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Cotton is the mainstay of Pakistan’s economy with large area of Punjab and Sindh under its cultivation. Bollworms infestation is a big threat to cotton production as it causes severe yield losses. The development of transgenic cotton having Bt (Bacillus thuringiensis) gene producing δ-endotoxin was a success story to get control of bollworm infestation. Dark side of this story was the progressive development of resistant insects against this toxin. The spatio-temporal expression of the transgene is considered one of the reasons for resistant pests. Though the transgene is under the control of constitutive promoter, but expression is not consistent and stable throughout the growing season.Therefore, the present study was planned to examine the basis of the variable expression of Bt gene in the genetic background of local cotton accessions. In this research Bt cotton genotypes were screened at both DNA (qualitative) and protein (quantitative) level to identify the level of expression in various accessions with different genetic background. The results obtained revealed that FH-142 and VH-295 were the highest Cry1Ac expressing genotypes while IUB-222 and MNH-886 were the least. A set of 10 selected Bt genotypes were used to study the season long expression. The plants were sampled at different growth stages (i.e. 30, 60, 90, 120 and 150 days after sowing - DAS) and in different parts (Leaves and Bolls). Intra-plant expression variability was also assessed from upper, middle and lower canopy leaves. Enzyme Linked Immuno Sorbent Assay (ELISA) was performed for the quantification of Cry1Ac gene in the sampled tissues at protein level. The results revealed that 30 DAS leaves had the highest concentration while 150 DAS had the least expression. A gradual decrease in the expression was observed throughout the growing season among all the genotypes with the age of the plant. Leaves expressed higher expression as compared to bolls and other reproductive parts. Upper canopy leaves had considerable amount of δ-endotoxin protein whereas, middle canopy and lower canopy leaves showed a lower level of toxin. Same genotypes were exposed to three different sowing dates to analyse the effect of sowing dates on transgene expression. Significant effects were noticed among all the three sowing dates for Cry1Ac gene concentration. The expression of the transgene significantly reduced at 3rd sowing date due to an increase in temperature (i.e., 38 °C). The results provided the better understanding of Cry1Ac behavior in cotton plant under local climatic conditions. The inheritance pattern of the Cry gene and its associated yield and fiber quality traits were studied in the subsequent generations (P1, P2, F1, F2, BC1 and BC2). Two high Cry1Ac protein expressing genotypes (FH-142 and VH-295) were crossed with two low Cry1Ac protein expressing genotypes (IUB-222 and MNH-886) and with two non-Bt genotypes (VH-144 and MNH-786). Chi-square analysis for Cry1Ac gene revealed that its inheritance was controlled by single homozygous dominant gene. Most of the yield and fiber quality traits showed the involvement of additive, non-additive and epistatic type of gene action. The study provided guidelines for the development of better performing Bt cotton genotypes with standard toxin expression. Development of high expressing Cry1Ac genotypes with better yield and fiber quality traits is the ultimate solution to avoid insect pest losses and increased cotton production.
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