ABSTRACT
BACKGROUND: Insects have developed resistance against Bt-transgenic plants. A multi-barrier defense system to weaken their resistance development is now necessary. One such approach is to use fusion protein genes to increase resistance in plants by introducing more Bt genes in combination. The locating the target protein at the point of insect attack will be more effective. It will not mean that the non-green parts of the plants are free of toxic proteins, but it will inflict more damage on the insects because they are at maximum activity in the green parts of plants. RESULTS: Successful cloning was achieved by the amplification of Cry2A, Cry1Ac, and a transit peptide. The appropriate polymerase chain reaction amplification and digested products confirmed that Cry1Ac and Cry2A were successfully cloned in the correct orientation. The appearance of a blue color in sections of infiltrated leaves after 72 hours confirmed the successful expression of the construct in the plant expression system. The overall transformation efficiency was calculated to be 0.7%. The amplification of Cry1Ac-Cry2A and Tp2 showed the successful integration of target genes into the genome of cotton plants. A maximum of 0.673 µg/g tissue of Cry1Ac and 0.568 µg/g tissue of Cry2A was observed in transgenic plants. We obtained 100% mortality in the target insect after 72 hours of feeding the 2nd instar larvae with transgenic plants. The appearance of a yellow color in transgenic cross sections, while absent in the control, through phase contrast microscopy indicated chloroplast localization of the target protein. CONCLUSION: Locating the target protein at the point of insect attack increases insect mortality when compared with that of other transgenic plants. The results of this study will also be of great value from a biosafety point of view.
Subject(s)
Animals , Bacterial Proteins/genetics , Recombinant Fusion Proteins , Chloroplasts/genetics , Insect Control/methods , Gossypium/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Lepidoptera , Bacillus thuringiensis , Bacterial Proteins/analysis , Insecticide Resistance/genetics , Immunohistochemistry , Gene Expression/genetics , Chloroplasts/metabolism , Polymerase Chain Reaction , Microscopy, Phase-Contrast , Plants, Genetically Modified , Cloning, Molecular , DNA Primers , Plant Leaves/genetics , Transgenes/physiology , Endotoxins/analysis , Gene Fusion , Hemolysin Proteins/analysis , Insecticides , LarvaABSTRACT
Advanced generations of different transgenic lines of indica basmati rice (Basmati-370) expressing two unrelated Bt genes, cry1Ac and cry2A were evaluated for resistance to Yellow Stem Borer (YSB) and Rice Leaf Folder (RLF) under field conditions compared to control lines over three years (2003-2005). Homozygous lines were selected and analyzed for insect resistance, morphological, physiochemical properties and risk assessment studies. After artificial infestation of target insects, the transgenic plants showed significant resistance. Data were recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines showed up to 100 and 96 percent resistance against yellow stem borer at vegetative and flowering stages, respectively. Natural damage of rice leaf folder was also observed during the year 2005. The transgenic plants were 98 percent more resistant as compared to untransformed control plants. Variations in some morphological characteristics, e.g., the average number of tillers, plant height and maturity were also observed. Transgenic lines produced 40 percent more grains than control plants. All these characteristics were stably inherited in advanced generations. The transgenic lines had no significant effect on non-target insects (insects belonging to orders other than Lepidoptera and Diptera) in field or under storage conditions. Chances of pollen-mediated gene flow were recorded at a rate of 0.14 percent.