THESIS Submitted in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy

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1 Mansoura University Faculty of Agriculture Agric. Botany Department EFFECT OF SOME ANTIOXIDANTS ON SEED QUALITY AND YIELD OF FABA BEAN UNDER SALINITY STRESS By MOHAMED TAHA ABD AL-RAHMAN ZALAMA B. Sc. Agricultural Science (Agronomy), Faculty of Agric., Mansoura University (1999) M. Sc. Agricultural Science (Agronomy), Faculty of Agric., Mansoura University (2007) THESIS Submitted in Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Prof. Dr. ZEIN EL-ABEDIN ABDEL- HAMID MOHAMED Prof. of Agric. Botany, Fac. of Agric., Mansoura University In (Agric. Botany) Supervisors Prof. Dr. MOHEB TAHA SAKR Prof. of Plant Physiology, Fac. of Agric., Mansoura University Prof. Dr. MAROUAH ISMAIL ATTA Chief Researcher, Seed Tech. Res. Department, Field Crops Institute 2014

2 Mansoura University Faculty of Agriculture Agric. Botany Department SUPERVISION Title of Thesis: Effect of some antioxidants on seed quality and yield of faba bean under salinity stress. The Researcher: Mohamed Taha Abd Al-Rahman Zalama Supervision Committee: No. Name Position Signature Prof. Dr. Moheb Taha Sakr Prof. Dr. Zein El-Abedin Abdel- Hamid Mohamed Prof. Dr. Marouah Ismail Atta Prof. of Plant Physiology, Faculty of Agric., Mansoura University. Prof. of Agric. Botany, Faculty of Agric., Mansoura University. Chief Researcher, Seed Tech. Res. Department, Field Crops Institute. Date of discussion: / / 2014 Head of Department Vice Dean Dean Prof. Dr. Zein El-Abedin A. Mohamed Prof. Dr. Yaser Mohamed Shabana Prof. Dr. Yaser Mokhtar El-Hadidi

3 Mansoura University Faculty of Agriculture Agric. Botany Department APPROVAL SHEET Title of Thesis: Effect of some antioxidants on seed quality and yield of faba bean under salinity stress. The Researcher: Mohamed Taha Abd Al-Rahman Zalama Supervision Committee: No. Name Position Signature Prof. Dr. Prof. of Plant Physiology, 1 Faculty of Agric., Moheb Taha Sakr Mansoura University. 2 3 Prof. Dr. Zein El-Abedin Abdel-Hamid Mohamed Prof. Dr. Marouah Ismail Atta Approval Committee: Prof. of Agric. Botany, Faculty of Agric., Mansoura University. Chief Researcher, Seed Tech. Res. Department, Field Crops Institute. No. Name Position Signature Prof. Dr. Moheb Taha Sakr Prof. Dr. Hosny Mohamed Abd El- Dayem Prof. Dr. Mahmoud Mohamed Darwish Prof. Dr. Zein El-Abedin Abdel-Hamid Mohamed Date of Discussion : / / 2014 Prof. of Plant Physiology, Faculty of Agric., Mansoura University. Prof. of Agric. Botany, Faculty of Agric. Moshtohor, Banha University. Prof. of Plant Physiology, Faculty of Agric., Mansoura University. Prof. of Agric. Botany, Faculty of Agric., Mansoura University. Head of Department Vice Dean Dean Prof. Dr. Zein El-Abedin A. Mohamed Prof. Dr. Yaser Mohamed Shabana Prof. Dr. Yaser Mokhtar El-Hadidi

4 Endorsement I certify / Mohamed Taha Abd Al- Rahman Zalama - registered for a PhD, Department of Agricultural Botany - Faculty of Agriculture - Mansoura University, doctoral thesis that modern and not published before. Title of English:- "Effect of some antioxidants on seed quality and yield of faba bean under salinity stress" Research published:- Number of research 1 Title of research Response of faba bean plants to application of some growth promoters under salinity stress conditions. Magazine or periodicals published by Search J. Plant production, Mansoura Univ., Vol. 5 (1): 79-94, 2014 The student's name Mohamed Taha Abd Al- Rahman Zalama Registered PhD, Department of Agricultural Botany

5 ACKNOWLEDGMENT First of all, I would like to express my Praise to "ALLAH" who gave me the power patience and help me to finish this work. I extend my deep thanks and sincere gratitude to Prof. Dr./Moheb Taha Sakr, Prof of Plant Physiology, Faculty of Agric. Mansoura University for his supervision, kind encouragement, scientific guidance, research facilities, critically reading the manuscript and his personal assistance, help me to right directions, offering all facilities at preparation of the manuscript and his great effort since the beginning of his work till finishing it. In fact without his help, this thesis would not have been possible. I would like to express my deep thanks to Prof. Dr./ Zein El- Abedin Abdel-Hamid Mohamed, Prof. Of Agric., Head of Botany Dept., Faculty of Agric., Mansoura University, for his supervision, continuous guidance, great support during this thesis. I deeply thank to Prof. Dr./ MAROUAH I. ATTA, Prof. of Agronomy Field Crop, Researches, Institute, Agric. Res. Center, Giza for great support, continuous encouragement, valuable directions, and providing facilities during the executing of this study. Thanks are also extended to all staff members of Botany Department, Faculty of Agriculture, Mansoura University and Research Unit Seed Technology, Mansoura for their support and encouragement during executing of this study. Lastly, greeting and faithful thanks to my beloved family that I have had the honor of them, my father soul " TAHA" I wish to him the mercy and forgiveness of God and I said to him I will always love you and think of you every step of the way. Great thanks also to my lovely mother, my lonely son Taha, my wife, my sisters and my friends for their continuous encouragement and support, which enable me to complete this work. Mohamed Taha Abd Al-Rahman Zalama

