Evaluation of Genetic Variations in Maize Seedlings ... (SDS) is a universally used method in biomedical research;[13]concludedthatelectrophoresis(SDS-PAGE)of proteinscanbeeconomicallyusedtoassessgeneticvariation

Research ArticleEvaluation of Genetic Variations in Maize Seedlings Exposed toElectric Field Based on Protein and DNA Markers

Asma A AL-Huqail1 and Ekram Abdelhaliem12

1Plant Physiology Botany and Microbiology Department Science College King Saud UniversityPO Box 22452 Riyadh 11495 Saudi Arabia2Plant Genetic and Molecular Cytogenetic Botany Department Science College Zagazig UniversityPO Box 44519 Zagazig 308213 Egypt

Correspondence should be addressed to Ekram Abdelhaliem ekramesagmailcom

Received 6 April 2015 Accepted 20 May 2015

Academic Editor Pengjun Shi

Copyright copy 2015 A A AL-Huqail and E Abdelhaliem This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

The current study analyzed proteins and nuclear DNA of electric fields (ELF) exposed and nonexposedmaize seedlings for differentexposure periods using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) isozymes random amplifiedpolymorphic DNA (RAPD) and comet assay respectively SDS-PAGE analysis revealed total of 46 polypeptides bands withdifferent molecular weights ranging from 18620 to 3600KDa It generated distinctive polymorphism value of 8462 Leucine-aminopeptidase peroxidase and catalase isozymes showed the highest values of polymorphism (100) based on zymogramsnumber relative front (119877119891) and optical intensity while esterase isozyme generated polymorphism value of 8333 Amino acidswere analyzed using high-performance liquid chromatography which revealed the presence of 17 amino acids of variable contentsranging from 2265 to 2809 RAPD revealed that 78 amplified DNA products had highly polymorphism value (9508) basedon band numbers with variable sizes ranging from 120 to 992 base pairs and band intensity Comet assay recorded the highestextent of nuclear DNA damage as percentage of tailed DNA (238) and tail moment unit (536) at ELF exposure of maize nucleifor 5 daysThe current study concluded that the longer ELF exposing periods had genotoxic stress onmacromolecules ofmaize cellsand biomarkers used should be augmented for reliable estimates of genotoxicity after exposure of economic plants to ELF stressors

1 Introduction

Plants are unique in their ability to serve as in situ monitorsfor environmental genotoxins that inflict damage to DNAand cause genotoxic stress which can reduce plant genomestability growth and productivity [1] Maize or corn (Zeamays) is a plant belonging to the family of grasses (Poaceae)and is one of the most important cereal crops worldwideas human nutrient a basic element of animal feed and rawmaterial for manufacture of many industrial products [2]Maize is the oldest plant to have a fully established gene mapwith the basic genome consisting of 10 chromosomes and isan excellent plant for the detection of genotoxins mutagenicand clastogenic substances in the environment [3]

As the result of the development of industrialization andtechnology an increasing number ofman-made electric fields

(ELFs) have appeared in living plant environment that pro-duce in all places by numerous sources including nearby highvoltage transmission lines primary and secondary overheadutility distribution lines and the electrical grounding system[4] ELF is one kind of stress which can cause more riskof disorders in the function of biological systems such asmorphology uncoiling immune defense and regulation ofthe cell division [5] The final effect of ELF in the numberof flowering plants was dependent on voltage and time ofexposition of tissues as well as on the electric field polarity[6] The study of [7] mentioned that the electric field ofan extremely low frequency induces electrical potentialsand resultant current flows in the aqueous medium thatsurrounds the living cells leading to various physiologicaland biochemical responses The study of [8] evaluated theeffect of electric field intensity and exposing time on some

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 874906 15 pageshttpdxdoiorg1011552015874906

2 BioMed Research International

physiological properties of maize grains The study of [9]concluded that an electric current directed to the plant cellsmay increase the capacity of some enzymes associated withgenes already inside the nucleus The results of [10] showedthat the electrostatic fields with certain intensity can increasethe content of free radicals in plant cells

It is important for detection of genotoxicity and muta-genic potential of various types of environmental stresssuch as ELF on crop plants to understand their biologicalconsequences and their molecular action on protein andDNAof plant cell by introducing biochemical molecular andmolecular cytogenetic assays The biochemical assays relatedto protein-based markers such as nonenzymatic protein(storage proteins) enzymatic proteins (isozymes which areone of themost commonly used proteinmarkers) and aminoacids composition [11] these markers detect the codingregions and the variations at the gene product level

Proteins are primary gene products of active structuralgenes their size and amino acids sequence are the directresults of nucleotide sequences of the genes hence anyobserved variation in protein systems induced by any muta-gen is considered a mirror for genetic variations [12] Varia-tion in theDNAcoding sequences frequently causes variationin the primary conformation of the proteins Determinationof protein molecular weight (MW) via polyacrylamide gelelectrophoresis (PAGE) in the presence of sodium dodecylsulfate (SDS) is a universally used method in biomedicalresearch [13] concluded that electrophoresis (SDS-PAGE) ofproteins can be economically used to assess genetic variationand relation in germplasm and also to differentiate mutantsfrom their parent genotypes Some studies used SDS-PAGEfor detection of alterations in protein profiles occurringduring exposure to electric field [5 14]

Isozymes are direct gene products that show differencesin protein-coding genes and arise from multiple gene locicoding for structurally distinct polypeptide chains The dif-ferent proteins that make up a set of isozymes can catalyzethe same reaction but differ in molecular weight theirkinetics substrate affinity number of subunits and aminoacid sequences electric charge or electrophoretic mobility[11 15] Protein variants in isozyme analysis are separatedchemically by nondenaturing native polyacrylamide gel elec-trophoresis (Native PAGE) resulting in different bandingpatterns (zymograms) and electrophoretic mobilities thatvisualized using an enzyme-specific staining mixture whichincludes a substrate cofactor and oxidized salt [11] Isozymeanalysis is a powerful biochemical technique with numerousapplications in mutagenic potential in plant cell [15 16]

Recently advances in molecular biology have led tothe development of a number of selective and sensitiveassays for detection of the variations at the DNA level anddifferences can clearly be shown when comparing DNAfingerprints from individuals exposed andor nonexposed togenotoxic agents [17] Molecular assays related to DNA-basedmolecular techniques like Random Amplified PolymorphicDNA (RAPD) are a very sensitive method of screeningfor nucleotide sequence polymorphisms that are randomlydistributed throughout the genome in both coding and

noncoding regions and repeated or single copy (unique)sequences [18] RAPD-PCR technique has been successfullyused to detect DNA damage and mutations in some plantspecies induced by various types of genotoxic agents [19 20]

In recent years a new molecular cytogenetic-based assaythe Comet or single cell gel electrophoresis (SCGE) assaycan theoretically be applied to every type of eukaryotic cellincluding plant cells for detecting DNA damage inducedby genotoxic agents [1] The basic principle of this assayis to determine the DNA breaks by measuring the DNAdamage which is quantified by the proportion of DNA whichmigrates out of the nuclei towards the anode when individualcells or isolated nuclei embedded in single cell agarose gelelectrophoresis (comet assay) [21] After electrophoresis adamaged nucleus takes on the appearance of ldquocomet-likerdquoshape (with a head the nuclear region and a tail whichcontains DNA fragments that migrate from the nucleus) [21]

The objective of this study to evaluate the possible influ-ence of electric field intensity and exposing time on Zea maysseedlings on the levels of biochemical analyses of proteinsusing SDS-PAGE isozymes amino acids composition andon the levels of DNA damage using RAPD-PCR and cometassay

