Journal of Cell and Molecular Biology 8(2): 87-94, 2010 Research ArticleHaliç University, Printed in Turkey. http://jcmb.halic.edu.tr

A study on the occurrence of genetically modified soybean and maizefeed products in the Jordanian market

Hiyam Al-ROUSAN¹, Nisreen Al-HMOUD¹,², Bassam HAYEK² and Mohammed IBRAHIM¹,²*

¹Royal Scientific Society, Amman, Jordan²Princess Sumaya University for Technology, Amman, Jordan(* author for correspondence; prof_mibrahim@yahoo.com)

Received: 08 August 2010; Accepted: 05 November 2010

AbstractThere are concerns on the bio-safety of genetically modified (GM) products and indications on transfer of GM sequences from GM feed products to the food chain. Thus the idea of this study was aimed to survey the status of occurrence of genetically modified soybean and maize feed products sold in the Jordanian market by qualitative polymerase chain reaction based methods. Genomic DNA was extracted by Cetyltrimethylammonium bromide (CTAB) method from maize and soybean-based feed products. The molecular genomic analysis was designed to identify plant species specific genes of maize (zein) and soybean (lectin), expression control specific systems 35S promoter and nos terminator, and specific transgenic events. According to the results obtained in this analysis, 100% of soy and 18.18% of maize used in production of feed were genetically modified and were unlabeled. The results of present investigation confirmed the occurrence of the genetic event hsp70 exon1/intron1 region of MON810 in the identified genetically modified maize feed products. The possibility of transfer of GM sequences from feed to food chain is discussed.

Keywords: GM detection, GM soy bean, GM maize, 35S promoter, nested PCR, food chain

Genetik Olarak Modifiye Edilmiş Soya Fasülyesi ve Mısırın Oluşturulması

ÖzetGenetiği değiştirilmiş (GD) ürünler ve GD dizilerinin GD beslenme ürünlerinden beslenme zincirlerine transferi üzerine belirtiler, biyo-güvenlik açısından kaygılar yaratır. Bu sebeple, bu çalışmanın fikri Ürdün marketinde satılan genetiği değiştirilmiş soya fasulyesi ve mısır yiyecek ürünlerinin ortaya çıkması durumunu kalitatif polmeraz zincir reaksiyonu tabanlı metodlar ile araştırmaktı. Mısır ve soya fasulyesi beslenme ürünlerinden genomik DNA, setiltrimetilamonyum bromid (CTAB) metodu ile izole edildi. Moleküler genomik analiz mısır (zein) ve soya fasulyesi (lectin) bitki türlerine spesifik genleri, 35S promotör ve nos terminatör gibi ifade kontrollü spesifik sistemleri ve spesifik transgenik olayları tayin etmek için tasarlandı. Bu analizden elde edilen sonuçlara göre, soyaların %100’ünün ve mısır besin örneklerinin %18.2’sinin genetiği değiştirildi. İçiçe (nested) PZR deneyleri, genetiği değiştirilmiş mısır besin ürünlerinde MON810 olayının hsp 70 ekson 1/intron 1bölgesinde meydana geldiğini doğruladı, ancak genetiği değiştirilmiş soya fasulyesi besin ürünlerinde Roundup-ReadyTM’nin Cp4 EPSPS’ye özgü olayına rastlanmadı.

Anahtar sözcükler: GD tespiti, GD soya fasulyesi, GD mısır, 35S promoter, içiçe PZR

