Field Trials And Genetic Stability Analysis Of Insect-resistant Transgenic Bt Maize

In the last two decades, remarkable achievements has been made on successful cultivation of a large number of genetic engineering crops, including those with resistance to insect, disease, herbicide and those with high yield and quality. However, after obtaining one transgenic plant, much work had to do, for their offspring, on pure line identification, genetic stability analysis, agronomic and economic traits selection and germplasm conservation in commercialization, all the above work was fuscous on tracking survey to the interesting gene and selection/identification of large number of progenies. Therefore, at the time on development of genetically modified crops, more and more attention paid to combination of transgenic and conventional breeding technology, and genetic expression and variation test of GM crops offspring in the field also strengthen. As a result, crop innovation and improvement by genetic engineering would make it possible to enable transgenic plants to commercialization, and effective expression and genetic stability were the key issues on the process.There were some national reports on Bt-transgenic maize and their genetic stability analysis, but they were concerned about performance of transformed plants and gene function, as well as gene usage evaluation of foreign Bt-transgenic maize, mainly in the early generations. Besides, only a few articles has been reported on identification of Bt maize harboring genes with intellectual property, and it was notice that there was few story about genetic stability analysis of inbred line high generation of insect-resistant Bt maize.The applied plant materials were high generations of inbred insect-resistant maize harboring cry1Ah transformed by Pollen tube pathway and insect-resistant maize harboring cry1Ah and cry1Ie by gene gun. Indoor and field resistance identification on corn borer, plus a series of molecular detection including PCR,RT-PCR,Southern blot,Western blot and ELISA has been served to genetic stability and pattern analysis. Results were as follows:1. Positive plants were confirmed by PCR. Bt gene was proved by Southern blot that it was integrated into plant genome, further it was transcripted and expressed in plant cells detected by RT-PCR, Western blot respectively. Genetic analysis showed that Bt gene was single-locus dominant inheritance, conforming to Mendelian genetic pattern. Three foreign genes, cry1Ah, cry1Ie and 2mG2-epsps, was complete linkage and coseparation in transgenic maize genome, namely they were transferred to offspring like single gene locus.2. Cry1Ah expression varied among plants from the same transformed event, but statistical analysis indicated that the difference was not significant, while there was significant difference among different transformed events. And Cry1Ah expression varied among different tissues from the same stage of one plant, higher in the bract and leaf, lowest in the pollen (bract>leaf>stalk>tassel>tassel handle>ear tip>seed>filament>pollen). Bt toxic protein expression possessed temporal and special specificity, Cry1Ah expression varied in different stage of leaf and stalk, taking on dynamic downtrend.3. Foreign gene expression varied in progenies from different transformed events hybridized with different conventional maize, largely related to foreign gene expression of one parent and gene type of the other. Therefore, it required transgenic inbred lines with high foreign gene expression to be parent. Resistance analysis on corn borer and foreign expression detection showed that transmission frequency of mega gamete was higher than andro gamete, so that choosing resistant parent to crossbred would make it possible to obtain high expression progenies.4. Indoor and field resistance analysis on corn borer demonstrated that, not only maize harboring cry1Ah and their progenies, but also transgenic maize harboring cry1Ah and cry1Ie were highly resistant to corn borer, besides the very resistance was able to transfer from generation to generation. Therefore it was effective to control Asia corn borer by means of transforming foreign Bt genes. Variety pattern of Cry1Ah protein expression detected through ELISA was on the whole consistent with resistance analysis, indicated that combination of indoor/field resistance analysis and molecular detection would illuminate resistance on corn borer more precisely, and the results showed that Cry1Ah protein expression was significant relative to Fracture rate of tassel handle and indoor larva death ratio in 7 days, and extremely significant relative to leaf-feeding level and tunnel length per 100 plants. So it made clear that Bt protein content was the internal cause of resistance to corn borer.5. Comparing conventional maize and transgenic maize harboring single cry1Ah or harboring cry1Ah and cry1Ie and their progenies, statistical analysis indicated that there was significant difference in kernals per row and bare tip length, yet indistinct difference in plant height, height, width, line number of ear and g/1000 seeds. Shorter bald tip and increasing in grains per ear indicated the possible product raising.6. Comparing conventional maize and transgenic maize harboring single cry1Ah, both in experimental field and wilderness, there was indistinct difference in geminating rate, growing vigor, plant type, bearing period and plant height, except for yield. In wilderness, maize geminating rate was only 5%, and weeds coverage was higher than that of maize (significant difference or extremely significant difference), showing that survival competitiveness of maize was much weaker. Germinating test showed that there was indistinct difference between conventional maize and progenies of transgenic maize harboring cry1Ah.7. Five pure lines of transgenic maize harboring cry1Ah and 1 transgenic maize harboring cry1Ah and cry1Ie have been selected.