Logo Impetus. PCR Results. 2. PCR detection of maize DNA and MON 810. DNA in DNA etracted from purified Bt-maize pollen standard (Genetic-ID) (Fig. 2). 3.
Development of PCR Methods for the Detection of airborne Pollen of GMO-maize and GMO-Pollen Monitoring in the vicinity of a Nature Reserve in Germany F. Hofmann (1); R. Epp (2); A. Kalchschmid (3); L. Kruse (2); U. Kuhn (4); B. Maisch (3); E. Müller (3); U. Schlechtriemen (5); W. von der Ohe (6); W. Wosniok (7) Ökologiebüro, TIEM Integrierte Umweltüberwachung GbR, D-28205 Bremen; (2 ) Impetus GmbH & Co. Bioscience KG, D-27572 Bremerhaven; (3) Genetic ID (Europe) AG, D-86167 Augsburg; (4) Büro Kuhn, D-28357 Bremen; (5) Sachverständigenbüro, TIEM Integrierte Umweltüberwachung GbR, D-37176 Nörten-Hardenberg; (6) Nds. Landesamt für Verbraucherschutz und Lebensmittelsicherheit, Institut für Bienenkunde D-29221 Celle; (7) Institut für Statistik, Universität Bremen, D-28355 Bremen
Pollen DNA extraction and PCR
Introduction The dispersal of airborne GMO pollen is of interest in conjunction with isolation distances for the cultivation of genetically modified plants in the vicinity of conventional cultivation, organic cultivation and nature reserves. For GMO monitoring the sampling of pollen by technical (aerosol sampling) and biological methods has been successfully standardised in the German Guideline VDI 4330 Part 3 and 4. Currently two corresponding Technical Specifications in CEN/TC 264 WG 29 are worked out. Whereas sampling and quantitative microscopic analysis of pollen is highly standardized, DNA analysis of pollen matrices to detect GMO input by PCR is considered as difficult and was so far not achieved satisfactorily. Complexity of pollen matrices, low concentration of target pollen in pollen samples and low DNA extraction efficiency due to e.g. inhibiting substances in aerosol samples represent main problems. Now two PCR based methods for the detection of GMO-maize pollen developed independently by two experienced GMO testing laboratories were compared in conjunction with a study of the dispersion of Mon 810 maize pollen in the direct vicinity of a nature reserve in Brandenburg, Germany.
Pollen sampling
PMF/Sigma 2
Technical and biological pollen sampling has been performed in 2007 according to VDI Guideline 4330 parts 3 (2007) & 4 (2006) using 8 passive samplers (pollen mass filter PMF/Sigma-2) and 6 honey bee colonies over the flowering period of maize (mid of July until mid of August) at 3 locations in a nature reserve area in Brandenburg, Germany, where maize and GMO-maize (MON 810) has been grown in the vicinity.
For quality assurance towards standardization, the performance of the analytical procedures was assured step by step: (1) At first both labs determined their general PCR performance using Mon 810 maize DNA solution, (2) following this purified Bt-maize pollen gained directly from the anthers in the field were analyzed (purified Bt-maize pollen standard, suspensions) before (3) finally the pollen samples collected in the nature reserve were analyzed.
PCR Results 1. Detection of Bt-maize MON 810: PCR performance of both laboratories using Mon 810 maize DNA solution (Fig. 1)
Bee colonies
Sample preparation The technical sampler PMF provides a complex bio aerosol matrix containing pollen. Disturbing aerosol components may lead to inhibition problems in subsequent DNA analysis. Thus separation techniques were applied for purification of PMF pollen samples and enrichment of maize pollen in these samples (micro-sieving cascade, fraction 60-120 µm). Honey bee baskets represent a very inhomogeneous matrix, that needs homogenization for further analysis. Although honey bees are attracted mainly by many other plants, maize pollen is sampled regularly, depending on general pollen supply, with resulting concentrations of maize pollen below 15% by separation techniques (as see above) has been applied.
Pollen samples were extracted using a CTAB-based method with prolonged lysis time and additional column purification (Impetus) or mechanical pollen disruption (BeatBeater) followed by SDS-Lyse and alcohol precipitation (Genetic ID). For RT-PCR detection of maize DNA, HMG gene specific primers (Impetus) and Adh gene specific primers (Genetic ID) were used. RT-PCR detection of Mon 810 DNA was performed with primers published by Hernandez, M. et al. 2003, Transgenic Res.12:179-189 (Impetus) and primers published by the CRL (http://gmo-crl.jrc.it/summaries/Mon810_validation_report.pdf) (Genetic ID).
PMF bio aerosol sample
Maize pollen
2. PCR detection of maize DNA and MON 810 DNA in DNA etracted from purified Bt-maize pollen standard (Genetic-ID) (Fig. 2). The graphs show the transition range of the linear sector towards detection limit. For GMO monitoring, it is very important to know the probabilities of falsenegative results. Therefore, results should always be documented with all replicates including those below detection. Fig. 2 3. PCR analysis of DNA extracted from the pollen samples collected in the nature reserve (Genetic ID and Impetus). Tab. 2 documents the conforming results of both labs on the GMO content. Fig. 3 illustrates the consistent results of the PCR analysis of both pollen matrices.
Logo Impetus
Honey bee baskets
Microscopic pollen analysis Microscopic pollen analysis was performed to identify different pollen species and to quantify the maize pollen in the samples. Tab. 1 shows, that maize pollen was found in considerable amounts at all sites and in all samples (bio aerosol samples and pollen baskets). Therefore a remarkable exposure of maize pollen even in the mid of the nature reserve has to be stated.
Fig. 3
Conclusions The results of both laboratories have been in close accordance and analogous results were received for the technical and the biological pollen samplers. The study showed that now suitable methods for GMO-pollen monitoring are available as successful performance of PCR of pollen samples and detection of GMO pollen entry in a nature reserve could be demonstrated. PCR analysis of pollen samples (especially bio aerosols) is considered as quite difficult and requires experienced analysts, therefore certified reference material of pollen matrices is desirable for standardisation and a broader application of GMO pollen monitoring. The results of the study showed high levels of maize pollen and MON810 exposure in the nature reserve. Therefore isolation distances for the cultivation of GMO maize are not only needed in respect to coexisting conventional and organic cultivation but also for nature reserves because of the precautionary principle.
