Development Of Bt Cotton Genotypes Using Pakistani Cultivars

More than 50 counties around the world including China and Pakistan are cultivating the cotton for their textile need.Due to its economic importance for cotton cultivating countries,it is termed as"White Gold".In addition to its importance,cotton faces many problems like insect pests,weeds and CLCuD especially in Pakistan.In past insects have been controlled by insecticides but CLCuD is still obnoxious disease for cotton causing lot of economic losses.Although conventional breeding methods provided many breakthrough in cotton breeding but cotton biotechnology has much more potential to cater with insect pests and diseases of cotton.Bt cotton is proof and miracle of cotton biotechnology.However,since the introduction of Bt cotton,some resistance breakdown against lepidopteron insects has been reported around the world.Therefore,it is imperative to develop cotton genotypes with multi-barrier defense system with high Bt toxin level along with considerable tolerance against CLCuD.In this scenario,current study was planned to develop multiple gene cassette constructs harboring either CrylAc or Cry2Ab4 with different promoter combination i.e.CaMV35S,NOS and PS-1 and a supplementary study was also conducted to unveil the biochemical bases of CLCuD for the sustainable cotton breeding.The tobacco was used as model system for characterization the multiple gene cassette constructs.Successful construction of genes cassettes were achieved and confirmed by appropriate PCR amplifications,sequencing and digested products.Two kinds of constructs having two gene cassettes and three genes cassettes were assembled in plant expression vector and transformed in tobacco.Successful expression of Bt proteins in transgenic tobacco plants was confirmed by the immunostrip assay.The RT-PCR and qRT-PCR confirmed the high expression of Cry1Ac and Cry2Ab4 genes in three cassettes expression vector as compared to two cassettes expression vector.qRT-PCR showed that the relative expression of Cry1Ac gene in tobacco plants with three cassettes expression vector was 1.5 folds higher than two cassettes.Similarly,expression of Cry2Ab4 in three-cassette vector was 3.5 fold high than two cassettes of Cry2Ab4.S-12,Mac-7,EG1 and EG2 genotypes and their FI and F2 hybrids were used for biochemical analysis under CLCuD stress.Disease rating scale revealed that Mac-7 was found to be highly tolerant genotype for CLCuD with no symptoms of virus.Biochemical analysis also reveled that Mac-7 have high activity of SOD,POD,CAT with high contents of total chlorophyll,carotenoids and total soluble sugar.Among the F1 hybrids all plants of cross Mac-7 × EG1 showed the symptoms of CLCuD of severity level 2.While all F1 plants of cross Mac-7 x EG2 showed CLCuD symptoms of severity level 3.Plants of F2 generation of both crosses showed varying degree of severity level and also different activity of biochemical at different severity level.Correlation analysis reveled that TSP has maximum positive and highly significant correlation with PAL and PPO.However,maximum negative and significant correlation was observed between POD and Chl"a".In conclusion it is suggested by the current study that driving the Bt genes with different promoter,staking the multiple gene cassettes in a expression vector coupled with revealing the biochemical basis of CLCuD could yield the encouraging results to deploy the sustainable resistance against insect pests and CLCuD.