The first document has the questions which should be answered based on the research paper by Szekacs et al 2010
:Detection of Cry1Ab toxin in the leaves of MON810 transgenic maize, that is attached .
ORIGINAL PAPER Detection of Cry1Ab toxin in the leaves of MON 810 transgenic maize András Székács & Éva Lauber & Eszter Takács & Béla Darvas Received: 30 October 2009 /Revised: 4 December 2009 /Accepted: 4 December 2009 /Published online: 21 January 2010 # Springer-Verlag 2010 Abstract The distribution of Cry1Ab toxin was detected in the leaves of genetically modified maize of genetic event MON 810 by enzyme-linked immunosorbent assay. Cry1Ab toxin contents in the leaves at reproductive (milk, R3) phenological stage were measured to be between 3,878 and 11,148 ng Cry1Ab toxin/g fresh weight. Toxin content was significantly lesser (significant difference (SD)= 1,823 ng Cry1Ab toxin/g fresh leaf weight, p <0.01) in leaves at the lowest leaf level, than at higher leaf levels, probably due to partial leaf necrotisation. A substantial (up to 22%) plant-to-plant variation in Cry1Ab contents in leaves was observed. When studying toxin distribution within the cross and longitudinal sections of single leaves, lesser variability was detected diagonally, with approxi- mately 20% higher toxin concentrations at or near the leaf vein. More significant variability (SD=2,220 ng Cry1Ab toxin/g fresh leaf weight, p <0.01) was seen lengthwise along the leaf, starting at 1,892 ng Cry1Ab toxin/g fresh weight at the sheath and rising to maximum concentration at the middle of the lamella. Cry1Ab toxin content may suffer significant (SD=2,230 ng Cry1Ab toxin/g fresh leaf weight, p <0.01) decreases in the leaf due to necrotisation. The results indicate that the longitudinal dimension of the leaf has more significance for sampling purposes than the diagonal position. Keywords Genetically modified organism (GMO) . MON 810 maize . Cry1Ab toxin . Enzyme-linked immunosorbent assay (ELISA) Introduction European corn borer ( Ostrinia nubilalis , Hübner) resistant first generation genetically modified (GM) plants express a foreign gene ( cry ) for protein derivatives of crystalline endotoxins (Cry toxins) produced by Bacillus thurigiensis (Bt) var. kurstaki (Berliner) [ 1 , 2 ]. Such transgenic GM maize lines produce a truncated version of Cry1Ab proteins (genetic events SYN-EV176-9, SYN-BT011-1 , MON 810 ). Since this Cry toxin is considered an insecticidal active ingredient of these plants, and since characteristics of the GM plants possibly influenced by environmental factors constitute an essential part in their environmental risk assessment [ 3 – 5 ], it is of crucial importance to possess proper methods for toxin monitoring. The most com- monly used formats are lateral flow devices and 96-well microplate-based enzyme-linked immunosorbent assay (ELISA) [ 6 ], and various innovative analytical techniques have also been developed for quantitative or qualitative detection of Cry1Ab protein including microsphere-based immunoassays [ 7 – 9 ] or an immunomagnetic electrochem- ical sensor [ 10 ]. The use of ELISAs for Cry1Ab monitoring has been reported extensively [ 6 , 7 , 11 – 20 ]. Although MON 810 cultivars (Agrigold, Asgrow, DeKalb, Garst, Golden Harvest, Monsanto, Pioneer, etc.) were registered in the USA and Europe as early as in 1996 and 1998, respectively, Cry1Ab toxin production of the cultivars to date has not been properly explored. The Monsanto documents [ 21 – 24 ] mostly refer to data (Table 1 )fromf ie ld trials in Europe and the U.S. from 1994 to 1996 as non- published studies. These sources mention Cry1Ab toxin content expressed in the first, second and third leaf levels (the lowest leaf level assigned as first level), 9,870, 8,430 and 4,910 ng Cry1Ab/g fresh weight, respectively. None- theless, this limited and partial information presented by A. Székács ( * ) : É. Lauber : E. Takács : B. Darvas Department of Ecotoxicology and Environmental Analysis, Plant Protection Institute, Hungarian Academy of Sciences, Herman Ottó u. 15, 1022 Budapest, Hungary e-mail: [email protected] Anal Bioanal Chem (2010) 396:2203 – 2211 DOI 10.1007/s00216-009-3384-6