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Introduction

Genetically modified (GM) soybean and maize crops have become a reality in agriculture, and are used in the production of food and feed (Borlaug, 2000; Demont and Tollens, 2004; Flachowsky et al., 2005; Vain, 2006; Al-Hmoud et al., 2010). There are several GM maize and soybean products available worldwide, for example Roundup ReadyTM soy resistant to Roundup herbicide and MON810 YieldGard Corn which expresses Bt toxin (Aulrich et al., 2001; Greiner and Konietzny, 2008; Ujhelyi et al., 2008). Though, there are global debates concerning the safety of genetically modified (GM) products currently used in food and feed. In this respect, a recent study on rats fed on three main commercialized genetically modified (GM) maize (NK 603, MON 810, MON 863), which are used in food and feed, showed effects mostly associated with the kidney and liver, other effects were also noticed in the heart, adrenal glands, spleen and haematopoietic system (De Vendômois et al., 2009). The results of other investigation showed that the transgenic crops expressing Cry1Ab protein at 5000 ppb may affect food consumption or learning processes of bees, and thereby may impact honey bee foraging efficiency (Ramirez-Romero et al., 2008). Moreover, there are scientific debates on the effects of GM crop cultivation on the environment considering the impacts caused by cultivation practices of modern agricultural systems (Sanvido et al., 2007; Kawata et al., 2009; Knispel and McLachlan, 2010). In this connection, Dunfield and Germida (2004) showed microbial diversity can sometimes be altered when associated with transgenic plants; however, they emphasized that their results indicated these effects are minor in comparison with environmental factors such as sampling date and field site. Moreover and as a consequence of wide use of GM plants, there are apprehension about horizontal gene transfer (HGT) of GM genetic elements to living cells and followed by its expression and as a result natural ecosystems could be affected (van den Eede et al., 2004; Cerdeira and Duke, 2006). In this respect, there are contradictory reports on possible transfer of GM elements to other organisms. An early investigation

could not confirm long-term persistence of transgenic elements under field conditions for up to 2 years and also no construct-specific sequences were detected by dot blot hybridizations of bacterial isolates (Gebhard and Smalla, 1999). The results of other study showed that transgenic DNA could not be detected in milk from cows receiving up to 26.1% of their diet as herbicide (glyphosate)-tolerant soyabean meal (Phipps et al., 2002). A more recent study, carried out by Agodi and coworkers in order to develop a valid protocol by Polymerase Chain Reaction (PCR) and multi-component analysis for the detection of specific DNA sequences in milk, it focused on GM maize and soybean to assess the stability of transgenic DNA after pasteurization treatment and determine the presence of GM DNA sequences in milk samples collected from the Italian market (Agodi et al., 2006). Their results from the screening of 60 samples of 12 different milk brands demonstrated the presence of GM maize sequences in 15 (25%) and of GM soybean sequences in 7 samples (11.7%). Based on these findings the objectives of our study are to survey the status of GM feed in the Jordanian markets and to identify the genetic events of identified GM maize and soybean. Thus, the results will form the base for future studies in Jordan for investigating possible transfer of GM sequences in the food chain.

Materials and methods

Maize and soybean feed products

Maize (11 samples) and soy (9 samples) used in preparation of feed were obtained during the period from January 2010 and June 2010 from Department of Animal Wealth Laboratories/Ministry of Agriculture, Food and Drug Testing Administration, Royal Scientific Society Testing Laboratories, and local market in Amman, Jordan. Whereas standard maize blank (ERM-BF413a), genetically modified MON 810 maize (BF413f), standard soy blank (BF410a) and genetically modified Roundup ReadyTM soy (ERM-BF410e) were provided by Dr. Eric Kubler, University of Applied Sciences Northwestern Switzerland. These samples are originally purchased from European Commission, DG JRC, IRMM, Belgium.

Genetically modified feed products

Genomic DNA extraction

Cetyltrimethylammonium bromide (CTAB) method was used for Genomic DNA extraction from ground feed samples of maize and soybean; the concentration and purity of extracted DNA were determined according to the reported methods (Querci et al., 2006; Al-Hmoud et al., 2010).

Primers

The primers and their sequences used in the PCR amplification experiments were reported in previous work (Al-Hmoud et al., 2010). The primers were obtained from Alpha DNA/Canada.

