Italian Journal of Food Safety 2014; volume 3:1628

Monitoring the presence due to its gelling properties that result in the dissolved being unstable. For Correspondence: Maria Giovanna Tilocca, of genetically modified potato this reason, the two components must be Dipartimento di Igiene degli Alimenti, Istituto EH92-527-1 (BPS-25271-9) separated. Separating and amy- Zooprofilattico Sperimentale della Sardegna, via in commercial processed food lose in potato starch requires energy and Vienna 2, 09123 Sassari, . water consumption which makes it uneco- Tel./Fax: +39.079.2892332. E-mail: [email protected] Maria Giovanna Tilocca,1 nomical. Although potatoes by their nature produce a mixture of amylose and amy- Gianfranca Serratrice,1 Key words: Solanum tuberosum, Amflora, lopectin, Amflora was genetically engineered Maria Agostina Oggiano,1 Amylopectin, GBSS, RT PCR. to produce only the amylopectin component 1 2 Maria Rosalba Mancuso, Ilaria Mascia, of starch. Amflora was developed by silenc- Received for publication: 30 April 2013. 1 1 Edoardo Marongiu, Bruna Vodret ing the expression of the starch synthase Revision received: 17 September 2013. 1Dipartimento di Igiene degli Alimenti, protein (GBSS), using antisense strategy to Accepted for publication: 26 September 2013. Istituto Zooprofilattico Sperimentale eliminate the expression of amylose. A gene This work is licensed under a Creative Commons 2 conferring kanamycin resistance (nptII) della Sardegna, Sassari; Dipartimento Attribution 3.0 License (by-nc 3.0). di Agraria, Università degli Studi was used as a selectable marker. Since di Sassari, Italy Amflora potato is not fit for human consump- ©Copyright M.G. Tilocca et al., 2014 tion, it is solely grown to improve the indus- Licensee PAGEPress, Italy trial applications of the potato (paper indus- Italian Journal of Food Safety 2014; 3:1628 try, textile industry, in adhesives and in con- doi:10.4081/ijfs.2014.1628 Abstract struction materials) (Abdallah, 2010). The placing of the GM potato EH92-527-1 (BPS-25271-9) on the market for cultivation The Amflora (EH92-527-1) potato is a and industrial uses has been approved by the genetically modified (GM) potato in which real-time PCR method for detection of T-nos Commission Decision 2010/135/EU, in accor- only starch of the amylopectin form is pro- (Permingeat et al., 2002). dance with Directive 2001/18/EC of the duced. This has been achieved by interven- and of the Council ing with the biosynthesis of starch in this (, 2010a); whereas variety of potato. The Amflora potato is sole- the Commission Decision 2010/136/EU, has Results ly grown for the purposes of enhancing its authorised the placing on the market of feed industrial application. Although the Amflora produced from GM potato EH92-527-1 (BPS- potato is not fit for human consumption, the The amplification plot showed the pres- 25271-9) and the adventitious and technical- ence of an 88bp fragment of the UGPase gene presence of the potato itself or any of its ly unavoidable presence of the potato in food from Solanum tuberosum in all samples derived products in the food chain cannot be and other feed products, under Regulation examined (Figure 1a). The PCR screening of excluded, it should be considerate adventi- (EC) No 1829/2003 of the European nos terminator (T-nos) DNA sequence, of tious or technically unavoidable and can be Parliament and of the Council (Commission nopaline synthase from tume- accepted in a proportion no higher than Decision, 2010b). faciens, confirmed the total absence of 0.9%. To achieve the goal of our work we The aim of this study was to screen com- Amflora potato in food matrices investigated, analysed forty-five potato-derived products mercial processed containing potato derived as showed in Figure 1b and Table 2. to evaluate transgenic potato presence by products from both nation and international real time polymerase chain reaction, obtain- markets, in order to monitor and verify the ing negative results. In order to verify the adventitious presence of GM Amflora. correct application of the law and to assure the quality for the consumer, it is necessary Discussion and Conclusions to continue GM monitoring to verify the adventitious presence itself in food. Materials and Methods In order to verify the correct application of the law, it is required to constantly monitor- Forty-five samples of potato-derived prod- ing food matrices to safeguard the con- ucts from different markets were investigat- sumers. The European Regulations set the Introduction ed (Table 1). DNA extraction from frozen and labelling requirements for all the GM organ- dried potato was carried out in accordance ism-containing products (food and feed), Amflora (EH92-527-1) is a genetically with CTAB method validated by European with a tolerance threshold established at modified (GM) potato variety, Solanum Union Reference Laboratory for GM food and 0.9% for authorised GM organisms and at tuberosum L. of the chemical company BASF feed. The DNA of each sample was examined 0.5% for GM organisms under authorisation (Ludwigshafen, ). The potato has a to verify potato-DNA by Real Time PCR by procedure (Regulation EC N.1829/2003; modified starch composition. In the conven- amplification of UDP-glucose pyrophosphory- European Commission, 2003). Amflora is tional potato starch is composed of two main lase (UGPase) gene (Savini et al., 2006, been marketed for industrial use but not parts amylose and amylopectin, 20 and 80% 2010). The potential presence of GM potato authorised for human consumption, thus its respectively. While amylopectin is the in food matrices was detected by PCR presence can only be accepted with a toler- required starch component for industrial screening for the nos terminator (T-nos) ance threshold below 0.9%, as an adventi- purposes due to its thickening properties, DNA sequence of nopaline synthase from tious presence. The method used for DNA- amylose is the unwanted starch component Agrobacterium tumefaciens, according to extraction of starch products from food

