IET Nanobiotechnology

Research Article

Synthesis and characterisation of metalnanoparticles and their effects on seedgermination and seedling growth incommercially important Eruca sativa

IET Nanobiotechnol., pp. 1–7& The Institution of Engineering and Technology 2016

ISSN 1751-8741Received on 13th May 2015Revised on 8th October 2015Accepted on 27th October 2015doi: 10.1049/iet-nbt.2015.0039www.ietdl.org

Mehreen Zaka1, Bilal Haider Abbasi1 ✉, Latif-ur Rahman2, Afzal Shah2, Muhammad Zia1

1Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan2Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan

✉ E-mail: bhabbasi@qau.edu.pk

Abstract: The synthesis, characterisation and application of metal nanoparticles have become an important and attractivebranch of nanotechnology. In current study, metallic nanoparticles of silver, copper, and gold were synthesised usingenvironment friendly method (polyols process), and applied on medicinally important plant: Eruca sativa. Effects ofapplication of these nanoparticles were evaluated on seed germination frequency and biochemical parameters of planttissues. Seeds of E. sativa were germinated on Murashige and Skoog (MS) medium incorporated with variouscombinations of nanoparticles suspension (30 µg/ml). Phytotoxicity study showed that nanoparticles could inducestress in plants by manipulating the endogenous mechanisms. In response to these stresses, plants release variousdefensive compounds; known as antioxidant secondary metabolites. These plants derived secondary metaboliteshaving a great potential in treating the common human ailments. In the authors study, small-sized nanoparticlesshowed higher toxicity levels and enhanced secondary metabolites production, total protein content, total flavonoidscontent and total phenolics content.

1 Introduction

Nanotechnology is a very versatile field covering almost all existingbranches of science. Nanotechnology is famed as 21st centuryscience, as it has found applications in physics, chemistry,biology and many other fields [1]. In the last decade manyadvances in nano-biotechnology were noted. Nanotechnology hasthe potential to improve the agriculture with new tools andenhance the plants ability to absorb nutrients. In recent years,scientists have started focusing on employment of nanoparticles inagriculture for the betterment of crop quality and also forincreased growth and disease control in plants. Studies onbiological effects of nanoparticles in higher plants are increasingday-by-day.

Eruca sativa commonly known as rocket plant is the well-knownfast growing herb of family Brassicaceae. Despite of its popular useas vegetable, rocket plant is also considered as useful medicinal plantsince ancient times [2]. Its phytochemistry is very rich and it is agood source of fibres, flavonoids, carotenoids, vitamin C andglucosinolates [3]. It has shown anti-tumour, anti-ulcer andhepatoprotective activities [4, 5].

Plant tissue culture is considered as promising alternate toconventional breeding. It establishes high-frequency regenerationprotocols and improves the quality of economically importantplants. E. sativa has shown adaptability to in vitro regenerationprotocols [6]. Tissue culture techniques well explained the need ofbiotechnology by solving food security and agricultural productionissues, and highlighted the use of biotechnological techniques forhaving genetically improved varieties [7].

Stress enhanced secondary metabolites content in medicinalplants. Elicitors are reported to effect production of thesesecondary metabolites positively [8]. Recently, exploitation ofnanomaterials is reported to effect biosynthesis of economicallyand commercially viable secondary metabolites in medicinal plantspecies [9]. An increase in shoot/root ratio was observed byapplying different nanoparticles in soil. As the change in growth

behaviour was observed after the certain time period, so it wasassumed that nanoparticles do not directly influence plants butindirectly alters the mechanisms [10].

As plants possess cell walls as a primary interacting site forforeign particles, so entry of nanoparticles become difficult. Themechanism by which nanoparticles enters the plants is still poorlydefined. Yet, nanoparticles have ability to magnify changes in cellstructures and molecules and also the defensive mechanisms.Effects of nanoparticles depend on its physical and chemicalproperties and include solubility of toxic nanoparticles andproduction of reactive oxygen species [11].