2 Materials and Methods

21 Plant Material and Germination Maize grains (hybridnumber 323 from Agronomy Research Department FieldCrops Institute Agriculture Research Center Giza Egypt)were used as plant material in this study Grains werescreened for viability and uniformity size and divided intotwo groups (A and B) 50 grains of each group were sterilizedand germinated until reached seven-day-old seedlings inearthenware pot 60 cm in diameter containing soil obtainedfrom the agriculture field Seedlings of group (A) are exposedto the electric field exposure system while seedling of group(B) was maintained without exposure (unexposure samples)

22 The Electric Field (ELF) Exposure In the laboratory themaize seedlings of group (A) were exposed to an alternat-ing electric field of 50Hz frequency and 6 kVm strengthgenerated between two parallel aluminum electrodes of 60 times50 times 2 cm dimensions fixed horizontally above and belowseedlings for 1 3 and 5 days The electric field was deriveddirectly from 50Hz high voltage setup transformer manufac-tured by the ldquoCenter of Scientific and Electronic EquipmentMaintenance Faculty of Science Cairo Universityrdquo Maizeseedlings exposed to ELF for 1 3 and 5 days termed asthree treatments (T1 T3 and T5) while seedlings of group(B) which non-exposed to the electric field termed as zerotreatment (T0) Ten seedlings from exposed and nonexposedseedlings were harvested and thoroughly cleaned with freshwater followed by distilled water for quantitative removalof any foreign particles and then dried in air conditionsuntil completely dried and then subjected to biochemical andmolecular analyses

BioMed Research International 3

23 Biochemical Analysis Using SDS-PAGE Dried leaves ofexposed and nonexposedmaize seedlings were used for SDS-PAGE isozymes and amino acid analyses

231 Preparation of Leaf Powder and Defatted PreparationThe dried leaves of ELF exposed and nonexposed maizeseedling were milled to leaf powder and defatted accordingto methods described by [22]

232 Extraction of Proteins and SDS-PAGE Analysis Theprotein extraction technique employed was similar to theextraction technique described by [23] Sample buffer wasadded to 02 g seed flour as extraction liquid and mixedthoroughly in an Eppendorf tube by vortexingThe extractionbuffer contained the following components (final concentra-tion) 05M Tris-HCl pH 68 25 SDS 5 urea and 5 2-mercaptoethanol Before centrifugation at 10000 g for 5minat 4∘C the sample buffer was boiled for 5min SDS-PAGEwas performed using a standard method on a vertical slabgel Bromophenol blue was added to the supernatant as atracking dye to watch the movement of proteins on the gelProtein profiling of samples was performed using SDSPAGEas described by [24] Seed proteins were analyzed by SDS-PAGE on 10 polyacrylamide gel After electrophoresis theprotein bands were visualized by staining with Coomassiebrilliant blue G-250 Marker proteins (Fermentas) were usedas references The bands produced in the electropherogramwere scored and their molecular weights were compared tothe standard Pharmacia protein marker

233 Protein Imaging and Data Analysis Gel photographyand documentation were carried out using the Bio-Radgel documentation system The number of bands revealedon each gel lane was counted and compared using theGel Pro-Analyzer software Quantitative variations in bandnumber and concentration were estimated using the Bio-Radvideo densitometer Model Gel Doc 2000 With regard tovariation in protein banding patterns electropherograms ofeach exposed and nonexposed sample were scored for thepresence or absence of bands

24 Biochemical Analysis of Amino Acids Composition UsingHigh-Performance Liquid Chromatography (HPLC) Freeamino acids (AAs) were extracted from fine powders ofdried leaves for each exposure time as described by [25] withsome modifications Amino acid analyses were performedby HPLC after hydrolysis of samples with 6N HCl at 110∘Cunder vacuum for 24 h on an amino acid analyzer (AppliedBiosystems 421 amino acid analyzer Foster City CA USA) asdescribed by [26] The results of the analysis were expressedas the nitrogen (N) content of the sample g100 g of crudeprotein (N times 625) The quality of amino acid compositionwas tested using the essential amino acid index (EAAI) andthe amino acids were quantified by comparing the peakarea with corresponding amino acid standard solutions usingthe Spectra Physics Data System program (Santa Clara CAUSA)

25 Biochemical Analysis of Isozymes

251 Extraction PAGE Technique and Isozyme StainingMethods Four isozymes leucine-aminopeptidase (LAP)esterase (EST) peroxidase (PER) and catalase (CAT) wereused in this experimentThe dried leaves of ELF exposed andnonexposedmaize seedling were separately milled and defat-ted according to methods described by [22] Approximately04 g powdered seed was crushed with acid washed sand and400mL extraction buffer Extraction buffer consisted of 01MTris-HCl (pH 75) containing 20 sucrose as described by[27] The samples were then centrifuged at 15000 g for 15minat 4∘C supernatants were collected and used directly forisozyme analyses in a separate vial Each sample was appliedto vertical polyacrylamide gel electrophoresis (45 stacking9 separating gel) using a mini gel apparatus in Tris-glycine(pH 83) buffer as described by [27] Visualization of enzymeactivity after electrophoresis was achieved by histochemicallystaining the gels The gels were stained for LAP EST PERand (CAT) separately with specific activity stain solutions asdescribed by [28ndash31] respectively Gels were photographedusing the Vilber Lourmat gel documentation system

252 Gel Scoring and Identification of Zymogram Following[32] isozyme banding patterns were recorded on the basisof number the relative front (119877119891) values of zymograms ongel electrophoresis and their optical intensitiesThe 119877119891 valueof each respective band on schematic isozyme patterns wasdetermined to allow precise comparisons among the varioustreatment exposures of maize seedlings to electric field Datawere scored as the presence or absence of zymogram of aunique pattern

26 Detection of DNA Damage Based onRAPD-PCR Technique

261 Isolation of Genomic DNA Genomic DNA from thedried leaves of ELF exposed and nonexposed maize seedlingwas extracted using the hexadecyltrimethylammonium bro-mide method as described by [33]

262 Qualitative and Quantitative Analyses of ExtractedDNA DNA yield was measured using a UV-visible spectro-photometer (PerkinElmer Waltham MA USA) at 260 nmDNA purity was determined by calculating the absorbanceratio at A260280 nm Polysaccharide contamination wasassessed by calculating the absorbance ratio at A260230 nm[34] For quality and yield assessments electrophoresiswas performed for all DNA samples on 08 agarose gelsthat were stained with ethidium bromide the bands wereobserved using a gel documentation system (Alpha InnotechSan Leandro CA USA) and compared with a known stan-dard lambda DNA marker sample

263 PCR Amplification Using Random Primers of RAPDThe PCR reaction mixture contained 25 120583L 10x buffer with15mMMgCl2 (Fermentas Vinius Lithuania) with 025mMeach dNTP (Sigma St Louis MO USA) 03 120583M primer


