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Vol 22, No. 10;Oct 2015

Physiological, biochemical and molecular responses of wheat (Triticum aestivum L.) to abiotic stress Osman Gamal1, 2* and Quronfuahl Afnan2 1- Agricultural Genetic Engineering Research Institute (AGERI), Giza, Egypt. 2- Biology Department, Faculty of Applied Sciences, Umm Al-Qura University, Makkah, Saudi Arabia. *Corresponding author: email address: [email protected]; Fax: +966 2 5270000 Ext. 3233. T:00966530760365 Abstract This investigation was carried out on two Egyptian cultivars of Triticum aestivum L. namely: Sakha93 and Giza168. The Plants were grown in hydroponic solution and treated by ion-deficient cultures under osmotic potential and Pb concentrations. The results indicated that: The effect of osmotic potential and lead was variable on the length of roots and shoots. .Under the lower Osmotic Potential (Ψs) and Pb concentration, the chlorophyll content in all plants was increased. Conversely, the chl. a / b ratio was decreased. Generally, the amount of soluble sugars in both shoot and root were increased under low s and presence of Pb element. Therefore, the s had main role on soluble sugars of all plant organs and the (s × Pb) factor had the secondary role. The decreased s and Pb concentration led to an increase in free amino acids of roots, particularly in Sakha93. The same was true in case of shoot, but with increased Pb concentration. The content of soluble proteins was variable among cultivars, where more increased in Sakha93 than Giza 168. The total soluble proteins were increased in response to low s and Pb, except in roots of Giza168, which were increased under high Pb concentration. In addition, the s had the predominant role on soluble proteins. Keywords: Wheat; Lead; Salt stress. Introduction Salinity, drought and presence of heavy metals such as lead prevailing in arid and semi – arid habitats are among the common abiotic stresses that adversely affect growth, photosynthetic efficiency and yield of crops. Excess salinity causes osmotic stress, ion imbalance and ion toxicity, which induced disturbance of plant metabolism. In addition, lead contamination, especially in rural areas induces disturbance in ion uptake by plants and toxicity. However, plants have evolved a cultivar of protective mechanisms to allow them to cope with these unfavorable environmental conditions for survival and growth. These mechanisms included the accumulation of ions and osmolytes (Osman et al., 2004; Youssef, 2009: Munshi Abdulla and Osman Gamal 2010). Salinity is one of the most severe environmental factors that may impair crop productivity. Salt stress is one of the major

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stresses especially in arid and semi-arid regions and can severely limit plant growth and productivity (Osman et al.,2007Koca et al., 2007: Abouseadaa et al., 2015).Therefore, soil salinity affects on plants by three major ways: (a) osmotic stress and physiological drought, decreasing water availability;(b)toxicity, particularly with Na + and Cl− ions; and (c) alteration of cellular ionic balance, i.e., mineral deficiency (Kirst, 1989; Hopkins, 1999;Osman 2012 Osman et al., 2013). The ability of plants to survive under high salt conditions is important for the ecological distribution of plant species and agriculture in semi-arid, arid, and salinized regions. This variable trait is dependent on many factors including plant species. Hence, plant tolerance to salt stress depends on a complex of interrelated systems ensuring plant adaptation on metabolic and gene levels including ionic homeostasis, synthesis of osmolytes, compartmentation of toxic ions, and structures preventing the generation of reactive oxygen species (ROS)(Flowers and Colmer, 2008). On the other hand, in many arid and semi-arid areas, the toxic effects of heavy metals have caused a considerable reduction in crop yield. Among the heavy metals, lead is the most common heavy metal causing environmental pollution and toxicities on soil fertility. Lead is not an essential element for plants, but most of plants absorb it when it is present in their environment, especially in rural areas when the soil is polluted by automotive exhaust and in fields contaminated with fertilizers containing heavy metal impurities. (Lamhamdi et al., 2013; Abdelkalik et al., 2012; El-gharead 2012; Osman et al., 2014). Consequently, lead can cause a broad range of physiological and biochemical dysfunctions on seed germination, plant growth, water status, nitrate assimilation and followed by impaired plant metabolism (Seregin and Kosevnikova, 2008). Although, lead transport from plant roots to shoots is usually limited (Huang and Cunningham, 1996), photosynthesis is especially affected by lead exposure chlorophyll contents, photosynthetic rate and CO2 assimilation are strongly decreased (Bazzaz et al., 1975). Also, Ca +2, Fe+2 and Zn+2 levels decrease in the root tips after lead exposure (Eun et al., 2002). The heavy metal uptake by different species of crops differs significantly based on their genetic features (Peris et al., 2007).Therefore, the toxic effects of heavy metals in different crops may also differ significantly (Le´on et al., 2002). It has been demonstrated that increasing salinities in soil can improve heavy metals mobilization and promote the metals uptake by crops (Usman et al., 2005). The ability of a plant to take up significant quantities of Pb depends upon its concentration in soil and its bioavailability. The aim of this research study is to understand the strategies of Triticum aestivum L.cultivars (Sakha93, and Giza168) to overcome the osmotic potential (salinity), lead and their interaction stresses. MATERIALS AND METHODS The investigated plants included two cultivars of Triticuma estivum L. were grown in wooden trays containing sawdust suitable for germination of seeds. Crop Science Department- Agricultural Research Center- Dokki- Giza- Egypt supplied two cultivars (Sakha93 and Giza168) of experimental seeds. Plant preparation 146

