Expression Analyses, Efficacy Studies and Environmental Risk Assessment of Insecticidal Proteins Produced in Transgenic Plants

Insect pests are a serious threat to agricultural crops all over the world as they cause \ud huge losses to crop yield and economy. Due to low innate immunity in plants, synthetic \ud insecticides have been used to cope with the pest problem since decades. These \ud chemicals are expensive, broad spectrum and hazardous to health and environment. \ud Moreover, most of the target insect species have developed resistance against the \ud available insecticides. Therefore, search for alternative methods of insect pest control \ud has been a priority in agricultural research. One of the successful methods is to develop \ud Insect Resistant, Genetically Modified Crops (IR-GMCs) expressing insecticidal \ud proteins. IR-GMCs have been rapidly adopted around the world as they are target \ud specific, environment friendly, cost effective and easy to use. However, two major risks \ud of growing IR-GMCs have been proposed; resistance development against insecticidal \ud toxins (e.g. Cry proteins) expressed in plants and adverse effects to beneficial, non\ud target arthropods (NTAs). NTAs include important species that contribute directly to \ud agricultural ecology via biological control, pollination and decomposition of organic \ud materials. Pakistan officially adopted Bt-cotton in 2010 and became one of the leading \ud countries in terms of area under cultivation by 2013. Variation in the expression of Bt \ud proteins have been associated with reduced efficacy of the IR crops and also resistance \ud development in the target insect pests. Therefore, one of the objectives of this study was \ud expression profiling of first batch of approved, locally bred Bt-cotton varieties through \ud ELISA and efficacy analysis against Helicoverpa armigera. \ud While Bt cotton varieties are effective in controlling several key lepidopteran pests, they \ud are less effective against some Lepidoptera species and provide no protection against \ud non-lepidopteran pests. In addition, the initial cases of resistance to Cry1Ac have been \ud observed in target insect pests in many countries. It is thus indicated to explore \ud alternative source for toxins that can be incorporated in future transgenic plants. The peptide ?-Hexatoxin-Hv1a (Hvt) is one of the most studied spider venom toxins. Its \ud insecticidal potential has been reported for species belonging to different arthropod \ud orders including Lepidoptera, Diptera and Orthoptera. The gene encoding for Hvt has \ud thus been transformed into cotton and tobacco to protect the plants from damage by \ud Lepidopteran pests. The second objective of the study therefore, was to evaluate the expression of ?-Hexatoxin-Hv1a gene in transgenic plants, and the toxicity of plant expressed and purified Hvt against target lepidopteran insects and four non-target arthropod species. Bollgard IITM cotton plants and purified Cry2Ab2 protein were \ud included in the study as comparators. \ud In studies with locally bred Bt cotton, expression of Cry1Ab/1Ac protein were found to \ud vary significantly (P