05U Taq DNA polymerase (Sigma) and 50 ng templateDNA The PCR reaction was performed in a Palm Cyclerapparatus (Corbett Research) using the following methodinitial denaturation of 4min at 95∘C followed by 40 cyclesof 1min at 95∘C 1min at 38∘C and 2min at 72∘C withfinal extension at 72∘C for 10min and a hold temperatureof 4∘C A total of 20 random DNA oligonucleotide primers(10-mer) were independently used in the PCR reactions(University of British Columbia Canada) according to [35]with somemodificationsOnly 5 primers (P-02 06 08 10 and14) successfully generated reproducible DNA amplificationproducts For DNA amplification the PCR was run for 35cycles which consisted of a denaturation step (1min at 95∘C)annealing step (1min at 35∘C) and elongation step (2minat 72∘C) After 34 cycles a final extension period was added(5min at 72∘C) Amplification products were electrophoresedon 15 agarose gel (Sigma) in TAE buffer (004M Tris-acetate 1mMEDTA pH 8)The gel was run at 100V constantvoltage for 1 h Gels were stained with 02 120583gmL ethidiumbromide for 15minThe PCR products were visualized undera UV light transilluminator The 100-base pair DNA ladder(Gibco-BRL Grand Island NY USA) was loaded into thefirst lane of each gel to evaluate band sizes The gels werephotographed under UV light using a gel documentationsystem (Bio-Rad Hercules CA USA)

264 Scoring andDataAnalyses After separating PCRprod-ucts by agarose gel electrophoresis the gels were visualizedusing a Photo Print (Vilber Lourmat France) imaging systemQuantitative variations in numbers and sizes of bands as wellas their optical intensities were analyzed using Bio-One D++software (Vilber Lourmat France) The RAPD bands werescored as the presence or absence of DNA bands

27 Detection of DNA Damage Based on Single Cell GelElectrophoresis (Comet Assay) Technique

271 Isolation of Nuclei The nuclei suspension was used inthe alkaline single cell gel electrophoresis assay as describedby [36] Dried leaves of ELF exposed and nonexposed ofmaize seedlings were placed in a small Petri dish containing200120583L of cold 400mM Tris-HCl buffer pH 75 (on ice)Using a razor blade the leaf was gently sliced into a ldquofringerdquoto release nuclei into the buffer under yellow light Eachslide previously coated with 1 normal melting point (NMP)agarose and dried was covered with a mixture of 55120583L ofnuclear suspension and 55120583L of low melting point (LMP)agarose (1 prepared with phosphate-buffered saline) at40∘C and cover slipped The slide was placed on ice forat least 5min and coverslip was removed Then 110 120583L ofLMP agarose (05) was placed on the slide and coverslipwas mounted again After 5min on ice the coverslip wasremoved

272 Single Cell Gel Electrophoresis (SCGE) Slides of SCGEwere prepared as described by [36] The slides with the plantcell nuclei were placed in a horizontal gel electrophoresistank containing freshly prepared cold electrophoresis buffer

T0 T1 T3 T5 M (kDa)250

148

98

64

59

36

Figure 1 SDS-PAGE banding patterns of proteins of ELF exposedand nonexposed maize seedlings

(300mM NaOH 1mM EDTA pH gt 13) and incubated for15min Electrophoresis was performed at 16 V 300mA for30min at 4∘C Then the gels were neutralized by washingthree times in 400mM Tris-HCl pH 75 and stained withethidium bromide (20120583gmL) for 5min After staining thegels were dipped in ice-cold distilled water and immediatelyanalyzed

273 Imaging and Analysis Software In each slide DNA-damage extent of 50 randomly chosen cells was analyzed andassessed quantitatively by visual scoring or using the fluores-cence microscope with an excitation filter of 546 nm and abarrier filter of 590 nm using computerized image analysissystem (Komet Version 31 Kinetic Imaging Liverpool UK)Percentage of nuclei with tails the relative tail length tailDNA (TD relative percentage of DNA in the comet tail)and tail moment (TM integrated value of density multipliedby migration distance) were used as parameters of DNAdamage

3 Results

31 SDS-PAGE Analysis The proteins electrophoretic band-ing patterns of ELF exposed and non-exposed maize leavesusing SDS-PAGE exhibited distinctive quantitative and qual-itative alterations compared to nonexposed one (T0) as shownin Table 1 and Figure 1 These protein alterations based onchanges in polypeptides molecular weights (MWs) bandsintensities fractionation of some bands appearance of newbands (unique bands) and disappearance of some bands(polymorphic bands) SDS-PAGE analysis revealed total of 46polypeptides bands with different bands intensities andMWsthat ranged from 18620 to 3600KDaOut of which 22 bandswere polymorphic with 8462 polymorphism (16 bandswere unique with value of 3404 6 bands were nonuniquewith value of 1277) and 4 bands were monomorphic bands


Table 1 Electrophoretic banding analysis of proteins of ELF exposed and nonexposed maize seedlings

Rows Mol weights

Electric field treatmentsLanes

Band typesLane T0 Lane T1 Lane T3 Lane T5

KDa KDa KDa KDa 1 18620 1 227 1 440 1 921 1 1054 M2 18015 0 mdash 0 mdash 0 mdash 1 1180 U3 15500 0 mdash 0 mdash 0 mdash 1 110 U4 15127 0 mdash 1 1200 0 mdash 0 mdash U5 14631 1 1040 0 mdash 1 1120 1 190 Non-U6 13138 0 mdash 0 mdash 0 mdash 1 166 U7 13019 1 100 1 109 1 162 0 mdash Non-U8 11879 0 mdash 0 mdash 0 mdash 1 111 U9 11671 1 2210 1 1960 1 2160 1 2421 M10 10642 1 1040 1 1460 1 1690 1 1790 M11 9684 0 mdash 0 mdash 1 514 1 618 Non-U12 8906 1 2830 0 mdash 0 mdash 0 mdash U13 8529 1 3230 1 2250 1 2660 1 2800 M14 8016 1 270 0 mdash 0 mdash 0 mdash U15 7884 0 mdash 1 1266 1 514 1 262 Non-U16 7555 0 mdash 0 mdash 0 mdash 1 1590 U17 6927 1 466 0 mdash 0 mdash 0 mdash U18 6666 1 1340 0 mdash 0 mdash 0 mdash U19 6477 0 mdash 0 mdash 1 2150 0 mdash U20 6345 0 mdash 1 1260 0 0 mdash U21 6291 0 mdash 0 mdash 0 mdash 1 2130 U22 6211 1 939 0 mdash 0 mdash 0 mdash Non-U23 6145 1 2250 1 2010 0 mdash 0 mdash Non-U24 6123 0 mdash 0 mdash 1 2150 0 mdash U25 6091 0 mdash 0 mdash 0 mdash 1 1730 U26 3600 0 mdash 0 mdash 0 mdash 1 597 UNumber of polypeptidebands of each lane 12 9 11 15

Total polypeptide bands 47 of total bands of eachlane 2553 1915 2340 3192

Types and frequency ofpolypeptide bands

Unique (U) (Non-U) Polymorphic Monomorphic (M) Polymorphism Number Number Number Number

16 3404 6 1277 4681 22 4 851 8462

with value of 3404 It also generated considerable valueof polymorphism reaching 8462 SDS-PAGE generated 8unique bands at the electric field treatment after 5 days (T5)with MWs (18015 15500 13138 11879 7555 6291 6091and 3600KDa) compared to nonexposed treatment (T0)which recorded 5 unique bands with MWs (14631 89068016 6927 and 6666KDa) It also generated 2 unique bandsat the electric field treatments (T1 and T3) with MWs (151276345 KDa for T1 and 6477 6123 KDa for T3) These uniquebands can be used as markers for the protein alterations atthese treatments The maximum and minimum number of

bands (15 bands with value 3192 and 9 bands with value1915 resp) was found at ELF treatment (T5 and T1 resp)compared to 12 bands with value of 2553 at nonexposedtreatment (T0)