6 LIST OF CONTENTS SUBJECT CONTENTS i. INTRODUCTION. 1 ii. REVIEW OF LITERATURE. 3 A-Effect of salinity stress on: 3 - Growth parameters and yield of faba bean plants. 3 - Photosynthesis. 9 Page - Non-enzymatic antioxidants Free proline content Na + and K + content. 16 B-Effects of applying antioxidants on growth, yield observation and biochemical constituents. 18 C-Role of non-enzymatic antioxidants on alleviating and mitigation the harmful effects of salinity stress. 24 iii. MATERIALS AND METHODES Pot experiments Field experiments laboratory experiment. 43 iv. RESULTS. 45 POT EXPERIAMENT: 45 Vegetative Growth parameters of faba bean plants: 45 Yield components of faba bean plant: 66 Biochemical constituents in faba bean plant: 72 - Photosynthetic pigments Proline, ascorbic and phenols contents Na + and K + contents Na + /K + ratio Protein percentage (%). 95

7 LIST OF CONTENTS v FIELD EXPERIMENT: 98 - Growth parameters of faba bean plant Yield of faba bean plant and its components. 100 Laboratory experiment: Quality of faba bean seeds. 103 DISCUSSION. Pot experiment. 106 A - Effect of salinity stress on: Vegetative growth parameters Shoot and Root length Shoot and Root fresh and dry weights Total leaf area / plant Yield and it`s components Photosynthetic pigments Proline accumulation Activity of non-enzymatic antioxidants (Ascorbic acid and Total phenol) Na + and K + content and Na + / K + ratio. 114 B- Effect of applying non-enzymatic antioxidants on growth, yield parameters and biochemical constituents: Salicylic acid (SA) Ascorbic acid (ASA) and Tocopherol (TOCO) Humic acid (HA) Yeast extract. 118 C: Role of non-enzymatic antioxidants on alleviating and mitigation the harmful effects of salinity stress: Salicylic acid (SA) Ascorbic acid (ASA) Tocopherol (TOCO). 124

8 LIST OF CONTENTS - Humic acid (HA) Yeast extract. 126 Vi SUMMARY AND CONCLUSION Pot experiment Field experiment Laboratory experiment Recommendation. 137 vii REFERENCES viii ARABIC SUMMARY. ٧-١

9 LIST OF TABLES LIST OF TABLES No. Title Page A. Chemical analysis of yeast extract after Mahmoued (2001). 37 B. 1(a) 1(b) 1(c) 2(a) 2(b) 2(c) 3(a) Soil and Water Analysis Inst., Mansoura Lab., Agric. R. Center (ARC). Growth parameters of faba bean (Shoot length/cm) grown under salinity stress levels, applied antioxidants (presoaking) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Growth parameters of faba bean (Shoot length/cm) grown under salinity stress levels, applied antioxidants (foliar spraying) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Growth parameters of faba bean (Shoot length/cm) grown under salinity stress levels, applied antioxidants (Presoaking and Foliar spraying) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Growth parameters of faba bean (Root length/cm) grown under salinity stress levels, applied antioxidants (Presoaking) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Growth parameters of faba bean (Root length/cm) grown under salinity stress levels, applied antioxidants (Foliar spraying) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Growth parameters of faba bean (Root length/cm) grown under salinity stress levels, applied antioxidants (Presoaking and Foliar spraying) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Growth parameters of faba bean (Shoot fresh wt. g/plant) grown under salinity stress levels, applied antioxidants (Presoaking) and their interactions during the two growing seasons (2010/2011 and 2011/2012)