Chemical composition of nanoparticles is very much responsiblefor the toxic effects of metal nanoparticles (MNPs) on plants andalso the stress caused by surface, shape and size of particle [12].Moreover, toxic nanoparticles may increase the production ofreactive oxygen species and hydroxyl radicals that damage the cellmembranes and as a result permeability is altered. As a result, entryof nanoparticles into plant cells become easier and stress inducedby the particles results in secondary metabolites production [13].

Up to now, only few studies have been reported on application ofnanomaterials in plant tissue culture. According to Safavi [14],nanosilver and nano-titanium dioxide can be used as antimicrobialagent in plant tissue culture medium.

No report is available on application of MNPs in tissue culturemedia to evaluate the seed germination frequency and plant growthin Eruca. Hence, we have evaluated the effect of chemicallysynthesised copper (Cu), silver (Ag) and gold (Au) MNPs ongrowth parameters and secondary metabolites content in E. sativa.

2 Materials and methods

2.1 Plant source and surface sterilisation

Seeds of E. sativa were provided by Dr. Bilal Haider Abbasi,Department of Biotechnology, Islamabad, Pakistan. Seeds were

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surface sterilised according to the protocol of Abbasi et al. [15], i.e.seeds were sterilised by immersion in 0.1% mercuric chloride for 1min followed by three time washing with distilled water.

2.2 Metal nanoparticles

MNPs of Au, Cu and Ag were synthesised by environment friendlypolyols process. Reagents used were Cu chloride (CuCl2) (98%), Agnitrate (AgNO3) (99%), hydrogen Au chloride (HAuCl4) (99%),ethylene glycol (C2H6O2) (98%), polyethyleneimine (2%). All ofthese chemicals were purchased from Thermo fisher scientific Inc.(USA), except polyethyleneimine that was obtained from AcrosOrganics.

2.2.1 Preparation of Cu nanoparticles: About 10 mlpolyethyleneimine (2%) was added to 20 ml of CuCl2 solution(1 mM). This mixture was purged with argon blow for 30 min. Thesolution was heated in oven at 175°C for 30 min. The appearanceof bluish black colour showed the formation of Cu nanoparticlesthat was confirmed by ultraviolet (UV)–visible spectroscopy.

2.2.2 Preparation of Ag nanoparticles: About 10 ml ofpolyethyleneimine was added to 20 ml of AgNO3 solution (1 mM)and was heated at 150°C for 15 min, the appearance of blackishcolour shows the formation of Ag nanoparticles that was alsoconfirmed by UV–visible spectroscopy.

2.2.3 Preparation of Au nanoparticles: For preparation of Aunanoparticles 10 ml polyethyleneimine was poured to 20 ml ofHAuCl4 (1 mM) solution and was heated at 100°C for 15 min.The appearance of yellowish back colour evidenced the formationof Au nanoparticles that was confirmed by UV–visiblespectrophotometry.

All the three nanoparticles Cu, Ag and Au were stable for sixmonths. These nanoparticles were characterised by usingcharacterisation techniques which include UV spectrophotometer(Cary 100, Varian, Shimadzu, Tokyo Japan), X-ray diffraction(XRD) (Brucker SMART APEX diffractometer) and transmissionelectron microscopy (TEM) (Philips, Holland Tecnai 20).

2.3 Seed germination protocol

MNPs were suspended directly in distilled water using sonication.30 µg/ml suspension was prepared of each MNPs by sonication indistilled water for 30 min according to the protocol described bySavithramma et al. [16]. This suspension was added to MS0medium in concentration of 3 ml/30 ml of MS medium with thehelp of a micropipette. Sterilised E. sativa seeds were incubatedinto conical flasks containing autoclaved solidified MS media.Seed germination was observed in ∼2–4 days and first data onseed germination was collected after 14 days of inoculation. Datawas collected after every 2 weeks and the experiment wasconducted for 42 days, i.e. 6 weeks.

2.4 Seed germination parameters

2.4.1 Percentage germination frequency: The percentagegermination was recorded after every 2 weeks. Seeds were takenas germinated when radicle had emerged from seed coat [17]

percentage germination (%) = number of germinated seeds

× 100/total number of seeds

2.4.2 Root and shoot length: Root and shoot length of seedswere recorded after every 2 weeks starting from inoculation date.Mean root and shoot length were compared in the form of bar charts.