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In the preliminary germination test, a control experiment of the untreated seeds was carried out for comparing both the rate and the amount of germination. Glass Petri-dishes (11 cm diameter) were used for germination tests. Each dish contained ten seeds conveniently spaced over chemically pure filter paper. The filter paper which served as an embedding medium for germinating seeds was kept visibly moist during the test and addition of 15 ml of distilled water was enough to keep the filter paper visibly moist during the test. Preliminary germination tests performed before experimentation indicated a high germination percentage, reaching about 100% in these seeds. Ten days after seed germination, healthy seedlings were transferred to grow in growth chamber at optimum germination conditions (at 25˚C) in full strength hydroponic cultures Hoagland and Arnon (1950)which were contained in three replicates of plastic pots (3 individuals/pot). The cultures were kept covered during the experimental periods to prevent direct evaporation in incubator with air circulation under light condition (supplied by 60 watt incandescent bulbs, yielding 1500 - 2000 lux at culture level just about the compensation point). The cultures were constantly aerated with humid air introduced pumped through fine capillary tubes. The culture solution was periodically replaced by draining through siphoning tubes kept in place throughout the experimental period. Salinity Levels (Osmotic Potential, Ψs) and Lead (Pb) in the Culture Solution Thirty-day-old plants were transferred into pure distilled water culture (expressing deficiency in macro- and micro- nutrient elements). The water content of each pot (replicate) was treated with solutions of (NaCl + CaCl2) in concentrations that yield different osmotic potentials (Ψs) and Pb in the culture solution: Ψs levels were chosen at 0 (control −0.3, −0.7 and −1.0 MPa. The concentrations of NaCl and CaCl2 in solutions prepared are based on the calculations explained by El-Sharkawi, (1968). Solutions having different water potentials with Pb element as Pb (NO3)2, were prepared by dissolving certain amounts of NaCl + CaCl2 in Pb solution. The treatment solutions prepared thus are of certain levels of treatment combinations. For each cultivar, another series of Pb solutions (0, 2, 5 and 10 ppm) at the same different levels of osmotic water potential (Ψs + Pb) were prepared (i.e. each treatment had three replicates). Statistical Data Treatments Statistical inference necessary to evaluate the significance of effects and the relative roles of the single factors: lead, Ψs and their interaction in the total response to different treatment combination included analyses of variance (F. values) coefficient of determination η2, and a simple linear correlation coefficient (r.) respectively (Ostle, 1963). The latter (η2) is a statistic used to evaluate the relative role (share) of single factors, as well as their mutual interactions in contributing to the total effect of treatment (combination) usually expressed as a percentage or fraction (El-Sharkawi and Springuel, 1977). These analyses were computerized by using the SPSS program.