10 LIST OF TABLES No. Title Page Growth parameters of faba bean (Shoot fresh wt. g/plant) grown under salinity stress levels, applied antioxidants (Foliar spraying).3(b) and their interactions during the two growing seasons (2010/ and 2011/2012). 3(c) Growth parameters of faba bean (Shoot fresh wt. g/plant) grown under salinity stress levels, applied antioxidants (Presoaking and Foliar spraying) and their interactions during the two growing 55 seasons (2010/2011 and 2011/2012). 4(a) Growth parameters of faba bean (Shoot dry wt. g/plant) grown under salinity stress levels, applied antioxidants (Presoaking) and their interactions during the two growing seasons (2010/2011 and /2012). 4(b) Growth parameters of faba bean (Shoot dry wt. g/plant) grown under salinity stress levels, applied antioxidants (Foliar spraying) and their interactions during the two growing seasons (2010/ and 2011/2012). 4(c) Growth parameters of faba bean (Shoot dry wt. g/plant) grown under salinity stress levels, applied antioxidants (Presoaking and Foliar spraying) and their interactions during the two growing 58 seasons (2010/2011 and 2011/2012). 5(a) Growth parameters of faba bean (Root fresh wt. g/plant) grown under salinity stress levels, applied antioxidants (Presoaking) and their interactions during the two growing seasons (2010/2011 and /2012). 5(b) Growth parameters of faba bean (Root fresh wt. g/plant) grown under salinity stress levels, applied antioxidants (Foliar spraying) and their interactions during the two growing seasons (2010/ and 2011/2012). 5(c) Growth parameters of faba bean (Root fresh wt. g/plant) grown under salinity stress levels, applied antioxidants (Presoaking and Foliar spraying) and their interactions during the two growing 61 seasons (2010/2011 and 2011/2012). 6(a) Growth parameters of faba bean (Root dry wt. g/plant) grown under salinity stress levels, applied antioxidants (Presoaking) and their interactions during the two growing seasons (2010/2011 and 2011/2012). 62

11 LIST OF TABLES No. Title Page 6(b) 6(c) Growth parameters of faba bean (Root dry wt. g/plant) grown under salinity stress levels, applied antioxidants (Foliar spraying) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Growth parameters of faba bean (Root dry wt. g/plant) grown under salinity stress levels, applied antioxidants (Presoaking and Foliar spraying) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Growth parameters of faba bean (Total Leaf Area cm2/ plant) grown under salinity stress levels, applied antioxidants (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) and their interactions during the two growing seasons (2010/2011 and 2011/2012). No. of pods / plant as affected by salinity stress levels, applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Pods weight / plant (g) as affected by salinity stress levels, applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) and their interactions during the two growing seasons (2010/2011 and 2011/2012). Seeds weight / plant (g) as affected by salinity stress levels, applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) and their interactions during the two growing seasons (2010/2011 and 2011/2012). No. of Seeds / plant as affected by salinity stress levels, applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) and their interactions during the two growing seasons (2010/2011 and 2011/2012). 100 Seed weight / plant (g) as affected by salinity stress levels, applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) and their interactions during the two growing seasons (2010/2011 and 2011/2012)

12 LIST OF TABLES No. Title Page 13 Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on chlorophyll (A) and chlorophyll (B) content (mg/g f.wt) in leaves of faba bean plant Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on carotenoids content (mg/g. f. wt) in the shoots of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on proline content (mg/g. f. wt) of faba bean fresh leaves. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying) as well as their interactions on ascorbic acid content (mg/ 100g f. wt) of faba bean leaves. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying) as well as their interactions on total phenol content (mg/ 100g f. wt) of faba bean leaves. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on Na + and K + content (mg/g. DW) in shoots of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying) as well as their interactions on Na + and K + content (mg/g. DW) in roots of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on Na + /K + ratio in shoots and roots of faba bean plant

13 LIST OF TABLES No. Title Page Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on Protein percentage (%) of faba bean plant. Growth parameters of faba bean (Plant height/cm, Plant fresh weight/g, Plant dry weight/g, Total Leaf Area cm2/ plant) as influenced by salinity stress levels, applied antioxidants and their interactions during the growing seasons 2010/2011. Yield parameters of faba bean as (No. of pods/plant, Weight of pods/plant, No. of seeds/plant, Weight of seeds/plant and Seed yield (Ardab/fad) as influenced by salinity stress levels, applied antioxidants and their interactions during the growing seasons 2010/2011. Yield parameters of faba bean as (100 seed weight and seed yield (Ardab/fad) as influenced by salinity stress levels, applied antioxidants and their interactions during the growing seasons 2010/2011. Effect of salinity stress levels and applied antioxidants as well as their interactions on germination percentage and speed of germination index of faba bean seeds. Effect of salinity stress levels and applied antioxidants as well as their interactions on plumule/radical length (cm) and seedling vigor index of faba bean seeds

14 LIST OF FIGURES LIST OF FIGURES No. Title Page Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on chlorophyll (A) content in leaves of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on chlorophyll (B) content (mg/g f.wt) in leaves of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on carotenoids content (mg/g. f. wt) in the leaves of faba bean plant Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on proline content (mg/g. f. wt) of faba bean fresh leaves. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying) as well as their interactions on ascorbic acid content (mg/ 100g f. wt) of faba bean leaves. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying) as well as their interactions on total phenol content (mg/ 100g f. wt) of faba bean leaves. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on Na + content (mg/g. DW) in shoots of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on K + content (mg/g. DW) in shoots of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying) as well as their interactions on Na + content (mg/g. DW) in roots of faba bean plant

15 LIST OF FIGURES Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying) as well as their interactions on K + content (mg/g. DW) in roots of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on Na + /K + ratio in shoots of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on Na + /K + ratio in roots of faba bean plant. Effect of salinity stress levels and applied antioxidants as (Presoaking, Foliar spraying or Presoaking and Foliar spraying together) as well as their interactions on Protein percentage (%) of faba bean plant