32 Isozymes Analyses LAP EST PER and CAT isozymesused in the current study showed clear polymorphismsbetween exposed maize seedlings to ELF treatment (T1T3 and T5) compared to nonexposed one as shown inTables 2ndash5 and Figure 2 These polymorphisms are based onthe number of zymograms the 119877119891 values and their optical


Table 2 Distribution of leucine-aminopeptidase (LAP) zymograms of ELF exposed and nonexposed maize seedlings

Rows 119877119891 valueElectric field treatments

Lane T0 Lane T1 Lane T3 Lane T5 Zymogram types119877119891 OD 119877119891 OD 119877119891 OD 119877119891 OD

1 020 radic 175 U2 025 radic 349 U3 033 radic 387 U4 034 radic 548 U5 035 radic 295 U6 065 radic 329 U7 066 radic 351 radic 343 radic 244 Non-U8 087 radic 208 U9 088 radic 363 radic 285 Non-U10 089 radic 299 UNumber of zymograms ineach lane 3 3 4 3

Total zymograms 13 of zymograms in eachlane 2308 2308 3077 2308

Frequency of isozymeticbands and Polymorphism

Unique (U) (Non-U) Polymorphic Monomorphic (M) of polymorphismNumber Number Number Number

8 6154 2 1538 10 7692 0 0 100

Table 3 Distribution of esterase (EST) zymograms of ELF exposed and nonexposed maize seedlings



1 013 radic 4360 U2 014 radic 5560 radic 5220 Non-U3 016 radic 3430 U4 034 radic 1080 U5 036 radic 1140 U6 038 radic 1170 U7 039 radic 1350 U8 057 radic 2580 radic 3300 radic 1920 radic 1710 M9 077 radic 2630 radic 1270 radic 1380 radic 1900 M10 085 radic 3040 U11 088 radic 2630 U12 091 radic 3240 UNumber of zymograms ineach lane 5 5 5 4

Number of totalzymograms 19

of zymograms in eachlane 2632 2632 2632 2105

Frequency of isozymeticbands and polymorphism


9 4737 1 526 10 5263 2 1053 8333


Table 4 Distribution of peroxidase (PER) zymograms of ELF exposed and nonexposed maize seedlings



1 007 radic 246 U2 008 radic 3630 U3 009 radic 237 radic 3000 Non-U4 034 radic 579 U5 035 radic 357 radic 255 Non-U6 036 radic 363 U7 060 radic 337 U8 067 radic 28 U9 073 radic 175 radic 508 Non-U10 078 radic 118 U11 080 radic 105 U12 088 radic 966 U13 105 radic 834 UNumber of zymograms ineach lane 3 4 3 6





10 5250 3 1875 13 8125 00 00 100

intensities A total of 63 different electrophoretic zymogramswere observed for the 4 isozymes LAP EST PER and CATanalyses generated 13 19 16 and 15 zymograms respectivelywith different 119877119891 values ranging from 020 to 089 for LAP013ndash091 for EST 007ndash105 for PER and 004ndash099 forCAT LAP EST PER and CAT analyses generated highernumber of zymograms (4 5 6 and 5 with values 30772632 375 and 3333 resp) at treatments (T3) (T0 T1and T3) (T5) and (T0) respectively while lower numberof zymograms (3 4 3 and 3 with values 2308 21051875 and 2000 resp) was scored at treatments (T0 T1and T3) (T5) (T0 and T3) and (T3 and T5) respectivelyOn the other hand LAP analysis revealed 10 polymorphiczymograms (8 unique and 2 nonunique zymograms) Thesezymograms generated a high value of polymorphism of 100while EST analysis revealed 10 polymorphic zymograms (9unique and 1 nonunique zymograms) and 2 monomorphiczymograms with a considerable polymorphism value of8333 Moreover PER analysis revealed 13 polymorphiczymograms (10 unique and 3 nonunique zymograms) whileCAT analysis revealed 12 polymorphic zymograms (10 uniqueand 2 nonunique zymograms)These two isozymes generateda high value of polymorphism of 100

33 Amino Acid Analysis The results of EAAs and NEAAscompositions of dried leaves of exposed and nonexposedmaize seedlings are shown in Table 6 HPLC analysis revealed

the presence of 17 amino acids 9 of which are essential inhumans The free amino acids contents were ELF exposuretime dependent as evident from their reduction by ELFtreatments (T1 T3 and T5) compared to zero treatment (T0)The highest content of total free amino acids (FAA) wasscored at T0 with value of 2809 while the lowest contentof FAA was at ELF treatment T5 with value of 2265respectively Total FEAA content (6273) was higher thanFNEAA (3827) The current data showed that the contentsof all types of FAAs were prevalent in control (T0) exceptthe NEAA proline was prevalent in ELF treatment T5Furthermore the EAA Arginine scored the highest contentamong all types of FAAs

34 Detection of DNA Damage Based on RAPD-PCR Tech-nique RAPD analysis was employed in the present study toevaluate the extent of theDNAalterations in ELF exposed andnonexposed maize leaves as shown in Table 7 and Figure 3Twenty random primers were used for the RAPD analysisin which only five primers of them (P-02 06 08 10 and14) succeeded to produce clear reproducible DNA bands andgave satisfactory results with many alterations in the RAPDprofiles In total Seventy-eight (78) amplified DNA productswere scored these bands varied in number of specific randomsequences band intensity and sizes ranging from 120 to992 bp An average of 1560 bands per primer was scoredRAPD analysis generated highly polymorphism values of


Table 5 Distribution of catalase (CAT) zymograms of ELF exposed and nonexposed maize seedlings



1 004 radic 2490 U2 006 radic 2990 U3 008 radic 2400 U4 009 radic 3240 radic 4070 Non-U5 010 radic 3370 U6 033 radic 2430 U7 034 radic 329 radic 6630 radic 5930 Non-U8 071 radic 3470 U9 075 radic 589 U10 088 radic 255 U11 091 radic 517 U12 099 radic 118 UNumber of zymograms ineach lane 5 4 3 3





10 6667 2 1333 12 8000 00 00 100

9516 and 7564 (59 in total) DNA polymorphic bands ofwhich 51 bands were unique with value 6535 and 7 bandswere nonunique with value 897 in addition to 3 bandswhich were monomorphic with value 385 The maximumnumber of gene products (23 bands) with value 2949 wasobserved at ELF treatmentT5 whereas theminimumnumberof bands (16) with value 2051 was at ELF treatment T1compared to zero treatment T0 at which RAPD analysisscored 21 bands with value 2692 On the other hand thehighest number of gene products (21 bands) was generatedby primer P-14 whereas the lowest number (13 bands) wasgenerated by the primers 02 and 06 Furthermore the highestvalue of polymorphism (100) was revealed by the primerminus10 and 14 whereas the lowest one (8750) is revealed byprimer 02

35 Detection of DNA Damage Based on Single Cell GelElectrophoresis (Comet Assay) Technique The present studyused the comet assay to evaluate DNA damage induced inmaize seedlings under stress of electric field exposure asshown in Table 8 and Figure 4 The higher percentage of tailDNA (TD ) and tail moments (TM) (fraction of migratedDNA multiplied by some measure of tail length) was usedas parameters of DNA damage The current data showedremarkable variations in the extent of DNA damage in Zeamays nuclei ELF treatment T5 showed the highest DNAmigration reached (tailed 15) with tail length 225120583m of

tailedDNA (238) and tail moment unit 536 this indicatedthat this ELF treatment had genotoxic stress increased dam-age of nDNA of maize cells compared to zero treatment T0which showed the lowest DNAmigration reached (tailed 4)with tail length 131 120583m tailed DNA 140 and tail momentunit 230