Monsanto is hard to judge without suitable specifications on cultivars and exact ELISA methods of determination applied. During the last decade, only a few papers were published about Cry1Ab toxin production of MON 810 cultivars (Table 2 ), even though the entire phenomenon of variable toxin production is still unclear. MON 810 cultivars produce Cry1Ab toxin in a tissue- and time-specific manner [ 25 – 27 ]. Soil quality, especially its nitrogen fertility also has a strong influence on Cry1Ab toxin expression [ 28 ], and multi-stack varieties have been reported to have expressed toxin content two fold higher than in single stack MON 810 cultivars [ 29 ]. As reported Cry1Ab toxin content often scatter over wide ranges within a single survey, concerns regarding non uniform expression levels of the transgene and plant-to- plant variation in Cry1Ab contents were raised [ 27 , 30 ]. Our aims in this work were to measure Cry1Ab toxin content in the leaves of MON 810 DeKalb cultivar DK-440 BTY under experimental cultivation and to assess Cry1Ab distribution among leaf levels and within individual leaves of using the validated commercial Abraxis Bt-Cry1Ab/Ac ELISA method. Materials and methods Seeds of MON 810 maize cultivar DK-440 BTY and its near isogenic variety DK-440 were kindly provided by Monsanto Hungária Ltd. (Budapest, Hungary). Both Bt maize and near isogenic varieties were grown at the Ecological Experimental Station of the Plant Protection Institute, Hungarian Academy of Sciences (Julianna- major, Nagykovácsi, Hungary). Cry1Ab toxin was obtained from three sources, purchased from Abraxis Inc. LLC (Warminster, PA, USA), acquired from Sándor Szobszlay (Department of Environmental Protection and Safety, Szent István University, Gödöll ő ,Hung a ry ) ,and received from Luke Masson (Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada) [ 31 ]. Chemicals were purchased from Sigma-Aldrich Ltd. (Budapest, Hungary) unless stated otherwise. Data were analysed using Statistica software by StatSoft Inc. (Tulsa, OK, USA). Standard curves for analytical determinations were calculated by simple linear regression in a narrow concentration range (up to 4 ng/mL) and sigmoid regression for a broader concentration range (up to 50 ng/mL) using the four-parameter logistic model [ 32 ]. Cry1Ab concentrations determined by linear reg- ression are expressed as mean±standard deviation (sd) unless stated otherwise. Statistical uniformity of detected concentrations was determined in unpaired two-sample Student's t -tests. Significant differences (SD) among Cry1Ab concentrations were calculated using one-way analysis of variance (ANOVA) with the probability level ( p )spec i f ied . Post-hoc analysis (Fisher's least significant difference (LSD test) was used to assign homogeneous groups (in which members are not statistically different from each other) at the probability level of the ANOVA tests in each experiment on Cry1Ab toxin content in leaves (cross sampling, longitudinal sampling and necrosis sampling measurements). Statistical grouping of the data in each experiment is assigned by capital letters in parentheses denoting separate homogeneous groups, but homogeneous groups in one experiment are not related to those in the other experiments. Enzyme-linked immunosorbent assays Cry1Ab toxin content in maize leaves was determined by commercial 96-well microplate format sandwich immuno- assay, Abraxis Bt-Cry1Ab/Ac ELISA kit (#PN 51001, Warminster, PA, USA). For sample preparation, approxi- mately 20 mg of ground maize samples were homogenised in 0.5 mL of manufacturer-provided extraction buffer, and were centrifuged at 12,000 rpm for 3 min. Samples were further diluted with phosphate-buffered saline (PBS) buffer as needed, at least 1:50. Immunoassays were carried out in 96-well ELISA microplates according to protocols provided by the manufacturers, in sequential steps, with a washing step with phosphate-buffered saline (PBS with 0.02% Tween 20) buffer (pH 7.4) in between each. Samples and calibrators were added to the plates in triplicates. Mono- clonal Cry1Ab-specific mouse antibodies in PBS buffer were added, after the addition of the samples and calibrators, and the plate was incubated for 30 min at room temperature, washed, followed by the addition of an anti- mouse IgG-HRP enzyme conjugate, and the plate was incubated for a further 30 min at room temperature. Finally, hydrogen peroxide as a substrate and tetramethyl benzidine as a chromophore diluted in citrate buffer (pH 5.0) were added. Colour development was measured in the kinetic mode at a wavelength of 650 nm, the colour reaction was stopped after 20 min by adding 4 M sulphuric acid, and the colour intensity was read at 450 nm wavelength on an iEMS microtiter plate reader (Labsystems, Helsinki, Finland). Calibrators were put on every microplate at concentrations between 0.25 and 4 ng/mL, assays were used for determination of the analyte concentration in the linear range of determination by linear regression. The limit of detection (LOD) claimed by the manufacturer, defined as the concentration corresponding to an assay signal of 3 standard deviations above the background signal (negative corn leaf sample) interpolated from a Cry1Ab standard curve by linear regression is 0.125 ng/mL Cry1Ab in corn leaf extract [ 33 ]. Consequently, the lower limit of detection in the plant samples was 156 ng Cry1Ab toxin/g fresh weight. 2204 A. Székács et al.
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