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Plant extracts analysis: At the end of experimentation (37day-old plants), average lengths of both root and shoot were measured, and average fresh weights of roots and shoots were immediately recorded. For extraction, it is important to freeze the tissues in liquid nitrogen immediately after detaching the tissues from the plants and grind the tissues into powder with a mortar. A known weight of powder sample was rapidly blended with 10 cm³ of ice cold distilled water The suspension was quantitatively transferred to centrifuge tubes and decanting the residue with distilled water. Centrifugation at 7000 rpm was carried out for 15 min. After the centrifugation, the supernatant was then transferred to 25 ml. Erlenmeyer flask and the supernatant was kept in deep freeze until analysis (El-Sharkawi et al., 2012). Measurement of chlorophyll content: At the same time, chlorophyll a and chlorophyll b were extracted from the leaves of Triticum cultivars by using 85% acetone and determined by using spectrophotometer according to Lichtenthaler (1987) as follows : Ch a = 12.25 A664 - 2.79 A648 , Ch. b = 21.50 A648 - 5.1 A66 SDS-PAGE of plantlet protein method Total plantlet proteins were extracted by grinded of o.5 g of healthy plantlet on liquid nitrogen in 0.2 MTris pH 8, 2% w/v SDS, 10% sucrose and 1% BME. Proteins were separated by SDS-PAGE according to Laemmli, 1970. Gel slab was scanned using gel proanalyzer ver. 3.3 (Media Cypermetics 93-97). The presence or absence of each band was treated as binary character in a data matrix i. e. coded 1 and 0 respectively Data were statistically analyzed by using gel Doc 2000 BioRad system. The supernatant containing total cellular protein was loaded onto 10% (w/v) SDSPAGE gels and run at 45 mA for 1 hour, then fixed and stained with Coomassie brilliant blue (Laemmli, 1970). Result Effect of salinity (s) and Pb on the chlorophyll pigments (a, b, a/b): In the studied plants the changes of chlorophyll a, chlorophyll b and chlorophyll a/b ratio under the effect of s and Pb were shown (Table 1). Chlorophyll content (Chl.): Apparently, the Chl.a consent in all plants was produced a higher values than the Chl.b content. The maximum values (3.5, 3.4 & 2.4 mg.g-1fresh weight) were recorded in all wheat cultivars (Giza168, Sakha93) respectively at lowest s (-1.0 MPa) in the absence of Pb (Table 1). Whereas, the minimum Chl.a contents ( 0.9, 1.4 and 0.7 mg.g-1 fresh weight, respectively) were existed in unstressed plants at low Pb concentration (2ppm). At the same Pb concentrations, the Chl.a content 148

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of cultivar sakha93 was increased at s(-0.3) MPa. Commonly, the presence of Pb were induced the Chl.a content in plants under high water potential and vice versa . The behavior of Chl.b content in plants was similar to Chl.a (Table 1). In Giza168, a high Chl.b contents (2.1 & 1.2 mg.g-1fresh weight ) were recorded at s(-1.0)MPa in the absence of Pb. The same was true in case of cultivar Sakha93 plants. Whereas, the treated plants of Sakha93 with Pb 2ppm and s (-0.3)MPa increased the Chl.b content. Meanwhile, the Chl.b in Giza168. Cultivars was the lowest under Pb (2ppm) and s(0 MPa), the absence of Pb at the same s level produced a low Chl.b content in Sakha93, as well as the same was existedat s(-0.7) MPa.The chlorophyll content (Chl.a+ Chl.b) in the three cultivars exhibited a high values under lowest s level (-1.0 MPa ) in the absence of Pb, likewise at Pb = 2ppm with s( - 0.3)MPa in case of Sakha93. The minimum values ( 1.4 ; 2.2 & 1.0 mg.g -1 fresh weight ) of Chl. content In Giza168 and Sakha93, respectively was found under low Pb (2ppm) concentration with non-stressed plants (Table 1). Table (1): ANOVA test showed the effect of Salinity, lead and their interaction on the, chlorophyll content and chlorophyll a/b ratio of investigated Triticum aestivum L. cultivars.