DNA amplifications

The reported PCR amplification conditions by Querci et al., (2006) were followed. The volume of PCR reaction mixture was 50 µl and contained: 5 µl of 10x PCR Buffer, 5 µl of 25 mM MgCl2, 0.25 µl of Taq DNA polymerase obtained as TopTaq TM PCR kit (Qiagen/Germany), 2.5 µl 4 mM dNTPs (Promega/Germany), 1.25 µl of 20µM of each primers, 32.75µl nuclease-free water and 2 µl of extracted DNA. The conditions for PCR amplifications experiments for CaMV 35S promoter and Nopaline synthase (nos) terminator primers used for detection of GM maize and soybean feed products were: 3 min initial denaturation at 95º C followed by 50 cycles of 25 s denaturation at 95ºC, 30 s annealing at 62ºC, 45 s extension at 72ºC and a final 7 min extension at 72ºC. Parameters for PCR amplifications experiments for maize specific gene (zein) were: 3 min initial denaturation at 95ºC followed by 40 cycles of 1 min denaturation at 96ºC, 1 min annealing at 60ºC, 1 min extension at 60ºC and a final 3 min extension at 72ºC. Parameters for PCR amplifications experiments for soy specific gene (lectin) were: 3 min initial denaturation at 95ºC followed by 40 cycles of 30 s denaturation at 95ºC, 30 s annealing at 63ºC, 30 s extension at 72ºC and a final 3 min extension at 72ºC.

Nested PCR

The amplification conditions of nested PCR

experiments were carried out according to the standard protocols (Querci et al., 2006). Parameters for PCR amplifications experiments for specific primers mg1/mg2 and mg3/mg4 used for detection of specific genetic event of MON810 GM maize were: 3 min initial denaturation at 95ºC followed by (35 cycles for mg1/mg2 and 40 cycles for mg3/mg4) of 45 s denaturation at 95ºC, 50 s annealing at 60ºC, 50 s extension at 72ºC and a final 3 min extension at 72ºC. Whereas Parameters for PCR amplifications experiments for primers GM05/GM09 and GM07/GM08 used for detection of RRS specific genetic event were: 3 min initial denaturation at 95ºC followed by 25 cycles for GM05/GM09 and 35 cycles for GM07/GM08 of 30 s denaturation at 95ºC, 30 s annealing at 60ºC, 40 s extension at 72ºC and a final 3 min extension at 72ºC.

Each run included positive control which represented DNA extracted from standard blank and GM maize or soy feed products (MON810 or Roundup ReadyTM soy), and No-template control containing all PCR mix component except DNA. The amplifications were performed in the Applied Biosystem Thermocycler 9902 with a heating lid.

Gel electrophoresis

The amplified DNA fragments and DNA marker ladder of 100 bp (Qiagen) were separated using 1.5% agarose gel and visualized under UV light after staining with ethidium bromide for molecular size determinations in base pair (bp) of DNA fragments (Sambrook and Russell, 2001).

Results

Maize and soybean food products

Twenty samples of maize and soy used in preparation of feed and available commercially in Amman, Jordan were obtained during the period from January to June 2010. 45% of studied feed samples were soy and 55% were maize. The majority of the collected feed products were of unknown origin (66.67 and 81.81% of soy and maize respectively); these represented 75% of 20 samples used in the study (Table 1).

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Molecular genomic analysis

Differences were observed in the concentrations and purities of extracted genomic DNA by CTAB method from feed samples. The lowest yield of extracted DNA was 1 µg/ml; whereas the highest yield was 44 µg/ml. The purity of extracted DNA showed variations between 1.42 and 1.76.

Specific primers were used to detect species specific genes of maize and soybean; these tests were performed for confirming the plant origin of the feed products. Species specific gene (zein) for maize was identified in all maize feed products, the size of detected amplified DNA fragment by primers ZEIN3F/ZEIN4R was 277 bp, whereas soy specific gene (lectin) was identified in soybean feed samples by primers GM03F/GM04R, the size of amplified DNA fragment was118 bp (Table 2).

