Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton

Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Final Health and Environmental Risk Assessment of Genetically Modified Soybean MON 87701

Soybean MON 87701 expresses the cry1Ac gene from Bacillus thuringiensis. The encoded Cry1Ac protein confers resistance against specific lepidopteran pests. Updated bioinformatics analyses of the inserted DNA and flanking sequences in soybean MON 87701 have not indicated a potential production of harmful toxins and allergens or polypeptides caused by the genetic modification. Genomic stability of the functional insert and consistent expression of the cry1Ac gene, have been shown over several generations of soybean MON 87701. Data from several field trials performed in USA, Canada, Chile and Argentina during 2005-2006 show that soybean MON 87701 is compositionally, morphologically and agronomically equivalent to its conventional counterpart and other commercial soybean cultivars. Subchronic feeding studies with rats as well as nutritional assessment with broilers have not revealed relevant adverse effects of MON 87701. These studies indicate that MON 87701 is nutritionally equivalent to and as safe as conventional soybean cultivars. The Cry1Ac protein produced in soybean MON 87701 do not show sequence resemblance to known toxins or IgE-dependent allergens, nor has the whole GM plant been reported to cause changes in IgE-mediated allergic reactions in patients reactive to soybean or in non-ectopic control individuals. Soybean is not cultivated in Norway, and there are no cross-compatible wild or weedy relatives of soybean in Europe. Based on current knowledge and considering the intended uses, which exclude cultivation, the VKM GMO Panel concludes that soybean MON 87701 with the Cry1Ac protein: - Is compositionally, morphologically and agronomically equivalent to its conventional counterpart and other commercial soybean cultivars - Is unlikely to introduce a toxic or allergenic potential in food or feed compared to conventional soybean cultivars - Is nutritionally equivalent to and as safe as its conventional counterpart and other conventional soybean cultivars - Does not represent an environmental risk in Norway.

Reconstruction and validation of three different binary vectors suitable for generation of genetically engineered Helicoverpa protected crops

Availability of a suitable plant transformation binary vector is necessary for the generation of transgenic crops with an adequate expression of transgenic proteins. Therefore, three binary vectors were constructed viz., pBK204, pBK205, and pBK206 harboring either a truncated or a full-length version of a Cry1Ac gene for the generation of Helicoverpa protected crops. Two different promoters viz., Arabidopsis Rubisco small subunit (AtSSU) gene promoter or CaMV35S promoters were used to regulate the various versions of Cry1Ac gene. The binary vectors were reconstructed either by the Gibson assembly method and others by ligating the restriction enzyme digested fragments. The reconstructed binary vectors were mobilized into Agrobacterium strain AGL1 and validated by Agrobacterium infiltration assays of Nicotiana benthamiana. The amount of Cry1Ac protein accumulated in the Agroinfiltrated tobacco leaves was quantified using the quantitative ELISA assay. The expression of the Cry1Ac protein in the tobacco leaves ranged from 0.25 to 0.26 µg /g fresh weight (FW) when transformed with these three constructs. Thus, the vectors constructed in this study appeared to be suitable for generation of Helicoverpa resistant transgenic crops by Agrobacterium-mediated genetic transformation method.

Bacillus thuringiensis Maize Expressing a Fusion Gene Cry1Ab/Cry1AcZM Does Not Harm Valued Pollen Feeders

The ladybird Propylea japonica, adults of the green lacewing Chrysoperla nipponensis and the honey bee Apis mellifera are common pollen feeders in many crop systems. They could therefore be directly exposed to Cry proteins in Bacillus thuringiensis (Bt)-transgenic crop fields by ingestion of pollen. They, or closely related species, are therefore often selected as surrogate test species in non-target risk assessment of Bt plants. In the current study, we evaluated the potential effects of the ingestion of Bt maize pollen containing the Cry1Ab/Cry1Ac fusion protein on various life-table parameters of the three pollen-feeding non-target species in laboratory feeding assays. The results showed that pupation rate and male adult fresh weight of P. japonica were significantly increased when fed pollen from Bt maize compared to control maize pollen, but other test life-table parameters were not affected. For the other two species, none of the tested life-table parameters (survival, pre-oviposition period, fecundity and adult fresh weight for C. nipponensis; survival and mean acinus diameter of hypopharyngeal glands for A. mellifera) differed between non-Bt and Bt maize pollen treatments. ELISA measurements confirmed the stability and uptake of the Cry protein by all three species during the feeding bioassays. In addition, a sensitive insect bioassay confirmed the bioactivity of the Cry1Ab/Cry1Ac protein in the Bt maize pollen used. Overall, the results suggested that the three pollen feeders are not sensitive to the Cry1Ab/Cry1Ac protein, and planting of the Bt maize variety will pose a negligible risk to P. japonica, adult C. nipponensis and adult A. mellifera.