2.4.3 Seedling vigour index: The seedling vigour index (VI)was calculated by using the method suggested by Abdul-Baki and

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Anderson [18] and expressed as index numbers [17]

seedling vigor index = [root length (cm)+ shoot length (cm)]

× germination (%)

2.5 Analytical methods

2.5.1 Determination of free radical scavenging activity by2,2-diphenyl-1-picrylhydrazyl (DPPH) method: The freeradical scavenging assay (FRSA) of methanolic extracts ofE. sativa was measured in terms of hydrogen donating or radicalscavenging ability using the stable radical DPPH. Protocol of Leeet al. [19] was followed with some modifications according towhich the test extracts were prepared in methanol, so the DPPHwas also prepared in methanol. DPPH solution was added insample solution according to the defined calculated concentrationsseparately. These solution mixtures were kept in dark for 30 min(incubation period) at room temperature. After 30 min, theabsorbance was measured at 515 nm using micro-plate reader.Lower absorbance of the reaction mixture indicated higher FRSA.Finally, the radical scavenging activity was calculated aspercentage of DPPH discolouration using the equation

% scavenging DPPH free radical = 100× (1− AE/AD)

where AE is the absorbance of the solution, when extract has beenadded at a particular level and AD is the absorbance of the DPPHsolution with nothing added (blank, without extract).

2.5.2 Evaluation of total antioxidant capacity (TAC) byphosphomolybdenum method: The TAC of the methanolextract was evaluated by the phosphomolybdenum methodaccording to the procedure described by Prieto et al. [20].According to this assay Mo (VI) is reduced to Mo (V) by theextract and green phosphate/Mo (V) complex is formed at acid pH.A 50 µl of extract was combined with 450 µl of reagent solution(0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mMammonium molybdate). The extract was then put into water bath at95°C for 90 min and then samples were cooled at room temperature.The absorbance of the reaction mixture was measured at 695 nmusing a spectrophotometer against blank. Methanol was used as theblank. The antioxidant activity is expressed as the number of gramequivalent of ascorbic acid [21]. Some of the compounds which arenormally not measured as antioxidants have some chain breakingantioxidant activity also, so TAC assay is helpful to measure allthese compounds collectively including complex interactionsoccurring during chain breaking antioxidants. Generally, TAC isdecreased under oxidative stress condition and administration ofchain breaking antioxidants increases antioxidant capacity [22].

2.5.3 Determination of total phenolics and total flavonoidscontent: Total phenolics content (TPC) was determined followingthe Folin–Ciocalteu (FC) method by Singleton and Rossi [23]. Brieflythree solutions were prepared for TPC activity. Ten times dilutedsolution of FC reagent in distilled water, 6% sodium carbonate(Na2CO3) solution in distilled water and 4 mg/ml solution of gallicacid in methanol were prepared. About 25, 20, 15, 10 and 5 µ/mlconcentrations of gallic acid were used as positive control and 20 µlof Dimethyl sulfoxide (DMSO) as negative control. Absorbancewas measured at 630 nm by using microplate reader.

Total flavonoids content (TFC) was determined by following theprotocol of Haq et al. [24]. Stock solutions of 10% aluminiumchloride in distilled water, 1 M potassium acetate in distilled waterand 4 mg/ml quercetin in methanol were prepared. About 20 µl ofmethanol was used as negative control and 40, 20, 10, 5 and 2.5µg/ml of final concentration of quercetin as positive control.Absorbance was recorded at 415 nm using microplate reader.

2.5.4 Estimation of total protein content: For total proteincontent estimation method of Lowry et al. [25] was used. Three

IET Nanobiotechnol., pp. 1–7& The Institution of Engineering and Technology 2016

reagents, i.e. A [1 g sodium–potassium (Na–K) tartrate × 4 H2O, 50g Na2CO3, 250 ml 1 N sodium hydroxide (NaOH)], B (2 g Na–Ktartrate × 4H2O, 1 g CuSO4.5H2O, 0.1 N NaOH) and C (FCreagent ten times diluted with distilled water) were used. About40 µl of protein extract was taken and 36 µl of reagent A wasadded and then incubated for 10 min at 50°C and after cooling thereaction mixture 4 µl of reagent B was added and again incubatedunder same conditions. After cooling reaction mixture at 25°C,120 µl of reagent C was added. The absorbance was then recordedat 650 nm using microplate reader. Standard curve of bovine serumalbumin was prepared at series of 25, 50, 75, 100 and 125 µg/ml used.