The results of PCR experiments for the detection of GM maize and soybean were obtained following

the amplification of transgenic DNA sequences by specific primers. It was possible to identify two GM maize out of eleven investigated maize feed products; whereas all tested soy feed products were genetically modified (Table 1). DNA fragment for the 35S promoter (123 bp), which is a common indicator of GM plant, was identified in GM maize and soy feed products (Figure 1). The results of nested PCR experiments showed that the detected GM maize feed products in this study contain the hsp70 exon1/intron1 region of maize MON810, this event was detected by primers mg1 and mg2 which gave amplicon of 401 bp, while the size of amplified DNA fragment from 401 bp amplicon by primers mg3 and mg4 was 187 bp (Figure 2). On the other hand it was not possible to detect Cp4 EPSPS event of soybean when using following primers: GMO5 and GMO9 which should give amplicon of 447 bp, and GMO7 and GMO8 which should give amplified DNA fragment of 169 bp.

Table 2. Sizes in base pairs (bp) of PCR amplified DNA fragments when using specific primers for detectionof GM soy and maize.

CaMV 35S promoter: p35S-cf3, Fp35S-cf4, R

Nopaline synthase (nos) terminator: HA-nos 118-f, FHA-nos 118-r, R

Maize specific(Zein): ZEIN3, FZEIN4, R

Soybean specific(lectin): GM03, FGM04, R

MON 810 specific (nested PCR): mg1 mg2

MON 810 specific (nested PCR): mg3mg4

Soybean RRS (nested PCR): GMO5GMO9

123

118

277

118

401

149

447

Detected

Not detected

Detected

Detected

Detected

Detected

Not detected

Primers Amplified DNA fragment detectedin this studyLength of amplified DNA fragment (bp)

Table 1. Country of origin of analyzed maize and soybean feed products. The number of identified GM maizeand soybean are shown between brackets.

Feed product category

Country of origin

No. of tested

samples

% of detected

GM Soybean Argentine 2(2)

Syria 1(1) 100 Unknown 6(6)

Maize USA 2(0) 18.18 Unknown 9(2)

Genetically modified feed products

Discussion

The detection of GMOs represents a new field of diagnostics, in which much progress has already been achieved with DNA assays. Currently PCR methods are considered the main assays used for detection of GMOs, this technology targets and amplifies a specific DNA sequences in GMOs products. This can be carried out by selecting the

target DNA sequences and the appropriate PCR conditions; hence one can achieve generic or specific detection of GMOs (Holst-Jensen and Berdal, 2004). Primers have been designed based on the regulatory sequence or structural gene in the inserted gene fragment; these designed primers possess some specific characteristics and can be used for product screening and product specificity detection (Querci et al., 2006, Al-Hmoud et al.,

Figure 1. Detection of PCR amplified 35 S sequence (123 bp) in maize and soybean products. Electrophoresis was performed on 1.5% agarose gel and run with 3 volt cm-1. Lane L, indicates the 100 base pair ladder, Lane1 represents PCR negative control, Lane 2 and 3 represent blanks of maize and soy, lanes 4 and 5 represent standard GM maize and soybean respectively, lanes 6 and 7 represent identified GM soy as representative of nine soy samples used in this study, lanes 8, 9, and10 represent non GM maize identified in this study, lane 11 and 12 represent identified GM maize in this study.

Figure 2. Detection of amplified nested products in maize feed products, 401 bp sequence identified by mg1/mg2 primers; 149 bp sequence identified by mg3/mg4 primers. Electrophoresis was performed on 1.5% agarose gel and run with 3 volt cm-1. Lane L indicates the 100 base pair ladder, lanes 2 and 3 represent MON 810 specific event detected in standard BF413f (5%), Lanes 4 and 5 represent detection of MON810 event in GM feed maize sample detected in this study.

