English abstract
Bacillus thuringiensis is a Gram-positive, aerobic bacterium commonly found in soil. This bacterium is characterized by its ability to produce crystalline protein inclusions during sporulation. The protein has insecticidal properties against various insect pest orders such as Lepidoptera, Diptera, Coleoptera, Hymenoptera and Homoptera. These insect pests cause variable losses on agricultural crops in Oman and worldwide. Many B. thuringiensis genes encoding toxic proteins have been cloned and sequenced and are commonly named as cry gene. In this project, B. thuringiensis gene cry1C encoding an insecticidal crystal protein (ICP) directed by the cauliflower mosaic virus (CMV) double 35S promoters was transferred into tobacco plants by Agrobacterium-mediated tissue culture with kanamycin selection. A total of 50.0% transformed and 77.0% control plantlets were obtained. Integration of cry1C gene into tobacco genome was confirmed by polymerase chain reaction (PCR) and southern blot analysis. The Cryic protein concentration was assayed by enzyme-linked immunosorbent assay (ELISA) and insects bioassay. Seventy percent of the transgenic plants of To generation gave an expected PCR amplicon of 450bp, but no such amplification was observed with control plants. All transgenic plants of Ti generation also showed the expected 450bp PCR amplified VA fragment. These findings were further confirmed by southern blot analysis which showed one hybridization band in the expected size range. No hybridization band was as observed in the control plants. The concentration of Cry1C protein varied considerably ranging from 0 to 96.8 ppb among the leaves of transgenic plants tested by ELISA. About 54% of the transgenic plants produced low level of Cry1C protein (<1 ppb). High level of Cry1C protein (>1ppb) was observed in about 24% of the transformed plants. No Cry1C protein was detected in 22% transgenic plants tested positive with PCR, There was 70.0% mortality of Spodoptera litura feeding on the transgenic plant expressing 96,8ppb of Cry1C protein. Transgenic plant expressing 69.3ppb of Crylc yielded only 16.7% mortality of S. litura. No mortality was recorded for Pseudococcus sp. (mealy bug) feeding on the same transgenic tobacco plants.