2.5.5 Superoxide dismutase (SOD) and peroxidase (POD)activities: SOD activity assay was performed according tomethod of Ullah et al. [26] with minor changes. Inhibition ofphotochemical reduction of nitro blue tetrazolium (NBT) by SODis the principle of assay. About 1 mM ethylenediaminetetraaceticacid, 130 mM methionine, 0.02 mM riboflavin, 0.75 mM NBT and50 mM phosphate buffer (pH 7) was used. Blank was prepared bymixing all the chemicals except enzyme extracts in the samequantity. Reaction mixture was exposed to fluorescent light for 7min. optical density (OD) was taken at 560 nm.

Activity of enzyme was calculated applying Lambert–Beer law

A = ELC

where A = absorbance; ε = extinction coefficient (6.39 mM−1 cm−1);L = length of each wall (0.25 cm); C = concentration of enzyme(value of C measured in nM/min/mgFW); and FW = fresh weightof the sample.

For POD activity method of Lagrimini [27] was used. About 27.5mM hydrogen peroxide (H2O2) (10×), 100 mM guaiacol (10×),distilled water, 1% polyvinyl-pyrrolidone. About 50 mMK-phosphate buffer of pH 7 were used as chemicals. Reactionmixture of 200 microlitre was prepared using these chemicals.Blank was prepared by mixing of 60 µl K-phosphate buffer, 20 µlguaiacol, 100 µl distilled water and 20 µl H2O2. Absorbance wasrecorded at 470 nm with a gap of 20 s and activity was calculatedaccording to the formula used for SOD activity.

Fig. 1 UV–visible spectra of

a Cu nanoparticles prepared by polyols process using C2H6O2 as a solventb Ag nanoparticles prepared by polyols process using C2H6O2 as a solventc Au nanoparticles prepared by polyols process using C2H6O2 as a solvent

Table 1 Data obtained from UV–visible spectroscopy, XRD and TEM

Nanoparticles Maximum absorbance for 1 mMsolution

lmax Average size,nm

Cu 0.26 550 15Ag 0.68 451 18Au 0.82 485 20

3 Results and discussion

The synthesised nanoparticles were characterised by UV–visiblespectroscopy, XRD and TEM. UV–visible spectra confirmed theformation of nanoparticles showing single Plasmon band havingmaximum wavelength closed to the value that have already beenpublished by Rahman et al. [28]. Fig. 1 shows UV–visible spectraof Cu (Fig. 1a), Ag (Fig. 1b) and Au (Fig. 1c) nanoparticles. Theirmaximum absorbance for 1 mM solution and maximumwavelengths are shown in Table 1. Such suspension wascentrifuged and the powdered nanoparticles were analysed byXRD as shown in Fig. 2. XRD helped us in calculating the size ofnanoparticles as well as their crystallinity. In Fig. 2a, 111 isconsidered as desired peak. In Fig. 2b, 111, 200 and 210 weretaken as the desired peaks, whereas in Fig. 2c 111 and 200 weretaken as desired peaks. Applying Debye Scherrer equation,average size of the nanoparticles was calculated as shown inTable 1. XRD also revealed that nanoparticles are crystalline andmostly to be cubic. Fig. 3 exhibits the TEM photographs of pureCu, Ag and Au nanoparticles. A typical TEM image of Cunanoparticles in Fig. 3a are seemed to be non-spherical; however,shapes of Ag and Au nanoparticles are spherical as shown inFigs. 3b and c. Interestingly, size shown by these images wassame as calculated from XRD. The size of nanoparticles evaluatedfrom XRD as well as TEM can be seen in Table 1.