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2010). In this study qualitative PCR methods were used

for the detection of GM maize and soybean used in feed production, specific primers were used to identify common sequence of 35S promoter and specific genetic events of GM products. Our results demonstrated the detection of lectin gene of soybean, in the nine samples confirming that tested samples were soybean products. 100% of soy samples were identified as carrying amplified DNA fragments of 35S promoter region, which is a common indicator of GM plant. However, as shown by nested PCR experiments when using specific primers GMO5 and GMO9, these samples could not be confirmed as carrier of specific transgenic Cp4 EPSPS event of Roundup-ReadyTM. It might be possible to suggest that the GM soy feed might be a carrier of other events. In this context it is worth mentioned the results of our previous investigation showed 33% GM soy were found in food sold unlabeled in Jordan’s market (Al-Hmoud et al., 2010).

It was possible in this study to identify the common GM 35S sequence and specific genetic event in 18.18% of studied maize feed samples. Nested PCR experiments showed that the GM maize used as feed contain the hsp70 exon1/intron1 region of maize MON810. The identification of this genetic event came as a result of detection of the amplified DNA fragment by primers mg1 and mg2 with molecular size equivalent to 401 bp, while the size of amplified DNA fragment by primers mg3 and mg4 was 149 bp. The obtained results in this study are believed being the first survey for GM feed products in Jordanian market. It is interesting to note the percentage of GM maize detected in present investigation in feed samples is similar to the percentage of GM maize detected in food samples reported in our previous study (Al-Hmoud et al., 2010), these results might suggest similar sources of maize are used for food and feed.

There are rising international concerns about possible potential risks of GM products, this is because of the results of various studies which have reported possible harmful effect might be caused from the use of GMO or their products (De Vendô-

mois et al., 2009, Séralini et al., 2007, 2009) and the possibility of transfer GM sequences through food chain. In this respect, a number of studies failed to identify GM sequences in DNA of milk, meat, or eggs derived from livestock receiving GM feed ingredients (Phipps et al., 2002). Other investigators showed that by using high sensitivity screening methodology it was possible to identify GM maize and GM soy sequences in milk samples obtained from cows fed on GM feed (Agodi et al., 2006). It is worth noting that there is a serious scientific debate about the criteria for testing significant adverse health effects arising from the consumption of GM food or feed products. Scientists are concerned about possible GM health risks, and are calling for more serious standardized tests such as those used for pesticides or drugs (Séralini et al., 2009).

The detected GM feed products in this study are not labeled, so there is need for implementation of labeling regulations for feed products containing materials derived from genetically modified organ-isms, similar to the regulations for labeling of GM food products (Gruere and Rao, 2007; Al-Hmoud et al., 2008). It is worth mentioned there are regula-tions issued by Ministry of Environment for GM food and feed products, yet the results of this study and other recent investigation (Al-Hmoud et al., 2010) showed presence of unlabeled genetically modified maize and soya food and feed products in the Jordanian market. These results show the impor-tance for implementing national policy for GM monitoring in Jordan. Recently the National GM Laboratory was established in Royal Scientific Society to perform GM monitoring in Jordan.

Furthermore, we aim that our study will be continued to identify the other genetic events of GM feed products and to start an investigation on the transfer of GM sequences from GM feed products to the food chain in Jordan and if possible in the region.

Conclusions

CTAB method was found appropriate for DNA extraction from maize and soybean used for feed; it gave adequate yield and purity of extracted DNA for PCR analysis. Common GM sequence indicator of 35S promoter was detected in 55 % unlabeled GM

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soy and maize used in feed products, specific trans-genic event of MON810 was detected 18.18% of GM maize used in feed production, whereas 100% of soy used in feed production were genetically modified. There is need for monitoring policy for the GM feed products and to investigate possible transfer of genetic events in food chain.

Acknowledgments

M. Ibrahim is grateful to the International Institute of Education for the fellowship. The research team is grateful to Dr. Eric Kubler, University of Applied Sciences Northwestern Switzerland for his assistance in providing the GMO standard samples of maize and soy.

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