4 Discussion

41 SDS-PAGE Analysis The present study observed thatSDS-PAGE analysis exhibited distinctive qualitative andquantitative alterations in electrophoretic SDS-proteins ofexposed and nonexposed maize seedlings These alterationsare based on variations in number of polypeptide bandsmolecular weights and intensities of polypeptides bandsas well as gain or loss of protein bands that led to highlevels of protein polymorphisms Electrophoretic analysis ofprotein provides information concerning the structural genesand their regulatory systems that control the biosyntheticpathways of that protein Each polypeptide band representsthe final products of transcriptional and translational eventsoccurring due to active structural genes [37] The changedprotein products caused by ELF exposure time may resultfrom base changes in DNA or altering protein sites orchanges in amino acid sequences or frame shift mutationsAdditionally they may serve as genetic markers because theycan be quite polymorphic and their variability is generallyhighly heritable [38]


0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es

Figure 2 Banding pattern (a) and schematic distribution (b) of LAP EST PER and CAT zymograms (119877119891 values) in ELF expose andnonexposed maize seedlings


Table 6 Essential and nonessential amino acid composition of ELF exposed and nonexposed maize seedlings

Free amino acids(120583mol100mg dry weight)

Electric field treatmentsT0 T1 T3 T5

Essential amino acids (EAA)Arginine 829 763 721 719Histidine 292 277 215 113Isoleucine 174 168 135 130Lysine 213 188 165 202Methionine 247 225 197 203Leucine 084 083 080 074Phenylalanine 134 122 113 119Threonine 206 189 159 142Valine 133 121 121 124Total of EAA at each treatment 2312 2136 1906 1826Sum 8180 = 6273 total of EAA at each treatment 2826 2611 2330 2232

Nonessential amino acids (NEAA)Alanine 268 246 185 178Aspartic acid 168 152 144 139Glutamic acid 148 125 105 101Cystein 233 045 046 039Proline 049 227 205 276Tyrosine 141 128 128 098Asparagine 238 221 214 212Glutamine 166 163 151 133Total of NEAA at each treatment 1411 1307 1178 1176Sum 5072 = 3827 total of NEAA at eachtreatment 2782 2577 2323 2319

Total number of EAAs andnon-EAAs 3723 3443 3084 3002

Sum 13252 2809 2598 2327 2265

The appearance of new bands (unique bands) may beexplained on the basis of mutational events at the regulatorysystem of unexpected gene(s) or on the basis of bandsubfractionation which could be attributed to duplicationof gene followed by the occurrence of point mutation thatencoded the fractionated band [16] or result from differentDNA structural changes (breaks transpositions deletionetc) which led to change in amino acids and consequentlyprotein formed [16] On the other hand the disappearance ofsome protein bands which led to formation of polymorphicbands could be attributed to the loss of genetic materialwhich may be due to the breaking of a small number ofpeptide bonds to formpolypeptides of shorter length than theoriginal protein [16]

Furthermore the changes in band intensity could beinterpreted on the basis of gene duplication or pointmutationthat leads to production of shorter and longer polypeptidechains and alteration in the structural genes which maybe due to the changes in regulator gene(s) expression [16]

The distinction protein polymorphisms shown between ELFexposed and nonexposed samples in the present study maybe resulting from insertions or deletions between mutatedsites of protein bands and could be used as biomarkersfor identification of ELF stressed plants [38] Additionallythe increase in ELF exposure time causes alterations in therelative mobility of bands intensities expression of newproteins and suppression of some proteins

42 Isozymes Analyses Because isozymes being proteins aredirect products of gene expression that show differences inprotein-coding genes and produced by different alleles andgene loci [11] Electrophoretic banding patterns (zymograms)generated can be effectively correlated to change in genesequence mutation and gene interaction with ELF [11] Thismay be explained by the presence of multilocus isozymeforms fromgene duplication throughmutation polyploidiza-tion and chromosomal aberrations [11]


Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949


Table 8 The extent of nuclear DNA damage of ELF exposed and nonexposed maize nuclei

Electric field treatments Tailed Untailed Tail length (120583m) Tail DNA Tail moment unitT0 4 96 131 140 230T1 6 94 172 197 339T3 10 90 193 219 423T5 15 85 225 238 536

T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100

(bp) T0 T1 T3 T5Ladder

P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100

(bp)T0 T1 T3 T5Ladder

P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)

Figure 3 RAPD products of DNA fragments extracted from ELF exposed and nonexposed maize seedlings using 5 random primers


T0 T1

T3 T5

Figure 4 Comets images showed varying extent of DNA damage of ELF exposed and nonexposed maize nuclei

The electrophoretic banding profiles generated by LAPEST PER and CAT isozymes revealed multiple bands ondifferent loci with different119877119891 values and unequal intensitiesThe 119877119891 value of each respective band for schematic isozymepatterns was determined to compare various ELF treat-mentsThe different electrophoretic mobilities of zymogramsresulted from the different sizes and shapes of enzymemolecules therefore their variations indicate genetic varia-tion induced due to ELF stress [27]

If an amino acid substitution occurs in a proteinmoleculedue to ELF stress net charge may be altered or conforma-tional changes in isozymes may occur consequently alteringthe migration rate of proteins in an electric field as well astheir electrophoretic mobilities as well as catalytic efficiencyand stability [11] Each band on a gel electrophoresis revealedin the current study can be assigned a descriptive value basedon either the netmigration of the band from the origin (an119877119891value) or its position relative to that of the band coded by themost common allele The changes in the mobility of enzymesin an electrical fieldmay be reflecting changes in the encodingDNA sequences On the other hand differences in isozymeticpatterns generated between different ELF treatments havebeen attributed to one or more of the following causes (i)changes in the level of isozyme expression (ii) genetic tran-scription of different isozyme loci and (iii) posttranslationalmodifications changing the electrophoretic mobility thesewere found in agreement with [11]

43 Amino Acid Analysis Amino acids analysis by HPLCshowed variations in the amount of free amino acids betweenELF exposed and nonexposed maize seedlings these vari-ations may reflect the oxidative stress induced by intensityof the electric fields treatments at certain exposing time [10]which may lead to biosynthesis or degradation of proteinby increasing the free radical oxygen content inside maizecells These oxidative proteins may undergo loss of activityor be denatured leading to oxidation of amino acid chainsand generating stable and reactive products such as proteinhydroperoxides which result in fragmentation of proteinsThe side chains of all the amino acid residues particularlycysteine and methionine are susceptible to oxidation bythe action of ROS [39] On the other hand these quan-titative variations of amino acids will affect DNA geneexpression synthesis of proteins activity of isozymes andredox-homeostasis Free amino acids especially proline canaccumulate in plant cells due to stress and serve as osmoticallyactive solute a ROS scavenger and a molecular chaperonestabilizing the structure of proteins thereby protecting cellsfrom damage caused by stress [40]

44 Detection of DNA Damage Based on RAPD-PCR Tech-nique RAPD assay used in the current study showed thatvarious ELF treatments and nonexposed one exhibited dis-tinctive qualitative and quantitative alterations in the RAPDprofiles based on number of gene products the amplifiedDNA sizes their intensities and appearance or disappearance


of DNA bands that led to generation high levels of DNApolymorphism The number of amplification products maybe related to the number and direction of genome sequencescomplementary to the primer High levels of polymorphismgenerated byRAPD in the present study are considered to be abetter parameter formeasuring genetic variation patterns andreflect heritable changes in the nucleotide sequences both incoding and noncoding regions [41]