Cultivar Content

Sakha93

Giza168

Source of variance

F

ɳ²

F

ɳ²

Pb

2.853

0.142

1.739

0.120

s

5.622**

0.693

5.364**

0.798

Pb ×s

3.045*

0.164

1.172

0.082

Pb

3.212*

0.135

2.593

0.143

s

14.842**

0.642

8.271**

0.740

Pb ×s

6.354**

0.223

2.050

0.117

Pb

4.687*

0.153

2.065

0.095

s

10.505**

0.525

7.849**

0.601

Pb ×s

15.242**

0.322

9.692**

0.304

Chl.a

Chl.b

Chl.a+b

D.F

Pb =3

Salinity =3

Pb ×

Salinty =15 *Significant at P < 0.05 level **Significant at P < 0.01 level Effect of salinity (s) and Pb on the chlorophyll pigments (a, b, a/b): In the studied plants the changes of chlorophyll a, chlorophyll b and chlorophyll a/b ratio under the effect of s and Pb were shown(Table 2)

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The chlorophyll a/b ratio in wheat leaves of tested three cultivars were varied (Table 2). The Chl. a/b of fresh leaves ranges from 1.66 to 2.23 in Giza168 and from 1.47 to 2.16 in Sakha93. The proportion of Chl.a to Chl.b greatly depends on the Chl. content of the cultivar under differents and Pb levels. Therefore, the decreased Chl. a/b ratio corresponding to the relatively high Chl.b content at ‫ــ‬0.3 MPa and Pb 2 ppm Sakha93 plants and shifted to low s( -1.0MPa ) in the absence of Pb. Whereas, the high Chl.a/b ratio was detected in unstressed Giza168 plants at Pb= 0ppm(control). Plant was shifted to lowest s at different Pb levels. This reflect the interactive effects of both s and Pb on the Chl.a and Chl.b formation in the studied Triticum cultivars. The effects of s, Pb and the interaction (Pb×s) on the Chl.a, Chl.b and Chl. a/b ratio in Sakha93 were significant except that the Pb on the Chl.a. In Giza168, the effect of s was significant on Chl.a, Chl. B and Chl. a/b ratio, as well as the effect of the (Pb × s) interaction on the Chl. a/b ratio (Table 2).The effect of s was dominant on Chl.a,b contents and a/b ratio( 2= 0.69, 0.64 and 0.52 for Sakha93, respectively),(2= 0.75 , 0.76 respectively) and(2= 0.80 , 0.74 and 0.60 for Giza168, respectively).Meanwhile, The (Pb ×s) interaction had the same role on the chlorophyll contents and ratio in Sakha93. This was true in the role of the interaction factor on the Chl. a/b ratios in Giza168 cultivars. Table (2): Correlation coefficient (r.) values between chlorophyll content(a&b) and metabolites (soluble sugars, amino acids and soluble proteins)of both root and shoot of different studied cultivars of Triticum aestivum L. under osmotic potential , lead stress and their interaction.

Ch a

Ch a

Ch a

Ch b

&

&

&

&

soluble

soluble

amino

soluble

sugars

proteins

acids

sugars

Pb

-0.967**

-0.123

-0.927*

-0.755

0.381

-0.805

s

-0.374

-0.385

-0.348

-0.308

-0.362

-0.471

Pb×s

-0.262

0.219

0.186

-0.253

0.292

0.176

Pb

-0.644

-0.926*

-0.595

-0.630

-0.878*

-0.529

s

0.621

0.692

0.708

0.451

0.521

0.540

Pb×s

0.015

0.185

0.020

0.011

0.124

-0.011

Cultivar

Contents

Source

Ch b &soluble proteins

Ch b & amino

D.F

acids

15.3.3 15.3,3

168

Giza

Sakha93

of variance

*Significant at P < 0.05 level **Significant at P < 0.01 level

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SDS-PAGE The result revealed that when samples 1, 2 and 3 treated with 0ppm, 0MPa showed a disappearance of band at 100 KDa for lane no 3 only. On the other hand, there were a disappeared band at 60 KDa but not present in control. In addition, there were a new band at 55KDa for lanes 1,5,6,7 and 8. Fig (1A). In Fig (1B) there were a new band at 100 KDa for lanes 4, 5, 6 and 7. In addition there were new additional band at 50 KDa for lanes 5, 6 and 7. A

