shoots and roots of A. gangeticus and A. oleracea. Hamid et al., (2010) studied the physiological response of (Phaseolus vulgaris L.) to different Pb concentrations, in which the data showed that there is a significant decreases of total chlorophyll, total protein, carbohydrate content, total phenolic content, nucleic acid contents as compared with control . Azad et al., (2011) reported the response of the toxic effect of Pb on Sunflower (Helianthus annus L.) they observed that decline in total chlorophyll ,Ca+[2], K+ in leaves, root and shoot proline, enzyme activities in leaves as compared with control. Kadhim (2011) observed the effect of Pb (2.5,5, and 10 mM) on mung bean, he showed that all concentrations of Pb caused increasing in chlorophyll content except at 5mM of Pb which decreased chlorophyll content significantly, and Pb at 2.5mM increased the proline and decreased protein content significantly. Borah and Devi, (2012) studied response of (Pisum sativum L.) to different Pb concentrations; they showed the decrease in chlorophyll a, b and total chlorophyll, while proline content of leaves was increased as compared to the control plants.

From the previous review mentioned above it is concluded that Pb has different effects on chemical components of plant species which may differ according to concentrations applied.

2.3 Interaction effects of SA and Pb

It seems that a limited number of studies are available in literature regarding the interaction effects of SA and Pb, which are referred in the following paragraphs.

Mishra and Choudhuri, (1997) found that SA application at a concentration of 0.1 or 0.2 mM reduced the inhibitory effect of Pb+[2] on seedling growth of two rice (Oryza sativa L.) cultivars, SA increased the fresh and dry mass of shoots and roots in both cultivars under Pb stress conditions. Jazi et al., (2011) refereed to the response of (Brassica napus Var.Okapi) to combination effect of two levels of SA ( 5,10µM) and seven levels of Pb (0.25, 0.5, 0.75, 1, 1.5, 2 mM), the results showed that increasing Pb concentrations reduced root and shoot length, leaf area, root and shoot dry weight, root and shoot fresh weight, specific leaf area and leaf weight ratio, application of SA significantly increased these traits, although specific leaf weight and leaf water content were significantly increased with an increase in the concentration of Pb. Tavakoli et al.,( 2011) observed that effect of SA ( 1, 5 and 10 µM) and Pb ( 0.005, 0.01 and 0.015 M) significantly increased the plant height, number of leaves, leaf area, fresh and dry weight as compared with those treated with Pb only in eggplants. Ratushnyak et al., (2012) studied the response of (Pisum sativum L.) to combination effects of Pb (0.25mg.l-[1]) and SA( 10-[4])M ,the data observed that all variants (Pb alone , SA alone and their combinations) increased plant height, number of leaves, number of tendrils without detect any statistically significant changes in leaf length and width .

Sinha et al., (1994) studied the combination effect of SA and Pb (5, 10, and 20 mM) in (Zea mays L.) on the inhibition of nitrate reductase activity by Pb in maize leaves, SA increased nitrogenous reductase activity in the absence of Pb, the results showed that SA decreased the inhibitory effect of the Pb, which was related to its concentration. The experiments demonstrated that the inhibitory effect of Pb on enzyme activity could be antagonized by SA. Mishra and Choudhuri (1999) studies the effect of exogenous application of SA (100µM) alleviating the effect of Pb (10µM) on two cultivars of rice (Oryza sativia L.) Ranta and IR36, it was found that SA ameliorated the increased leakage of electrolysis, injury index and the content of malondealhehyde caused by Pb stress, Pb decreased H2O2 contents while application of SA increased H2O2 in presence of Pb, SA ameliorated the damaging action of Pb on membranes. Jing et al., (2007) studied the response of rice (Oryza sativia L.) to exogenous application of SA and Pb, they showed that chlorophyll content in both SA pre-treated and non SA pre-treated seedlings significantly decreased in accordance with increasing Pb concentrations, while accumulation of H2O2 contents in leaves were increased in Pb exposed seedlings and also H2O2 contents significantly increased in leaves of SA pre-treated seedlings as compared to control plants, the H2O2 levels in leaves induced by SA pre-treatment were different along with the Pb concentrations.

From these previous limited studies it was found that SA decreased the damaging action of Pb on vegetative growth and chemical characteristics. However, as far as we are aware no published work is available about the interaction effect of SA and Pb on yield characteristics, which is investigated in this study.

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