16 INTRODUCTION INTRODUCTION Faba bean (Vicia faba L.) have a long tradition of cultivation in old world agriculture, being among the most ancient plants in cultivation and also among the easiest to grow. Faba bean is believed to have become part of the eastern Mediterranean diet around 6000 BC or earlier. Also, is still often grown as a cover crop to prevent erosion, because it can overwinter and because as a legume, they fix nitrogen in the soil. The whole dried seeds of faba bean contain (per 100g) 344 calories, 10.1% moisture, 1.3g fat, 59.4g total carbohydrate, 6.8g fiber, 3.0 g ash, 104mg Ca, 301mg P, 6.7mg Fe, 8mg Na, 1123mg K, 130mg b-carotene equivalent, 0.38 mg thiamine, 0.24mg riboflavin, 2.1mg niacin, and 162mg tryptophane. Flour contains: 340 calories, 12.4, % moisture, 25.5g protein, 1.5g fat, 58.8g total carbohydrate, 1.5g fiber, 1.8g ash, 66mg Ca, 354mg P, 6.3mg Fe, 0.42mg thiamine, 0.28mg riboflavin, and 2.7mg niacin. The fatty acid composition of broad bean oil has been reported as 88.6% unsaturated" (Duke, 1981). The amino acid content except for methionine is reasonably well balanced (Bond et al., 1985). In Egypt, area harvested of faba bean plants in 2005 was ( fad.) and decreased in 2011 to ( fad.), (FAO. 2013) 1. More, one of the most important factors which causes reduction of area harvested of faba bean plants is exposure of large areas of lands for salinity as well as sensitivity of faba bean to medium levels of salinity stress. According to FAO. 2008, more than 800 million hectares of used lands threw the world are affected with salt condition (saline and sodic soils). Theses genesis of lands may be natural or accelerated by using saline irrigation water (Lambers, 2003). Soil salinity threshold levels depended on a crop species, variety, developmental stage and environmental factors. The one of the most important a biotic stress factors is soil FAOSTAT FAO Statistics Division May 2013 ١

17 INTRODUCTION salinity. It causes great effect of development of growth, yields of crops (Chaparzadeh et al., 2004, Rahnama and Ebrahimzadeh, 2005) and causes great losses in yield crops (Smirnoff, 1998). Seed germination also affected by the excessive content of salt in soil solution, particularly in case of sensitive plants. Production of reactive oxygen species forms one of the biochemical response of plants to a biotic and biotic stress (Rahnama and Ebrahimzadeh, 2005). ROS also produced during physiological metabolic activity of plants (photosynthesis and respiration). The first line of defense against ROS in plants is a complex antioxidative system (Dixon and Paiva 1995, Yamasaki et al., 1997). This system consist of enzymes such as superoxide dismutase, catalase, peroxidase and low molecular compounds as ascorbate, glutathione, β-carotene, α- tocopherol or total phenol compounds (Malencic et al., 2003). Application of antioxidants is one of the most important ways to increasing salt tolerance of plants. Treatments of [Salicylic acid (SA), Ascorbic acid (ASA), Tochopherol (TOCO), Humic acid (HA) and Yeast extract] proved effective in reducing the adverse effect of salinity on growth, yield and chemical composition of faba bean plants. Because of that, the current study take place to investigate the applying of some natural antioxidants as (presoaking, foliar spraying or presoaking and spraying together) on improving the growth observation, yield and it`s components and seeds quality of faba bean, as well as reducing the harmful effects induced by salinity stress conditions

18 REVIEW OF LITERATURE REVIEW OF LITERATURE The review herein considers the previous and current studies on the influence of salinity stress ant selected antioxidants on vegetative growth characters, yield and it`s components, biochemical constituents and seed quality of faba bean (vicia faba, L) grown under salinity stress conditions. This review will be classified under the following topics: A: Effect of salinity stress on:- 1- Growth parameters and yield of faba bean plants: Salt in soil water inhibits plant growth for two reasons. First, it reduces the plant s ability to take up water, and this leads to slower growth. This is the osmotic or water-deficit effect of salinity. Second, it may enter the transpiration stream and eventually injure cells in the transpiring leaves, further reducing growth. This is the salt-specific or ion-excess effect of salinity. The two effects give rise to a two-phase growth response to salinity. The salt in the soil solution reduces leaf growth and, to a lesser extent, root growth (Munns, 2002&2003). The cellular and metabolic processes involved are in common to drought-affected plants. Neither Na + nor Cl - builds up in growing tissues at concentrations that inhibit growth: meristematic tissues are fed largely in the phloem, from which salt is effectively excluded; and rapidly elongating cells can accommodate the salt that arrives in the xylem within their expanding vacuoles. Rana Munns (2005) reported that growth response results from the toxic effect of salt inside the plant. The salt taken up by the plant concentrates in old leaves: continued transport into transpiring leaves over a long period eventually results in very high Na + and Cl - concentrations, and the leaves die. The cause of injury is probably the salt load exceeding the ability of cells to compartmentalize salts in the vacuole. Salts would then build up rapidly in the cytoplasm and inhibit enzyme activity. Alternatively, they might build up in the cell walls and dehydrate the cell. The initial growth reduction is caused by the osmotic effect of salt