These DNA polymorphisms may result from DNA struc-tural changes within base-pair sequences of DNA betweenoligonucleotide primer binding sites in the genome duringDNA replication or gene expression under genotoxic stressesof electric field such as a nucleotide substitution within atarget site base pair insertion or deletion of a DNA-fragmentwithin the amplified regions rearrangements of genomicDNA or inversions translocations and transpositions ofgenes which result in the loss or gain of DNA bands resultingin different DNA lengths and consequently highly levelpolymorphisms [18]

On the other hand appearance of new DNA bands(unique bands) is usually resulting from different DNAstructural changes (insertions of the amplified DNA bandsbreaks transpositions additions etc) while disappearanceof some bands (polymorphic bands) and band intensity maycorrelate with the level of photoproducts in DNA templateafter ELF exposure which can reduce the number of bindingsites for Taq polymerase and the starting copy number ofa particular DNA sequence within genome [17] or may bedue to the deletion of DNA segments The present findingsupports this claim that DNA polymorphisms detected byRAPD can be considered as a powerful biomarker assay fordetection of the genotoxic environmental stress like ELF

45 Detection of DNA Damage Based on Single Cell GelElectrophoresis (Comet Assay) Technique Comet assay datashowed remarkable variations in the extent of DNA damagein maize nuclei exposed to ELF treatments which reflectspecific action on nDNAdamage producing increasedmigra-tion of DNA fragments (comet tail) from the nucleus (comethead) This confirmed that the increased exposure of maizeseedlings to ELF led to produce DNA lesions in their cellsThis DNA damage may be directly as a results of depositionof energy in cells or indirectly as a result of free radicalformations as ROS and oxidative damage that they arecapable of inducing several major types of DNA lesions suchas DNA strand breaks deletion or insertion of base pairspyrimidine dimers cross-links and base modification suchas alkylation and oxidation leading to DNA damage [42]These lesions cause increased DNA migration losing higheramount of DNA as comets from ELF maize nuclei TheDNA damage results in various molecular and physiologicaleffects such as reduced protein synthesis cell membranedestruction and damage to mitochondrial proteins whichaffects growth and development of the whole organism [43]

5 Conclusions

The current study concluded that each ELF exposing timehad special interference with maize cells exhibiting wide

range of genetic and oxidative action on protein and DNAmacromolecules Also it concluded that the biomarkers usedshould be combined and augmented for reliable estimates ofgenetic variability after exposure of economic plants to ELFstressors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

The authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor the funding of this research through the Research GroupProject no RGP-VPP-231They alsowish to express their deepthanks to Professor Magda Hanafy Biophysics DepartmentScience College ZagazigUniversity for her effort in perform-ing ELF exposure

References

[1] J Maluszynska and J Juchimiuk ldquoPlant genotoxicity a molec-ular cytogenetic approach in plant bioassaysrdquo Arhiv za HigijenuRada i Toksikologiju vol 56 no 2 pp 177ndash184 2005

[2] M E Amin ldquoEffect of different nitrogen sources on growthyield and quality of fodder maize (Zea mays L)rdquo Journal of theSaudi Society of Agricultural Sciences vol 10 no 1 pp 17ndash232011

[3] W F Grant and E T Owens ldquoZea mays assays of chemi-calradiation genotoxicity for the study of environmental muta-gensrdquoMutation Research vol 613 no 1 pp 17ndash64 2006

[4] M Racuciu and J Rom ldquo50Hz frequency magnetic field effectson mitotic activity in the maize rootrdquo Romanian Journal ofBiophysics vol 21 no 1 pp 53ndash62 2011

[5] M S Hanafy H A Mohamed and E A Abd El-Hady ldquoEffectof low frequency electric field on growth characteristics andprotein molecular structure of wheat plantrdquo Romanian Journalof Biophysics vol 16 no 4 pp 253ndash271 2006

[6] M Filek M Hołda I Machackova and J Krekule ldquoThe effectof electric field on callus induction with rape hypocotylsrdquoZeitschrift fur Naturforschung Section C Journal of Biosciencesvol 60 no 11-12 pp 876ndash882 2005

[7] E Jovanov and A G Volkov ldquoPlant electrostimulation andaata acquisitionrdquo in Plant Electrophysiology Method and CellElectrophysic XII A G Volkov Ed chapter 2 pp 45ndash67Springer Berlin Germany 2012

[8] N T Sedighi M Abedi and S E Hosseini ldquoEffect of electricfield intensity and exposing time on some physiological prop-erties of maize seedrdquo European Journal of Experimental Biologyvol 3 no 3 pp 126ndash134 2013

[9] L M J AL-Shamma ldquoEffect of electric shock onmorphologicaltraits yield yield components and protein content of threevarieties of Faba beans (Vicia faba L)rdquo Iraqi Journal of Sciencevol 54 no 1 pp 86ndash96 2013

[10] Y Y Bai H U Axiang Y X Yucai and Y C Bai ldquoOriginalmechanism of biological effects of electrostatic field on cropseedsrdquo Transactions of the Chinese Society of Agricultural Engi-neering vol 19 no 2 pp 49ndash51 2003


[11] M Karaca ldquoIsozymes as biochemicalmarkers in plant geneticsrdquoInternational Journal of Agricultural Sciences vol 3 pp 851ndash8612013

[12] M A Hamoud A R El-Shanshory Y M Al-Sodany and MS G El-Karim ldquoGenetic diversity among Ipomoea carnea jacqPopulations from different habitats types in Nile-Delta regionof Egyptrdquo The Egyptian Journal of Experimental Biology vol 1pp 1ndash10 2005

[13] S Ranjan A Poosapati H Vardhan et al ldquoSeed storage proteinprofile of few leguminous grains grown in india using SDS-PAGErdquo International Journal of Advanced Biotechnology andResearch vol 4 no 4 pp 505ndash510 2013

[14] KDymek PDejmekV Panarese et al ldquoEffect of pulsed electricfield on the germination of barley seedsrdquo LWTmdashFood Scienceand Technology vol 47 no 1 pp 161ndash166 2012

[15] H W Hailu D H Kristiyanto A R A ALatawi and S MRaqib ldquoIsozyme electrophoresis and morphometri comparisonof reed (imperata cylindrical) adaptation to different altitudesrdquoInternational Journal of Innovative Research in Science Engineer-ing and Technology vol 3 no 5 pp 12387ndash12394 2014

[16] N R Abdel-Hameid M A M Elanany A A A Haiba andE A A AbdEl-Hady ldquoRelative mutagenicity of some foodpreservatives on plant cellsrdquo Australian Journal of Basic andApplied Sciences vol 5 no 12 pp 2817ndash2826 2011

[17] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques appliedto genotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006

[18] J Welsh and M McClelland ldquoGenomic fingerprinting usingarbitrarily primed PCR and a matrix of pairwise combinationsof primersrdquoNucleic Acids Research vol 19 no 19 pp 5275ndash52791991

[19] G KekecM S Sakcali and I Uzonur ldquoAssessment of genotoxiceffects of boron on wheat (Triticum aestivum L) and Bean(Phaseolus vulgaris L) by Using RAPD analysisrdquo Bulletin ofEnvironmental Contamination and Toxicology vol 84 no 6 pp759ndash764 2010

[20] D Gjorgieva T Kadifkova Panovska T Ruskovska K Bacevaand T Stafilov ldquoInfluence of heavy metal stress on antioxidantstatus and DNA damage in Urtica dioicardquo BioMed ResearchInternational vol 2013 Article ID 276417 6 pages 2013