B

8 7 6 5 4 3 2 1 C M KDa

8 7 6 5 4 3 2

225 150 100 75

1 M

KDa 225 150 100 75 50

50 25

25

Fig ( 1 ) Polyacrylamide gel electrophoresis (SDS-PAGE coomassie blue stained) A: showing total cellular proteins of lane M: Broad range protein marker lane C: Control , lane 1 : 2ppm Pb, lane 3 : 5ppm Pb, lane 4: 10ppm Pb, lane 5 : -0.3MPa, lane6 : -0.3MPa,2ppmPb, lane 7 : 0.3MPa,5ppmPb , lane 8: -0.3MPa,10ppmPb (T. aestivum v. Sakha93) B: showing total cellular proteins of lane M: Broad range protein marker lane C: Control , lane 1 : -0.7MPa, lane 2 : 0.7MPa,2ppm Pb, lane 3: 0.7MPa,5ppm Pb, lane 4 : -0.7MPa,10ppm Pb, lane5 : -1MPa lane 6 : 1MPa,2ppmPb , lane 7: 1MPa,5ppmPb and lane 8: 1MPa,10ppmPb (T. aestivum v. Sakha93) Giza 168 samples showed a new band at 100, 75, 35 and 30 KDa for all samples but absent at control Fig (2A). On the other hand Fig (2B) showed no significant differences between control and treated samples.

A

B

8 7 6 5 4 3 2 1 C M KDa

151

8 7 6 5 4 3 2 1 C M KDa

225 150 100 75

225 150 100 75

50

50

25

25

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Fig (2) Polyacrylamide gel electrophoresis (SDS-PAGE coomassie blue stained) A: showing total cellular proteins of lane M: Broad range protein marker lane C: Control , lane 1 : 2ppm Pb, lane 2 : 5ppm Pb, lane 3: 10ppm Pb, lane 4 : -0.3MPa, lane5 : -0.3MPa,2ppmPb, lane 6 : -0.3MPa,5ppmPb , lane 7: -0.3MPa,10ppmPb (T. aestivum v. Giza 168) B: showing total cellular proteins of lane M: Broad range protein marker lane C: Control , lane 1 : -0.7MPa, lane 2 : -0.7MPa,2ppm Pb, lane 3: 0.7MPa,5ppm Pb, lane 4 : -0.7MPa,10ppm Pb, lane5 : -1MPa lane 6 : -1MPa,2ppmPb , lane 7: 1MPa,5ppmPb

and

lane

8:

1MPa,10ppmPb

(T.

aestivum

v.

Giza

168) Discussion The plant growth is affected by the availability of photosynthesis which mainly depending on the photosynthetic pigments. The reduction of photosynthetic pigments (Chl.a and Chl.b) in plants may be attributed to toxic action of salinity and lead on the biosynthesis of pigments (Santos, 2004; Li et al., 2012). In the investigated Triticum aestivum L. cultivars, the photosynthetic pigments tended to a maximum values under low s levels in the absence of Pb. This indicates that, the photosynthetic apparatus of plants, hitherto is capable of adapting to the higher salinity (Murillo-Amador et al., 2006), whereas, the low Pb doses encourage the Chl. content (a and b) in Sakha93 Plants at high s levels. Conversely, the presence of relatively high Pb concentration induced the Chl.a and Chl.b content in the absence of osmotic stress. This agrees with John et al., (2009) and they concluded that, no significant changes in total chlorophyll under low Pb concentration, whereas higher lead concentration led to significant decrease in chlorophyll content. In this respect, the wheat cultivars may have a better defense system against limited Pb concentrations. The Chl.a / Chl.b ratio in Giza168 yielded a high values in the absence of salinity and Pb stresses, as well as, under moderate Pb level and low s level . In Sakha93, the same was true at moderate s levels and in control plants. Hence, the converted of Chl.a to Chl.b may explain the increase in the Chl.a/b ratio at salinity levels (Murillo-Amador et al., 2006) and Pb exposure. Therefore, s had a predominant role on the Chl.a and Chl.b and Chl. a/b ratio in all tested cultivars of wheat, while the (s×Pb) interaction had the secondary role in Chl.a/b ratio. The same role was released on Chl.a and Chl.b in Sakha93. Furthermore, photosynthesis is adversely affected by Pb which could be due to metal induced reductions in the levels of photosynthetic pigments (Mishra et al., 2006) In general, s, Pb and/or their interaction were effected on the correlations between chlorophyll content and main metabolites or between each of soluble sugars, free amino acids and total soluble proteins. These correlations were negative in case of chlorophyll with main metabolites and positive between the investigated metabolites with each other. This means that the chlorophyll pigment is probably attached to a protein, which gives protection as well as, in case of soluble sugars and free amino acids.

