| In China transgenic Bt(Bacillus thuringiensis) cotton has been broadly planted since1997, which makes the China second largest Bt cotton growing country with planting area of4.2million hectares in2013. For the control of H. armigera (Hubner)(Lepidoptera:Nocruidae) the planting of transgenic Bt cotton has been extensively successful and competent tool. Although, the cultivation of Bt crops on large-scale may be the source of evolution in pest resistance to Bt toxin. Therefore, the main object of this study was biosafety of transgenic Bt cotton after continuous cultivation of Bt cotton in Yellow River cotton growing region of China during2012-2013and appropriate understanding the evolution of resistance of H. armigera to Bt cotton. The present scientifically studies helps to understand the efficacy of different transgenic Bt cotton, expression level of Bt insecticidal proteins and their effect on the survival of H. armigera. Also in this study, we estimate the frequency of Cryl Ac resistance genes in H. armigera populations with logical monitoring and find variation in cadherin gene related with resistance in H. armigera. As well as this study will help to improve the biosafety Bt cotton crops. The main conclusions of the study were as follows:1. During three growing stages of Bt cotton, larval mortality of H. armigera were calculated on cotton leaf tissue of three transgenic Bt cotton varieties after five days of trail in2012and2013. The result indicates that transgenic Bt cotton are resistant to H. armigera during early stage of cotton but as plant grow up the mortality is declining with cotton crop age. The highest mortality%was recorded during seedling stage and less mortality%during boll stage of CCRI41and Bollgard Ⅱ but in CCR179the less mortality%was recorded in bud stage during2012-2013. During seedling stage, the higher mortality%was recorded in CCRI4187.97%and75.48%in2012-2013respectively. During bud stage, the maximum mortality%was recorded in Bollgard Ⅱ49.56%and54.47%in2012-2013, in boll stage; the highest mortality%was recorded39.51%in Bollgard Ⅱ in2012and highest mortality was recorded in CCRI4147.68%in2013. The data showed that bollworms were more susceptible to Bollgard Ⅱ and CCRI41than CCRI79. The average weight of each insect also calculated after five days of feeding on three transgenic Bt cotton varieties. The result indicates that Bt toxins which are present in the transgenic Bt cotton reduced the growth and development of the H. armigera larvae. Among all transgenic Bt cotton varieties the highest average of weight was recorded in Bollgard Ⅱ. These results suggested that differences in mortality%between developmental stages of Bt cotton in two years can directly affect the development of resistance in populations of H. armigera.2. B. thuringiensis produced insecticidal protein CrylAc which is an important natural biological agent for the control of H. armigera considered as one of the most important economic insect pests in many parts of the world. To quantify the expression level of CrylAc and Cry2Ab during different growth stages of cotton plant, three Bt cotton varieties CCRI41(Bt+CpTI), CCRI79(CrylAc) and Bollgard II (CrylAc+Cry2Ab) were planted in2012and2013. For the quantification of the CrylAc and Cry2Ab content Envirologix Qualiplate kit for CrylAc and Cry2Ab were used. The Result showed some clear significant differences in the amount of CrylAc protein present in various plant parts of transgenic Bt cotton throughout the growing season (P<0.05). The expression levels of CrylAc proteins in the leaves of transgenic Bt cotton was significantly higher than buds and bolls but gradually decreasing as plant grew. Result showed that in Bt cotton CCRI41(5/+CpTI) which is toxic to H. armigera the CrylAc and CpTI proteins together have synergistic effect which enhances the level of Cry lAc Protein. In2012, measure the level of Cry2Ab, result showed that in Bollgard II the Cry2Ab present at much higher level throughout the season in the plant compared with CrylAc. Data showed that at the same time in fruiting bodies the expression level of Cry2Ab is higher but the expression level of Cry2Ab was lower in leaves, in Bollgard II expression level of the two Cry proteins found to be different from one another. However, data proved that in Bollgard II both proteins were present throughout the season and the addition of Cry2Ab had no deleterious effect on levels of CrylAc. These results recommend that expression levels of insecticidal proteins are linked with the cotton growth.3. During2012and2013, the cotton fiber quality characters were tested to recognize the effect of different transgenic Bt cotton on the lint. Result shows that the fiber quality characters of transgenic cotton varieties were not significantly affected when compared with conventional non Bt variety. But in2013, fiber strength and micronaire are significantly affected. Finally it is concluded that there was no significant effect of transgenic Bt cotton on the fiber quality of cotton during2012and2013. To know the effect of transgenic Bt cotton on fiber qualities further research work will be carry on in future.4. For the biosafety and continuous cultivation of Bt cotton in Yellow River region of China, the most important to gain a timely understanding the evolution of resistance of H. armigera to Bt toxin and estimate the frequency of alleles conferring resistance to CrylAc toxin in field populations of H. armigera. Adult female moths of H. armigera were collected from Henan Province, Shandong Province and Hebei Province in2010-2013. The female moths trapped in the field used for screening through bioassay test of F1and F2generations. The females moths reared on a diet containing1.0μg/ml CrylAc to estimate the frequency of resistance alleles. In2010, totals58,43and12isofemale lines tested for the F1generation bioassay, total48,25and26isofemale lines for the F1generation bioassay in2011, total of96,36and17isofemale lines for the F1generation in2012, and342,146and40isofemale were screened out during2013from Henan, Shandong and Hebei Provinces respectively. Yellow River region is the largest growing region of cotton in China, result shows that resistance gene frequency was still very low, fluctuating and it did not increase significantly from2010to2013in Henan Province, Shandong Province and Hebei Province(2010:χ2=0.0001<χ200.052=5.99;2011:χ2=0.0001<χ20.053=5.99;2012:=0.0001<χ20.0052=5.99;2013:χ2=0.0001<χ20.0052=5.99).Result shows that in F, tests the relative average development rating (RADR) of H. armigera larvae had no substantial increase in Cry1Ac tolerance during the four years period. In H. armigera to keep away from further increases in Bt resistance frequency, it is necessary to introduce Bt cotton expressing several Bt toxins and put together this technology with other strategy for management of H. armigera.5. To identify the CrylAc binding protein in H. armigera, characterized the deletion mutation of field collected strain; which significantly monitor the changes in the frequency of resistance gene. We tried to identify the mutant in field populations of H. armigera, but no mutant was found in moths collected in2012-2013. This result is constant with the existing low gene frequency of Bt resistance in field population of H. armigera. The results shows sequencing of four types of H. armigera, the full length of H. armigera were5190bp that encoding1730Amino Acid proteins of the same length with bollworm cadherin sequence in NCBI registered bollworm cadherin (CAD accession AF519180.2). Sequences are of the same length and all sequences have no deletion mutations. Sequence alignments showed that amino acid sequences of four groups were different. |
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