[21] M Dikilitas A Kocyigit and F Yigit ldquoA molecular-based fastmethod to determine the extent of DNA damages in higherplants and fungirdquo African Journal of Biotechnology vol 8 no14 pp 3118ndash3127 2009

[22] M P Hojilla-Evangelista and R L Evangelista ldquoEffects ofcooking and screw-pressing on functional properties of CupheaPSR23 seed proteinsrdquo Journal of the American Oil ChemistsrsquoSociety vol 83 no 8 pp 713ndash718 2006

[23] R Saraswati T Matoh P Phupaibul et al ldquoIdentification ofSesbania species from electrophoretic patterns of seed proteinrdquoJournal of Tropical Agriculture vol 70 pp 282ndash285 1993

[24] U K Laemmli ldquoCleavage of structural proteins during theassembly of the head of bacteriophage T4rdquo Nature vol 227 no5259 pp 680ndash685 1970

[25] AA E-HAly EMHusseinMROmarA-AAbdel-Mongyand K A Abd-Elsalam ldquoEffect of amino acid content on thelevel of cotton seed colonization by mycoflorardquo InternationalJournal of Agriculture and Biology vol 13 no 1 pp 71ndash76 2011

[26] T Pirman V Stibilj J M A Stekar and E Combe ldquoAmino acidcomposition of beans and lentilrdquo Zbornik Biotehniske fakultete

Univerze v Ljubljani Kmetijstvo Zootehnika vol 78 no 1 pp57ndash68 2001

[27] D A N Majumder L Hassan M A Rahim et al ldquoAnalysisof genetic diversity in mango (Mangifera indica L) usingisozymetic polymorphismrdquo African Journal of Biotechnologyvol 11 pp 15310ndash15323 2012

[28] J G Scandalios ldquoGenetic control of multiple molecular formsof enzymes in plants a reviewrdquo Biochemical Genetics vol 3 no1 pp 37ndash79 1969

[29] C R Shaw and R Prasad ldquoStarch gel electrophoresis ofenzymesmdasha compilation of recipesrdquo Biochemical Genetics vol4 no 2 pp 297ndash320 1970

[30] R C Graham Jr U Lundholm and M J Karnovsky ldquoCyto-chemical demonstration of peroxidase activity with 3-amino-9-ethyl- carbazolerdquo The Journal of Histochemistry and Cytochem-istry vol 13 pp 150ndash152 1965

[31] J L BrewbakerM D Upadhya YMakinen and TMacdonaldldquoIsozyme polymorphism in flowering plants III Gel elec-trophoretic methods and applicationrdquo Physiologia Plantarumvol 21 no 5 pp 930ndash940 1968

[32] A A Mouemar and J Gasquez ldquoEnvironmental conditionsand isozyme polymorphism in Chenopodium album Lrdquo WeedResearch vol 23 no 3 pp 141ndash149 1983

[33] M K Mishra N S Rani A S Ram and H L Sreenath ldquoAsimplemethod of DNA extraction from coffee seeds suitable forPCR analysisrdquo African Journal of Biotechnology vol 7 no 4 pp409ndash413 2008

[34] K Wilson and J Walker Principles and Techniques of Bio-chemistry and Molecular Biology Cambridge University PressCambridge UK 2005

[35] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990

[36] J Juchimiuk A Gnys and J Maluszynska ldquoDNA damageinduced bymutagens in plant and human cell nuclei in acellularcomet assayrdquo Folia Histochemica et Cytobiologica vol 44 no 2pp 127ndash131 2006

[37] M Sadia S A Malik M A Rabbani and S R PearceldquoElectrophoretic characterization and the relationship betweensome Brassica speciesrdquo Electronic Journal of Biology vol 5 no1 pp 1ndash4 2009

[38] L Mondini A Noorani and M A Pagnotta ldquoAssessing plantgenetic diversity by molecular toolsrdquo Diversity vol 1 no 1 pp19ndash35 2009

[39] G S Sanghera P K Malhotra G S Sidhu V K Sharma BB Sharma and R Karan ldquoGenetic engineering of crop plantsfor enhanced antioxidants activityrdquo International Journal ofAdvanced Research Technology vol 2 no 5 pp 428ndash458 2013

[40] L Szabados and A Savoure ldquoProline a multifunctional aminoacidrdquo Trends in Plant Science vol 15 no 2 pp 89ndash97 2010

[41] S Lal K N Mistry S D Shah R Thaker and P B VaidyaldquoGenetic diversity assessment in nine cultivars of Catharanthusroseus from Central Gujarat (India) through RAPD ISSR andSSRmarkersrdquo Journal of Biological Research vol 1 pp 667ndash6752011

[42] P L Olive and J P Banath ldquoThe comet assay a method tomeasure DNAdamage in individual cellsrdquoNature Protocols vol1 no 1 pp 23ndash29 2006

[43] A B Britt ldquoMolecular genetics of DNA repair in higher plantsrdquoTrends in Plant Science vol 4 no 1 pp 20ndash25 1999


physiological properties of maize grains The study of [9]concluded that an electric current directed to the plant cellsmay increase the capacity of some enzymes associated withgenes already inside the nucleus The results of [10] showedthat the electrostatic fields with certain intensity can increasethe content of free radicals in plant cells

It is important for detection of genotoxicity and muta-genic potential of various types of environmental stresssuch as ELF on crop plants to understand their biologicalconsequences and their molecular action on protein andDNAof plant cell by introducing biochemical molecular andmolecular cytogenetic assays The biochemical assays relatedto protein-based markers such as nonenzymatic protein(storage proteins) enzymatic proteins (isozymes which areone of themost commonly used proteinmarkers) and aminoacids composition [11] these markers detect the codingregions and the variations at the gene product level

Proteins are primary gene products of active structuralgenes their size and amino acids sequence are the directresults of nucleotide sequences of the genes hence anyobserved variation in protein systems induced by any muta-gen is considered a mirror for genetic variations [12] Varia-tion in theDNAcoding sequences frequently causes variationin the primary conformation of the proteins Determinationof protein molecular weight (MW) via polyacrylamide gelelectrophoresis (PAGE) in the presence of sodium dodecylsulfate (SDS) is a universally used method in biomedicalresearch [13] concluded that electrophoresis (SDS-PAGE) ofproteins can be economically used to assess genetic variationand relation in germplasm and also to differentiate mutantsfrom their parent genotypes Some studies used SDS-PAGEfor detection of alterations in protein profiles occurringduring exposure to electric field [5 14]

Isozymes are direct gene products that show differencesin protein-coding genes and arise from multiple gene locicoding for structurally distinct polypeptide chains The dif-ferent proteins that make up a set of isozymes can catalyzethe same reaction but differ in molecular weight theirkinetics substrate affinity number of subunits and aminoacid sequences electric charge or electrophoretic mobility[11 15] Protein variants in isozyme analysis are separatedchemically by nondenaturing native polyacrylamide gel elec-trophoresis (Native PAGE) resulting in different bandingpatterns (zymograms) and electrophoretic mobilities thatvisualized using an enzyme-specific staining mixture whichincludes a substrate cofactor and oxidized salt [11] Isozymeanalysis is a powerful biochemical technique with numerousapplications in mutagenic potential in plant cell [15 16]