19 REVIEW OF LITERATURE outside the roots, and the subsequent growth reduction is caused by the inability to prevent salt from reaching toxic levels in transpiring leaves. Stress is known to induced oxidative stress in plant tissues through the increase in reactive oxygen species. Chloroplast are the major organelles producing the reactive oxygen species (ROS) such as, the superoxide radicals (O.- 2 ), hydrogen peroxide (H 2 O 2 ) and singlet oxygen (O - ) during photosynthesis (Asada, 1992; Apel and Hirt, 2004). Greenway and Munns (1980) reported that the effect of salinity on leaf area was greater than on dry weight, as salt accumulation in the shoot occurs via transpiration stream, which is highest in old leaves killing them. Salt stress induced injuries which can occur not only due to osmotic and oxidative effects, but also toxic and nutrient deficiency effects of salinity. Ayres and Westcot (1985) indicated that salinity stress delays germination but do not appreciably reduce the final percentage of germination. However, the effect of salinity on plant growth is related to the stage of plant development at which salinity is imposed. Epstein (1985) reported that water salinity is an environmental stress factor that inhibits growth and yield of glycophytic crop plants in many regions of the world, agreement with. Pasternak (1987) indicated that salt stress condition can affect several physiological processes, from seed germination to plant development. The ability of the plant response to saline stress can be hardly explained by the fact that salinity imposes both an ionic and osmotic stress. Katerji et al. (1992) stead that the yield of the saline treatments is about 28% lower but without a significant difference between the saline treatments. The yield components indicate that the decrease in yield is not due to the effect of salinity on the reproductive stage of the beans because no significant effect is observed on the number of grains per pod and the number of pods per plant. The decrease in yield appears to be caused by the difference in weight of the grains. No difference was observed in the height and number of the plants

20 REVIEW OF LITERATURE Thus, salinity affects the water stress of the plant and its gaseous exchanges: from the flowering stage onwards systematic differences were observed between the saline treatments and its control, which lead to a decrease of about 15 to 30% in stomatal conductance, depending on the salinity level. In addition, the salinity effect on leaf area and dry matter appeared 20 to 40 days later and finally caused a decrease of about 15%. The decrease in yield of grains was about 28%, although the average soil salinity, expressed as ECe, only equaled 2.4 ds/m for the most saline treatment. Munns and Cramer (1996) indicated that there are chemical signals coming from roots in dry or saline soil that reduce leaf growth. These are commonly referred to as root signals. Abscisic acid is the obvious candidate for this signal, as it is found in xylem sap and increases after drought and salinity stress. However, there is still no conclusive proof that abscisic acid (ABA) is the only signal from the roots. Moreover, the origin of the ABA in the xylem sap is not known, for it moves readily in the phloem and recirculates from leaves to roots. Bray (1997) supported that many changes were happened in plants in response to the osmotic stress and ionic imbalance caused by salinity (Bohnert et al., 1995). In addition, the oxidative stresses were occurred to result from exposure of plants to osmotic stress that also is responsible for the damage caused to plants were grown under high concentration of NaCl. The interaction and input among these components that may finally end in plant death (Smirnoff, 1993). Lin and Kao (2000) studied that the increasing concentrations of NaCl reduce root growth. This reduction is closely correlated with the increase in H 2 O 2 level. Also, they supported that Hydrogen peroxide content was increased with increasing salinity level. Gaballah and Gomaa (2004) found that plant dry weight of faba bean was reduced under salinity stress condition mostly at 6000 ppm. It was reduced by 57.3, 62.0, 59.0, 63.9 and 67.4% in untreated samples of faba bean (Giza Blanka, Giza 674, 717,461 and 634) respectively

21 REVIEW OF LITERATURE Sakr et al. (2004) reported that salinity affects all stages of soybean growth and development, as well as yield of plants. The yield is much more depressed by salt than vegetative growth. The reduction in seed yield is largely due to a decrease in seed set, which may be attributed to a decrease in the viability of pollen or in the receptivity of the stigmatic surface or both. Demiral and Turkan (2005) resulted that the high concentrations of salt resulting from brackish groundwater, natural processes or disarrangement in irrigated agriculture result in inhabitation of plant growth and yield. Also, salinity stress affected seeds germination, is one of the most critical phases of plant life, as pointed by (Abo-Kassem, 2007), which either induces a state of dormancy at low levels or completely inhibits germination at higher levels (Iqbal et al., 2006). Yamaguchi and Blumwald (2005) Studied that salinity can affect plant physiological processes resulting in reduced growth and yield. Tolerant genotypes of plants respond to salinity stress with complex changes in their physiological and molecular status (morsy et al., 2007). Pahlavani and mirlohi (2006) reported that genetic information regarding seed germination could help to increase seedling emergence in saline soils through breeding programs. Further more, reactive oxygen species (ROS) like superoxide, hydrogen peroxide and hydroxyl radicals are generated (Wahid et al., 2007). (ROS) are highly reactive in the absence of any protective mechanism. They can hardly destroy normal metabolism through oxidative damage to essential membrane lipids, proteins and pigments (Di-Baccio et al., 2004 and Cakmak, 2005). High salinity delayed radicals emergence and decreased germination percentage (Abo-Kassem, 2007). Wilson et al. (2006) pointed that salinity treatments results in a progressive decline in plant growth. Also relatively high Na concentration may cause stimulation to the growth of salt tolerant plants by its effect on generation of turgor and thereby cell expansion (Marschner, 1995)