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Conclusion It can be concluded that, Sakha93 cultivar was more adapted to decreased osmotic potential and had a capability to avoid Pb accumulation. Additionally, the presence of Ca - salts in the root medium counteracted the deleterious effects of Pb during the desert or arid and semi-arid lands. The SDSPAGE of protein indicated some variation in the profile of electrophoretic protein bands. Additionally, increasing amount of protein content possibly due to increasing expression of stress proteins may have helped in inducing less toxicity symptoms to lead therefore, more research emphasis need to be directed in the direction of DNA analysis. Finally the obtained results in this study suggest that growth of Sakha93 plants in the presence of Ca salts. References Bazzaz F.A., Carlson R.W., Rolfe G.L. (1975). The inhibition of corn and sunflower photosynthesis by lead. Physiologica Planturm, 34, 326–329. EL-Ghareeb D.K., Osman G.H., & El baz A.F. ( 2012). Isolation, cloning, and overexpression of vip3Aa gene isolated from a local Bacillus thuringiensis. Biocontrol Science & Technology, 22, 11-21. El-Sharkawi H.M. (1968). Water relations of some grasses with phreatophytic properties. Ph. D. Thesis Oklahoma State University, U.S.A. EL-Sharkawi H.M., Farghali K.A. &Tammam S.A. (2012). Interactive Effects of Sodocity and Salinity on the Nitrogenous Metabolites of Three Economic Plants. Journal of Botany Assiut University,41(2),265-280. El-Sharkawi, H.M., Springuel, I. (1977). Germination of some crop plant seeds under reduced water potential. Seed Science and Technology 5, 677–688. Eun S.O., Youn H.S., Lee Y. (2002). Lead disturbs microtubule organization in the root meristem of Zea mays. Physiologica Planturm, 110; 357–365. Flowers T.J., & Colmer T.D. (2008). Salinity Tolerance in Halophytes, New Phytology, 179, 945– 963. Heba H Abouseadaa., Gamal H Osman., Ahmed M Ramadan., Sameh E Hassanein., Mohamed T Abdelsattar., Yasser B Morsy., Hussien F Alameldin., Doaa K El-Ghareeb., Hanan A Nour-Eldin., Reda Salem., Adel A Gad., Soheir E Elkhodary., Maher M Shehata., Hala M Mahfouz., Hala F Eissa., & Ahmed Bahieldin. (2015). Development of transgenic wheat (Triticum aestivum L.) expressing avidin gene conferring resistance to stored product insects. BMC Plant Biology, 15,183-190.

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Hopkins W. G. (1999). Introduction to Plant Physiology, 2nd edition , New York: John Wiley & Sons Publication. Huang J.W., & Cunningham S.D. (1996). Lead phytoextraction: species variation in lead uptake and translocation. New Phytology. 134, 75–84. John R., Ahmad P., Gadgila K., SharmaS. (2009). Heavy metal toxicity: effect on plant growth, biochemical parameters and metal accumulation by Brassica juncea L. International Journal of PlantProduction,3, 65-76. Kadry Abdel Khalik., Gamal Osman., & Waeil Al-Amoudi. (2012). Genetic diversity and taxonomic relationships of some Ipomoea species based on analysis of RAPD-PCR and SDSPAGE of seed proteins. Australian Journal of Crop Science. 6 (6) 1088-1093. Kirst G. O. (1989). Salinity tolerance of eukaryotic marine algae. Annual Review of Plant Physiology and Plant Molecular Biology, 40, 21–53. Hoagland D.R. & Arnon D.I. (1950). The water-culture method for growing plants without soil. California Agricultural Experiment Station Circular, 347:1-32. KocaH., BorM.,Özdemir F.,Türkan İ. (2007). The effect of salt stress on lipid peroxidation, antioxidative enzymes and proline content of sesame cultivars . Environmental and Experimental Botany, 60(3), 344–351. Laemmli UK.(1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 15,227(5259):680–685. Lamhamdi M., El Galiou O.,Bakrim A.,Novoa-Mun J. C., Arias-Estevez M.,Aarab A.,Lafont, R. (2013). Effect of lead stress on mineral content and growth of wheat (Triticum aestivum) and spinach (Spinacia oleracea) seedlings. Saudi Journal of Biological Sciences, 20, 29–36. Le´onA.M.,PalmaJ.M.,CorpasF.J.,G´omezM.,RomeroPuertasM.C.,Chatterjee,D.,MateosR.M.,delR ´,L.A.,SandalioL.M. (2002). Antioxidative enzymes in cultivars of pepper plants with different sensitivity to cadmium. Plant Physiology Biochemistry, 40, 813–820. Lichtenthaler K., (1987). Chlorophylls and carotenoids: pigments of photosynthesis, Methods of Enzymology, 148, 350 -352. Li X.,Bu N.,Li Y.,Ma L.,Xin Sh.,Zhang L. (2012).Growth, photosynthesis and antioxidant responses of endophyte infected and non-infected rice under lead stress conditions. Journal of Hazardous Materials, 213– 214 :55– 61.