Recently advances in molecular biology have led tothe development of a number of selective and sensitiveassays for detection of the variations at the DNA level anddifferences can clearly be shown when comparing DNAfingerprints from individuals exposed andor nonexposed togenotoxic agents [17] Molecular assays related to DNA-basedmolecular techniques like Random Amplified PolymorphicDNA (RAPD) are a very sensitive method of screeningfor nucleotide sequence polymorphisms that are randomlydistributed throughout the genome in both coding and

noncoding regions and repeated or single copy (unique)sequences [18] RAPD-PCR technique has been successfullyused to detect DNA damage and mutations in some plantspecies induced by various types of genotoxic agents [19 20]

In recent years a new molecular cytogenetic-based assaythe Comet or single cell gel electrophoresis (SCGE) assaycan theoretically be applied to every type of eukaryotic cellincluding plant cells for detecting DNA damage inducedby genotoxic agents [1] The basic principle of this assayis to determine the DNA breaks by measuring the DNAdamage which is quantified by the proportion of DNA whichmigrates out of the nuclei towards the anode when individualcells or isolated nuclei embedded in single cell agarose gelelectrophoresis (comet assay) [21] After electrophoresis adamaged nucleus takes on the appearance of ldquocomet-likerdquoshape (with a head the nuclear region and a tail whichcontains DNA fragments that migrate from the nucleus) [21]

The objective of this study to evaluate the possible influ-ence of electric field intensity and exposing time on Zea maysseedlings on the levels of biochemical analyses of proteinsusing SDS-PAGE isozymes amino acids composition andon the levels of DNA damage using RAPD-PCR and cometassay

2 Materials and Methods

21 Plant Material and Germination Maize grains (hybridnumber 323 from Agronomy Research Department FieldCrops Institute Agriculture Research Center Giza Egypt)were used as plant material in this study Grains werescreened for viability and uniformity size and divided intotwo groups (A and B) 50 grains of each group were sterilizedand germinated until reached seven-day-old seedlings inearthenware pot 60 cm in diameter containing soil obtainedfrom the agriculture field Seedlings of group (A) are exposedto the electric field exposure system while seedling of group(B) was maintained without exposure (unexposure samples)

22 The Electric Field (ELF) Exposure In the laboratory themaize seedlings of group (A) were exposed to an alternat-ing electric field of 50Hz frequency and 6 kVm strengthgenerated between two parallel aluminum electrodes of 60 times50 times 2 cm dimensions fixed horizontally above and belowseedlings for 1 3 and 5 days The electric field was deriveddirectly from 50Hz high voltage setup transformer manufac-tured by the ldquoCenter of Scientific and Electronic EquipmentMaintenance Faculty of Science Cairo Universityrdquo Maizeseedlings exposed to ELF for 1 3 and 5 days termed asthree treatments (T1 T3 and T5) while seedlings of group(B) which non-exposed to the electric field termed as zerotreatment (T0) Ten seedlings from exposed and nonexposedseedlings were harvested and thoroughly cleaned with freshwater followed by distilled water for quantitative removalof any foreign particles and then dried in air conditionsuntil completely dried and then subjected to biochemical andmolecular analyses




















T0 T1 T3 T5 M (kDa)250

148

98

64

59

36




3 Results




Rows Mol weights







16 3404 6 1277 4681 22 4 851 8462












8 6154 2 1538 10 7692 0 0 100









9 4737 1 526 10 5263 2 1053 8333










10 5250 3 1875 13 8125 00 00 100














10 6667 2 1333 12 8000 00 00 100




4 Discussion



0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es









Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References

































































T0 T1 T3 T5 M (kDa)250

148

98

64

59

36




3 Results




Rows Mol weights







16 3404 6 1277 4681 22 4 851 8462












8 6154 2 1538 10 7692 0 0 100









9 4737 1 526 10 5263 2 1053 8333










10 5250 3 1875 13 8125 00 00 100














10 6667 2 1333 12 8000 00 00 100




4 Discussion



0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es









Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References




















































T0 T1 T3 T5 M (kDa)250

148

98

64

59

36




3 Results




Rows Mol weights







16 3404 6 1277 4681 22 4 851 8462












8 6154 2 1538 10 7692 0 0 100









9 4737 1 526 10 5263 2 1053 8333










10 5250 3 1875 13 8125 00 00 100














10 6667 2 1333 12 8000 00 00 100




4 Discussion



0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es









Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References
















































Rows Mol weights







16 3404 6 1277 4681 22 4 851 8462












8 6154 2 1538 10 7692 0 0 100









9 4737 1 526 10 5263 2 1053 8333










10 5250 3 1875 13 8125 00 00 100














10 6667 2 1333 12 8000 00 00 100




4 Discussion



0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es









Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References






















































8 6154 2 1538 10 7692 0 0 100









9 4737 1 526 10 5263 2 1053 8333










10 5250 3 1875 13 8125 00 00 100














10 6667 2 1333 12 8000 00 00 100




4 Discussion



0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es









Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References























































10 5250 3 1875 13 8125 00 00 100














10 6667 2 1333 12 8000 00 00 100




4 Discussion



0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es









Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References























































10 6667 2 1333 12 8000 00 00 100




4 Discussion



0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es









Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References















































0 1

1

2 3 4 50

Band 1

Zymograms Zymograms

ZymogramsZymograms

Band 2Band 3Band 4

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5

010203040506070809

0 1

1

2 3 4 50

Band 1Band 2Band 3

Band 4Band 5Band 6

02

04

06

08

12

0 1

1

2 3 4 50

Band 1Band 2

Band 3Band 4

02

04

06

08

12

T0 T1 T3T2 T0 T1 T3T2

T0 T1 T3T2 T0 T1 T3T2

(a) (b)

LAP-Rf

valu

esPE

R-Rf

valu

es

CAT-Rf

valu

esES

T-Rf

valu

es









Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References





















































Sum 13252 2809 2598 2327 2265






Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References















































Table7RA

PDprod

uctsof

DNAextractedfro

mEL

Fexpo

sedandno

nexp

osed

maize

seedlin

gsusing5rand

omprim

ers

Prim

ercode

Prim

erssequence

(51015840rarr

31015840)

Amplicon

leng

ths

(bp)

Electricfield

treatments

Polymorph

icband

sNum

bero

fmon

omorph

icband

s

of

total

polymorph

ismNum

bero

fscorableb

ands

Totalbands

Num

bero

fun

ique

band

s

Num

bero

fno

nuniqu

ebands

Num

bero

ftotal

polymorph

icband

s

Lane

T 0Lane

T 1Lane

T 2Lane

T 3P-02

GGACC

CAAC

C992ndash395

33

34

131667

53846

21539

77692

176

98750

P-06

ACCTG

AAC

GG

929ndash

450

43

33

131667

75385

176

99

6923

176

990

P-08

GTG

TGCCC

CA

865ndash426

33

44

141795

64286

21429

85714

1714

8889

P-10

GGTCT

ACA

CC

764ndash

214

54

35

172180

158824

1588

169412

mdashmdash

100

P-14

CTTCC

CCA

AG

800ndash

120

63

57

212692

188571

1476

199048

mdashmdash

100

OveralltotalofD

NAband

sineach

treatment

2116

1823

7851

6535

7897

587436

3385

9508

2692

2051

2308

2949




T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References

















































T0 T1 T3 T5Ladder

P-2

1000900800700600500400300200100


P-6

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-10

1000900800700600500400300200100


P-8

1000900800700600500400300200100

(bp)

T0 T1 T3 T5Ladder

P-14

1000900800700600500400300200100

(bp)



T0 T1

T3 T5











5 Conclusions





Acknowledgments


References















































T0 T1

T3 T5











5 Conclusions





Acknowledgments


References



















































5 Conclusions





Acknowledgments


References

















































































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