22 REVIEW OF LITERATURE Yaser et al. ( 2006) reported that under saline conditions, a non-uniform distribution of ions in the successive leaves within the shoots and between the leaf blade and sheath has been observed frequently. Salt treatment affects differently early growth stages of plants and has both osmotic and specific ion effects on plant growth (Dionisio-Sese and Tobita, 2000). Asin et al. (2007) emphasized that salinity stress caused significantly reduction in most vegetative growth parameters caused by the combined treatments could be due to the reduction in the cell size. Also, decrease in growth may due to drastic changes in ion relationship (Grossmann et al., 2006). More, hormonal control of cell division and elongation is evident in roots. Several studies have shown that salinity has differential effects on root elongation rates and lateral root initiation (Rubinigg et al., 2004). Salter et al. (2007) studied that increasing seawater level reduced the absorption of water leading to a drop in water content of tested plants. The inhibitory effect of seawater on growth parameters could be attributed to the osmotic effect of seawater salinity. Ahmed et al. (2008) reported that growth parameters (shoot length, fresh and dry weight) of faba bean plants generally affected by salinity stress. Control plants showed comparatively higher degree of shoot length than stressed plants by 45 and 90 days old plants. Moreover, fresh and dry weights of control plants were commonly higher than those for water stressed plants. These results may attributed to the effect of salinity stress on the water content of the leaves, as suggested by (Hu et al., 2007). Salinity stress may lower the soil water potential. Water deficit or osmotic also effect in plants might explain the reduction in plant growth (Munns, 2002). Moreover, number of pods/plant, number of seeds/plant and weight of seed yield/plant of faba bean plants were higher in control plants more than salt stressed plants. Gomaa et al. (2008) showed that the high level of salinity negatively affected shoot dry weight and leaf area irrespective of the treatment. Salinity can damage the plant through its osmotic effect (Dorais et al., 2001)

23 REVIEW OF LITERATURE Bekheta et al. (2009) improved that irrigation of faba bean seedlings with saline solutions (2,000 and 3,000 mg/l) resulted in significant reduction in shoot height and shoot fresh and dry weights compared with tap water. These findings are in agreement with those obtained in bean seedlings (Phaseolus vulgaris) by (Stoeva and Kaymakanova, 2008). Sakr and El-Metwally (2009) indicated that salinity stress affect all stages of saybean growth and yield and its components. The yield is much more depressed by salt and the highest salinity stress level (9000 mg/l) was the most effective in this regard. The depression effects of salinity on grain yield may be due to decreasing the leaf area and number per plant, resulting reduction in the supply of carbon assimilate due to decreasing the net photosynthetic rate and biomass accumulation. Abdelhamid (2010) discussed that poor quality of irrigation water may result in an increase in soil salinity. Salinity became a problem when enough salts accumulate in the root zone to negatively affect plant growth (Sumner, 1993). Consequently, faba bean plants subjected to such soil conditions took up high amounts of Na+, whereas the uptake of K+, Ca2+, and Mg2+ was considerably reduced. More, low Ca2+ /Na+ ratio in a saline medium plays a significant role in growth inhibition, in addition to causing significant changes in morphology and anatomy of plants. In addition, salinity had adverse effects on morphological parameters such as plant height, number of leaves, root length, and shoot/root weight ratio. Also, he cleared that increasing salt stress resulted in growth reduction in terms of significant reduction in plant height, branch number per plant, leaf number and leaves, dry weight per plant, pod number and pods, dry weight per plant, root, and shoot and total dry weight per plant in both salinity levels compared to control. Hameda (2011) studied that salinity stress is one of the most important a biotic stress factors limiting plant growth and productivity. High concentration of both NaCl and Na 2 So 4 hardly reduced growth in length of shoot and root and D. W. of all plant parts. More, crop growth, green matter and dry matter components were significantly affected with increasing water salinity. As the salinity was increased the green matter production ranged from g/pot to 7306 g/pot and dry matter production ranged too from g/pot to g/pot