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Mishra S., Srivastava S.,Tripathi R.D., Kumar R.,Seth C.S.,Gupta D.K. (2006). Lead detoxification by coontail (Ceratophyllum demersum L.)involves induction of phytochelatins and antioxidant system in response to its accumulation, Chemosphere, 65 ,1027–1039. Munshi Abdulla and Osman Gamal. (2010). Investigation on molecular phylogeny of some date palm (Phoenix dactylifra L.) cultivars by protein, RAPD and ISSR markers in Saudi Arabia. Australian Journal of Crop Science, 4 (1) 23- 28. Murillo-Amador B., Jones H. G., Kaya C., Aguilar R. L., Garc´ıa-Hern´andez J. L., TroyoDi´eguez E., Avila-Serrano N.Y., & Rueda-Puente E. (2006). Effects of foliar application of calcium nitrate on growth and physiological attributes of cowpea (Vigna unguiculata L.Walp.) grown under salt stress. Environmental and Experimental Botany, 58, 188–196. Osman G. (2012). Detection, Cloning and Bioinformatics Analysis of vip1/vip2 Genes from Local Bacillus thuringiensis. African Journal of Biotechnology, 11(54) 11678-11685. Osman G., Already R., Assaeedi A., Organji S., El-Ghareeb D., Abulreesh H., & Althubiani A. S. (2015). Bioinsecticide Bacillus thuringiensis a comprehensive Review. Egyptian Journal of Biological Pest Control. (25):1:271-288 Osman G. Hussein E. M, & Abdallah N. A. (2004). Characterization and Purification of a Chitinolytic Enzyme Active against Sesamia cretica (pink borer). Arab Journal of Biotechnology, 7, 65-74. Osman G., Mostafa S., & Sonya H. Mohamed. (2007). Antagonistic Activities of some Streptomyces isolates against some phytopathogenes Pakistanian Journal of Biotecnology. 4,(1-2) 65-71. Osman G., Munshi A. Altf F., & Mutawie H. (2013). Genetic variation and relationships of Zea mays and Sorghum species using RAPD-PCR and SDS-PAGE of seed proteins. African Journal of Biotechnology, 12(27), 4269-4276. Ostle, B. (1963). Statistics in research. Basic concepts and techniques for research workers. The Iowa State University Press, Ames, Iowa. Peris M., Mico C., Recatal L., Sanchez R., Sanchez J. (2007). Heavy metal contents in horticultural crops of are presentative area of the European Mediterranean region. Sci.TotalEnviron,378:42–48. Santos C.V. (2004). Regulation of chlorophyll biosynthesis and degradation by salt stress in sunflower leaves. Science Horticulture, 103: 93–99. Seregin I.V., Kosevnikova A.D. (2008). Roles of root and shoot tissues in transport and accumulation of cadmium, lead, nickel, and strontium. Russ. Journal of Plant Physiology, 55, 1– 22.

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Usman A.R.A., Kuzyakov Y., Stahr K. (2005). Effect of immobilizing substances and salinity on heavy metals availability to wheat grown on sewage sludge- contaminated soil. Soil Sediment Contam, 14, 329–344. Youssef A. M. (2009). Salt Tolerance Mechanisms in Some Halophytes from Saudi Arabia and Egypt. Research Journal of Agriculture and Biological Sciences,5(3), 191-206.

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