[Italian Journal of Food Safety 2014; 3:1628] [page 57] Short Communication

Table 1. Potato-derived products investigated and their country of origin. matrices is particularly suitable to provide a very good performance as confirmed by Potato-derived Number Country of origin products of samples (manufactured or produced) UGPase endogenous gene amplification, for all sample analysed (Figure 1a and Table 2). Raw potato 5 Italy An high-quality potato-DNA is essential in Potato flour 5 Italy/Germany order to achieve the subsequently real time Mashed potato (frozen and dried) 5 Italy/Germany/ PCR assays to verify the potential presence of GM material. Results are able to confirm Crisps 5 Italy/Germany the total absence of Amflora for the samples Frozen fries chips 5 Italy/Germany/Canada analysed so far. It is necessary to continue Frozen raw potatoes 5 Italy/Germany/Canada GM monitoring so as to assure consumers Bread-potato 5 Italy about the absence of Amflora in both local Homemade potato-sweet 5 Italy and imported food products. Homemade potato-pasta 5 Italy

Figure 1. Amplification plot of UDP-glucose pyrophosphorylase gene (a) and nos terminator DNA sequence (b) in all samples exam- ined.

[page 58] [Italian Journal of Food Safety 2014; 3:1628] Short Communication

Table 2. Results obtained by real time polymerase chain reaction by amplification of UDP-glucose pyrophosphorylase gene and nos ter- minator DNA sequence in all samples investigated. Potato-derived products Number of investigated UGPase number of nos terminator (T-nos) samples positive samples DNA sequence Raw potato 55Absence Potato flour 55Absence Mashed potato (frozen and dried) 55Absence Crisps 55Absence Frozen fries chips 55Absence Frozen raw potatoes 55Absence Bread-potato 55Absence Homemade potato-sweet 55Absence Homemade potato-pasta 55Absence UGPase, UDP-glucose pyrophosphorylase.

04/03/2010. and DNA extraction of potato. Method for References European Commission, 2010b. Commission DNA extraction from freeze and dried Decision authorising the placing on the potato tubers. Joint Research-European Abdallah N, 2010. Amflora great expectations market of feed produced from the geneti- Commission GMOs Unit cally modified potato EH92-527-1 (BPS- for GM crops in Europe. GM Crops 1:109- ed., Varese, Italy. Available from: 12. 25271-9) and the adventitious or techni- http://gmo-crl.jrc.ec.europa.eu/sum- European Commission, 2003. Regulation of cally unavoidable presence of the potato in the European Parliament and of the food and other feed products under maries/EH92-527-1-%20Validation Council of 22 September 2003 on geneti- Regulation (EC) N. 1829/2003 of the %20Report.pdf cally modified food and feed, European Parliament and of the Council, Savini C, Foti N, Mazzara M, Charles Delobel 1829/2003/EC. In: Official Journal, L 268, 2010/136/EU. In: Official Journal, L 53, C, Van Den Eede G, 2010. Event-specific 18/10/2003. 04/03/2010. method for the quantification of event European Commission, 2010a. Commission Permingeat HR, Reggiardo MI, Valleyos RH, EH92-527-1 potato using real-time PCR. Decision concerning the placing on the 2002. Detection and quantification of Validation report and protocol. PCR reac- market, in accordance with Directive in grains by multiplex and real- tions set up and amplifications conditions. 2001/18/EC of the European Parliament time PCR. J Agr Food Chem 50:4431-6. Joint Research-European Commission and of the Council, of a potato product Savini C, Foti N, Mazzara M, Charles Delobel (Solanum tuberosum L. line EH92-527-1) C, Van Den Eede G, 2006. Event-specific Biotechnology GMOs Unit ed., Varese, genetically modified for enhanced content method for the quantification of event Italy. Available from: http://gmo-crl.jrc.ec. of the amylopectin component of starch, EH92-527-1 potato using real-time PCR. europa.eu/gmomethods/docs/QT-EVE-ST- 2010/135/EU. In: Official Journal, L 53, Validation report and protocol-sampling 001.pdf

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