24 REVIEW OF LITERATURE High exogenous salt concentration affect seed germination, water deficit, cause ion imbalance of cellular ions resulting in ion toxicity and osmotic stress. Sakr et al. (2013) studied that to control the level of ROS and protect cells under stress conditions, plant tissues contain several enzymes scavenging ROS (SOD, CAT, peroxidases and glutathione peroxidase), and network of low molecular mass antioxidants (ascorbate, glutathione, phenolic compounds and tocopherols). Moreover that, the decrease in growth due to salinity may be attributed to an increase in respiration rate resulting from higher energy requirements. The reduction in shoot and root dry weight accumulation was in proportion to the external concentration of salt. This reduction might be attributed to; a decrease in either leaf number and leaf area of soybean. And/or a decrease in Co 2 uptake in leaves mainly because NaCl treatment, decrease stomatal conductance and consequently less Co 2 is available for carboxylation reaction in the photosynthesis apparatus. 2- Photosynthesis: Price and Hendry (1991) noticed that during water stress produced by salt stress, produced of reactive oxygen species (ROS) and reduction of chloroplast stromal volume. Jonas et al. (1992) noted that dry matter accumulation significantly decreased with increasing salinity under stress conditions where photosynthesis was reduced by closure of the stomata. Leung et al. (1994) reported that photosynthesis is a major key in metabolic pathway in plants, although it's an important target for the salt stress. While the abscisic acid produced in response to salt stress decreases turgor in guard cells and decrease the CO 2 available for photosynthesis. During salt stress, as well as water deficit, the concentration of CO2 in chloroplasts decreases because of a reduction in stomatal conductance, in spite of the apparent stability of CO2 concentration in intercellular spaces. Also, reduction in photosynthetic carbon assimilation was due to reduced stomatal conductance (Brugnoli and Lauteri, 1991)

25 REVIEW OF LITERATURE Allen (1995) reported that ROS can be generated due to salinity stress in the chloroplast by direct transfer of excitation energy from chlorophyll pigments to produce singlet oxygen O -, or by univalent oxygen reduction at photosystem I. (Foyer et al., 1994). Garg et al. (1998) reported that, in most crop species there is considerable reduction in chlorophyll content due to water deficit. Also, chlorophyll content of leaves decreased in general under salt stress and the oldest leaves start to develop chlorosis and fall with prolonged periods of salt stress as reported by (Agastian et al., 2000). Raza et al. (2006) reported that salt stress induces a great reduction in photosynthesis, this reduction depends on photosynthesizing tissue (leaf area) and photosynthetic pigments (Dubey, 2005). Eraslan et al. (2007) suggested that Carotenoids might play a great role as a free radical scavenger. Therefore, increasing of carotenoids induced by salinity stress could enhance plant capacity to reduce the damage caused by ROS, which in turn increased chlorophyll content of such plants. Ahmed et al. (2008) stead that control plants showed the highest chlorophyll a+b content at 90 days age, while stressed plants exhibited commonly reduction in chlorophyll content at 90 days age. Moreover, stressed plants showed higher values of carotenoid content than the control faba bean plants at 90 days age. Khosravinejad and Faboondia (2008) studied that the increase in oxidative stress caused by salinity stress could be resulted to increase of sodium concentration in plant tissue, which causes deterioration in chloroplast structure and an associate lose in chlorophyll. Stoeva and Kaymakanova (2008) reported that treating bean seedlings (Phaseolus vulgaris) with different concentrations of saline solutions resulted in reduction of the photosynthetic pigments. Yildirim et al. (2008) studied that The unchanged effect of salinity stress on chlorophyll content in some faba bean genotypes may be attributed to its high level of antioxidant content and may be responded as protection of these genotypes against

26 REVIEW OF LITERATURE chlorophyll degradation. In this agreement Sairam et al., (2002) reported that chlorophyll content is one of the important indicators of salt tolerance in crop plants. Ashraf (2009) studied that the generation of (ROS) can be specially high, when plants are exposed to salinity stress (Athar et al., 2008). ROS causes chlorophyll deterioration and membrane lipid peroxidation. So, accumulation of lipid peroxidation and chlorophyll retention are two oxidative stress indicators, which used as tested tools for determining salt tolerance in plants (Yildirim et al., 2008). Bekheta et al. (2009) showed that irrigation of faba bean seedlings with saline solutions (2000 and/or 3000 ppm) decreased significantly the photosynthetic pigments of faba bean leaves (chl. a, chl. b & carotenoids) and this reduction increased with increasing NaCl concentration. The inhibition of photosynthetic pigments of bean leaves irrigated with NaCl may be attributed to the inhibition of assimilate translocation. Cornella and Maria (2011) observed that salt stress decrease the assimilatory pigments content (with 20% for chl a, 11.8% for chl b and with 37.5% for carotenoids). Similar results were obtained by Kaydan et al. (2007), they observed that under the influence of salinity the photosynthetic pigments greatly decreased. 3- Non enzymatic antioxidants: Tanaka et al. (1994) induced that, the highest salt concentration normally impair the cellular electron transport within the different subcellular compartments and lead to the generation of reactive oxygen species (ROS) which led to enhancing antioxidants system against oxidative stress induced by salinity stress. Mckersie et al. (1996) reported that antioxidants have activity; there have been increasing interest in oxygen- containing free radicals in biological systems and their implied roles as causative agents in the etiology of variety of chronic disorders. Farther more, many of studies reported that the ratio of damages induced by oxidative cellular in plants exposed to a biotic stress is controlled by the capacity of antioxidative systems