3.1 Seed germination frequency

Seed germination and root elongation are easy and widely used inphytotoxicity test and it is simple, low cost and suitable to

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unstable chemicals or samples [29, 30]. Seed germinationfrequency was calculated at different time intervals such as 14, 28and 42 days (Table 2). Among all metals, Ag showed maximumpositive response and seed germination frequency was recorded as

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Fig. 2 XRD spectra of

a Cu nanoparticles solidified by centrifugationb Ag nanoparticles solidified by centrifugationc Au nanoparticles solidified by centrifugation

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Fig. 3 TEM images of

a Cu nanoparticles prepared by polyol process using C2H6O2 as a solvent and solidified by centrifugationb Ag nanoparticles prepared by polyol process using C2H6O2 as a solvent and solidified by centrifugationc Au nanoparticles prepared by polyol process using C2H6O2 as a solvent and solidified by centrifugation

Table 3 Effect of metal nanoparticles on root length, shoot length andseed VI of E. sativa values are average of three replications ± SE

Germinationperiod

Treatments Rootlength, cm

Shootlength, cm

VI

after 14 days MS0 1.9 ± 0.09 1.5 ± 0.07 340 ± 17Cu 1.7 ± 0.08 2 ± 0.1 296 ± 14.8Ag 2.3 ± 0.11 2.5 ± 0.12 484 ± 24.2Au 1.2 ± 0.06 1.2 ± 0.06 192 ± 9.6

after 28 days MS0 1.5 ± 0.07 2.5 ± 0.12 164 ± 8.2Cu 1.9 ± 0.09 2.8 ± 0.14 225.5 ± 11.27Ag 4.5 ± 0.22 6.3 ± 0.31 866.6 ± 43.33Au 2.3 ± 0.11 4.5 ± 0.22 498.6 ± 24.93

73%. As reported by El-Temsah and Joner [31], Ag nanoparticles ofdifferent sizes and in different concentrations effect the seedgermination frequency differently, small-sized particles exert moreinhibitory effects on seed germination. In present study, timeinterval was taken as limitation after expecting that with theprogression of time more aggregation of nanoparticles in cellcompartments happens, so there is a possibility of nanoparticles ofsame size to impact in an unexpected way. As reported by Leeet al. [32] Cu nanoparticles are toxic to two species of mung bean(Phaseolus radiatus) and wheat (Triticum aestivum), so in thepresent paper it also showed inhibitory effect and seed germinationwas 56%. Au also showed inhibitory effect and germinationfrequency was 53% but in case of Au as time increases thegermination frequency was also increased and toxicity of Audecreased. In previous reports, it is concluded that toxicity ofnanoparticles depends on two different actions, i.e. chemicalcomposition and release of toxic ions and stress caused by surfacesize or shape of nanoparticles [33]. Therefore, in the present paperit is also observed that Cu of small size and toxic nature bothcontributed in the inhibition of seed germination. Ag showedpositive response and results were almost such as the controlseeds. Au responds late, it showed inhibition in start and thenshowed positive response after 28 days but as time increased up to42 days again seed germination was inhibited due to the release oftoxic ions and stress caused by nanoparticles.

after 42 days MS0 5 ± 0.25 9.8 ± 0.49 1480 ± 74Cu 2.1 ± 0.10 4.3 ± 0.21 384 ± 19.2Ag 2.3 ± 0.11 6.7 ± 0.33 900 ± 45Au 2.1 ± 0.10 3.4 ± 0.17 330 ± 16.5

3.2 Root and shoot elongation and seed VI

It is inferred that Ag nanoparticles stimulated the root and shootlength and seed VI at different time periods such as 14, 28 and 42days with exception in root length after 42 days as root length wasdecreased again after 42 days (Table 3). Savithramma et al. [16]reported that Ag period of time at optimum concentration. Reasonmaybe Ag nanoparticles penetrated and induce new pores whichhelped influx the nutrients in seed for rapid growth. Chemicalcomposition of Ag nanoparticles and their precise size and shapedoes matter in the response showed by these particles.