27 REVIEW OF LITERATURE Sharma et al. (1996) demonstrated that SA is required for O 3 tolerance by maintaining the cellular redox state and allowing defense responses. While using an Arabidopsis genotype that accumulated high levels of SA, it was shown that SA activates an oxidative burst and a cell death pathway leading to O 3 sensitivity. SA plays an important role in the plant sensitivity to different types of a-biotic stress, as decided by (Rao and Davis, 1999). Mullineaux and Creissen (1997) reported that plants with the higher accumulation of antioxidants have a greater resistance to such oxidative damages. Shirasu et al. (1997) reported that salicylic acid play an essential role in the defense response against the pathogen attack in many plant species. Also, SA mediates the oxidative burst the leads to cell damage in the hypersensitive response and acts as a single for the development of the systemic acquired resistance. Many studies supported an activated role of SA in modulating the plant response to several a-biotic stresses, as reported by (Yalpani et al., 1994 and Senaratna et al., 2000). Dat et al. (1998) studied that treating seedling with exogenous SA increased their thermo tolerance and heat acclimation. Although, a known effect of SA is to participate in the increase of the temperature in thermogenic plants, as reported by (Raskin et al., 1987). Noctor and Foyer (1998) suggested that applying vitamin C increased growth parameters and enhancing development of plants, which led to regulation of the cell cycle, hydroxylation of proline and many basic processes of plant growth and development. Asada (1999) recorded that α-tocopherol is low molecular weight lipophilic antioxidants, which protect membrane from oxidative damage. Positive correlation between α-tocopherol and shoot or root growth in the two grass species, as pointed by (Zhang et al., 2000). Janda et al. (1999) recorded that pretreatment with SA increased antioxidant enzymes, which play an important role to induced chilling tolerance

28 REVIEW OF LITERATURE Matamoros et al. (1999) reported that accumulation of vitamin c found at concentration of 1-2 Mm in legume nodules. It's positively correlated with nodule effectiveness, (Dalton et al., 1993). Vitamin C has been proposed for a long time as a biological antioxidant. It existed in rather high concentrations in many cellular environments, such as the stroma of chloroplasts where in level is M. Moreover, vitamin C posses significant antioxidant activity in many qualitative studies. Evidenced by the example of 10 3 M. vitamin C inhibited the photo-oxidation by illuminated spinach chloroplast. Vitamin C reduces two equivalents of O - 2 produce H 2 O 2, derivative dehydro-ascorbic acid and also reacts with O 2 at a relatively fast rate. It plays an essential metabolic role for the operation of the ASC-GSH pathways. It also has significant effects that do not require the presence of APX. ASC can directly scavenge ROS and reduce ferric Lb and Lb. Arrigoni and De Tullio (2000) induced that the exogenous application of ascorbic acid increased the endogenous ascorbic acid. They concluded that, ascorbic acid plays an essential role as an antioxidant and protect the plant during oxidative damage by scavenging free radicals and active oxygen that are generated during salt stress conditions. Further more that, the inductive role played by vit. C in overcoming the detrimental effects of seawater and enhancing the capacity of treated plants to scavenge the free radicals produced as a result of salinity caused by seawater. This was associated by improvement of plant growth, water stress, carotenoids, endogenous vit. C and antioxidant enzymes activities. These indicated that, plants treatment with vit. C triggers some unknown physiological processes which subsequently lead to improvement of seed germination, growth and development of treated. Bellaire et al. (2000) studied that adaptation to high NaCl levels involves an increase in the antioxidant capacity such as ascorbic acid, salicylic acid and tocopherol of the cell to detoxify reactive oxygen species (ROS). Hernadez et al. (2000) indicated that antioxidant glutathione (GSH) content and its soluble compounds were involved in the salt tolerance. The increase in glutathione content due to vitamin C treatment enhanced salt tolerance of faba bean, may be hardly due to

29 REVIEW OF LITERATURE increased GSH synthesis or/and decreased rates of degradation, as associated by (Noctor and Foyer, 1998). Mittler (2002) induced that plants synthesis different types of defense system composed of non-enzymatic antioxidants, such as ascorbic acid and enzymatic antioxidants. Scavenging system having potential to put out (ROS) in stress tolerance plants, in agreement with those reported by (Sairam et al., 2005 and Koca et al., 2007). Pastori and Foyer (2002) reported that such environmental stress may cause an imbalance between antioxidants defense and the amount of activated oxygen species (AOS) resulting in oxidative stress. Silvana et al. (2003) reported that the adaptation to high NaCl (salt tolerance) by vitamin C involves an increase in the antioxidants capacity (GSH) of the cell to detoxify reactive oxygen species through both enzymatic and non enzymatic reactions. Also, in agreement with pervious studies, the adaptation to salt tolerance involves an increase in betaine and antioxidants (glutathione). In contrast salt stress produces increment in proline content, as reported by (Smironff, 1993). Jaleel et al. (2006) studied that to scavenge ROS; plants posses specific mechanisms, such as activation of antioxidant enzymes and non enzymatic antioxidant such as, carotenoids and ascorbic acid (Mittler, 2002). Salt tolerance positively correlated with a more efficient antioxidant system (Noreen and Ashraf, 2008). Sarwat and El-Sherif (2007) reported that differences in faba bean genotypes due to salinity stress could be observed through variations in the criteria of osmotic solutes (soluble carbohydrate, protein and total free amino acids). The increases in free amino acids content under salinity stress may be related to the breakdown of protein. The ability of some faba bean genotypes to absorb more water from the saline soil was linked with its ability to stimulate the synthesis of such osmotic solutes (Kerepesi and Galiba, 2000)