Cu and Au nanoparticles have reduced the root and shoot length inE. sativa and showed more stress than Ag nanoparticles. Theseresults are similar to the previously reported results [32]. After 42

Table 2 Effect of MNPs on in vitro seed germination of E. sativa valuesare an average of five replications ± SE

Serialnumber

Treatments Percentageseed

germinationafter 14 days

Percentageseed

germinationafter 28 days

Percentageseed

germinationafter 42 days

1 control(MS0)

76.6 ± 3.3 40 ± 2 100 ± 5

2 Cu 56.6 ± 2.8 46.6 ± 2.3 60 ± 33 Ag 73.3 ± 3.6 80 ± 4 100 ± 54 Au 53.3 ± 2.6 73.3 ± 3.6 60 ± 3

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days of inoculation, mostly root and shoot lengths were reduced ascompared with control which can be justified by the reason thatwith the passage of time more ions from particles were releasedand accumulated in roots and shoots more surface ions wereexposed and they were more toxic to plantlets.

3.3 Percentage DPPH radical scavenging activity andTAC

After exposing the E. sativa seeds to different nanoparticles,significant radical scavenging activity was noted in plantlets of

Fig. 4 Percentage DPPH radical scavenging activity and TAC of E. Sativaplantlets against different treatments of nanoparticles

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Fig. 5 Total flavonoids and TPC after 6 weeks of time intervalFig. 7 SOD activity and POD activity of fresh matter after 6 weeks ofE. Sativa treated with MNPs

E. sativa. TAC and DPPH radical scavenging activity of the plantletstreated with nanoparticles is shown in Fig. 4. Correlation wasestablished between radical scavenging activity and TAC. Agnanoparticles have reduced the antioxidant capacity compared withcontrol and Au nanoparticles again started increasing the capacityas compared with Ag nanoparticles.

The contradiction observed between DPPH radical scavengingactivity and TAC in Au treatment can have the reason that TAC iscollective value of antioxidants and numerous other compoundswhich are involved in some chain breaking antioxidant activity[22]. As we are applying MNPs and after a long period ofinoculation accumulation of these particles occurs and ions arereleased in cell compartments, so DPPH radical scavengingactivity can be lesser in Cu-treated seeds which have releasedmore ions and these ions already had scavenged the radicals.

3.4 TFC and TPC

As reported by Krishnaraj et al. [34], Ag nanoparticles enhanced thetotal phenol content in plants; however, AgNO3 producedconsiderably higher phenolic content. In current paper, totalphenolics and flavonoids content were lesser in Ag/Au treatedplantlets than Cu treatment (Fig. 5). It is reported earlier that Cunanoparticles are inducing more stress and thus more secondarymetabolites are biosynthesised for protection of plant cells.

Fig. 6 Total protein content of fresh matter after 6 weeks of E. Sativatreated with MNPs

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3.5 Total protein content, SOD and POD

As shown in Fig. 6, total protein content is comparatively lesser inplantlets treated with Cu nanoparticles and in Ag and Au treatedplantlets total protein content is comparable with control, i.e. MS0.Higher total protein content correlates with higher SOD and PODactivities. Stress could be the main cause behind this enhancedtotal protein content. SOD and POD activities were stimulated bynanoparticles. However, these parameters were hampered by Cuions presence in medium [35]. As shown in Fig. 7, higher levelsof SOD and POD activities indicating their protective role againstAu nanoparticles-induced stress [34].

4 Conclusions

It is concluded from the current paper that metals at nanoscaleshowed different behaviours than metals in bulk form. Chemicalcomposition, size and shape of nanoparticles matters a lot to affectplants [33]. Small-sized nanoparticles are more toxic than largersize nanoparticles and among all the combinations applied Cunanoparticles are more stress inducing than Ag and Aunanoparticles because of the fact that Cu at bulk level is also moretoxic than Au and Ag. These results can be helpful for futurestudies on phytotoxicity of various nanomaterials especiallyengineered nanoparticles under certain conditions. Up till nowthere are very few reports on using nanomaterials in plant tissueculture as a stress inducing factor. Using the results reported inthis paper further study in domain of ecotoxicity can be conductedto check environment friendly nanoparticles and nanoparticles withharmful effects.

5 Acknowledgments

The authors are grateful to Miss Shagufta Shafique, National Centrefor Bioinformatics and Mr. Tariq Khan, Department ofBiotechnology at Quaid-i-Azam University for their assistancethroughout the manuscript. Mehreen Zaka acknowledges thesupport provided by Higher Education Commission, Pakistan.

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