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The genus Helicoverpa comprises of 2 species in India—H. armigera and H. assulta. This paper compares partial CO-1 sequences of the two-field-collected species with a laboratory strain of H. armigera and Drosophila yakuba whose entire mitochondrial genome has been sequenced. The region sequenced in the study corresponds to 2111 to 2601 bp sequence of D. yakuba mitochondrial genome, i.e mid to near terminal segment of the CO-I region. When analyzed, at least 18 nucleotide and 8 amino acid substitutions were observed between the two species. Using this information, a specific PCR-RFLP tool was designed that distinguishes between the two species at the egg stage itself, thus, influencing pest control options significantly, especially in areas and crops where the two may occur simultaneously. Introduction The genus Helicoverpa comprises of only two species in India—H. armigera and H. assulta that are visually different as adults. H. armigera (Huebner) is a polyphagous pest, recorded on about 181 host plants and considered the most important pest of field crops in India 1 . Whereas H. assulta (Guenee) is an oligophagus pest that feeds largely on solanaceous crops, but not exclusively on food plants, such as tobacco (Nicotiana tobaccum) and Datura spp. 2 . It is common to encounter both the species together in vegetable ecosystems, such as tomato. H. assulta is a pest of predominantly dicots, while H. armigera is found on both dicots and monocots. The service of trained entomologist is required to differentiate the two species in the adult stage, applying taxonomic keys as described by Hardwick 3 and Mathews 4,5 . However, eggs and neonates are virtually indistinguishable in mixed populations. Studies on inter and intra specific variations in insects are often attempted using taxonomic, biochemical or molecular markers 6,7 . The taxonomic tools are often laborious, time-consuming and require considerable skill; whereas biochemical markers, such as esterase isozymes, are influenced by the environment, food plants (hosts) and stage of the insect. In contrast, molecular methods are relatively recent, reasonably accurate and could be complimented with other methods. Mitochondrial genome is maternally inherited 8 . Any change in the mitochondrial (mt) DNA is transmitted to the entire progeny. Evolutionary changes in conserved regions of the mt DNA spread rapidly within populations. If the changes have a functional significance in evolutionary adaptation, the change itself along with the extent of spread represents the adapted strains. Such changes, which can be micro evolutionary in nature, can be a function of selection pressures induced by suitability or unsuitability of host plants, insecticides, predators and parasitoids or other biotic and abiotic stresses. H. armigera (cotton bollworm) has been subjected to such pressures over the past decades. Though changes were speculated in terms of race or geographical variability, there has so far been no genetic evidence to indicate the same The cytochrome oxidase I (CO-I) region of mt DNA is the most studied region of the insect mitochondrial genome. CO-I is the terminal catalyst in mitochondrial respiratory chain and its biochemical role has been clearly elucidated. It is also the largest of the three mitochondrial encoded cytochrome oxidase subunits, which is one of the largest protein coding genes of metazoan mitochondrial genome 9 .
Indian Journal of Biotechnology 04/2006; 5(2):195-99. · 0.51 Impact Factor
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The quantitative levels of Cry1Ac and the seasonal decline in expression differed significantly among the eight commercial Bollgard hybrids tested. The Cry1Ac expression was found to be variable among the hybrids and also between different plant parts. The leaves of Bt-cotton plants were found to have the highest levels of Cry1Ac expression followed by squares, bolls and flowers. The toxin expression in the boll-rind, square bud and ovary of flowers was clearly inadequate to confer full protection to the fruiting parts. Increasing levels of Helicoverpa armigera survival were correlated with the toxin levels decreasing below 1.8 mg/g in the plant parts. Genotype-independent seasonal decline of the Cry1Ac toxin levels was observed in all the hybrids. Cry1Ac expression decreased consistently as the plant aged. The decline in Cry1Ac was more rapid in some hybrids compared to others. The choice of parental background appeared to be crucial for sustainable expression of the cry1Ac transgene. The implications of variability in Cry1Ac expression and the seasonal decline on bollworm management are discussed.
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The quantitative levels of Cry1Ac and the seasonal de-cline in expression differed significantly among the eight commercial Bollgard hybrids tested. The Cry1Ac ex-pression was found to be variable among the hybrids and also between different plant parts. The leaves of Bt-cotton plants were found to have the highest levels of Cry1Ac expression followed by squares, bolls and flowers. The toxin expression in the boll-rind, square bud and ovary of flowers was clearly inadequate to confer full protection to the fruiting parts. Increasing levels of Helicoverpa armigera survival were correlated with the toxin levels decreasing below 1.8 µ µg/g in the plant parts. Genotype-independent seasonal decline of the Cry1Ac toxin levels was observed in all the hybrids. Cry1Ac expression decreased consistently as the plant aged. The decline in Cry1Ac was more rapid in some hybrids compared to others. The choice of parental background appeared to be crucial for sustainable ex-pression of the cry1Ac transgene. The implications of variability in Cry1Ac expression and the seasonal decline on bollworm management are discussed. THREE Bt-cotton transgenic hybrids (Bollgard-MECH-12, Bollgard-MECH-162 and Bollgard-MECH-184) were offi-cially approved and released in 2002 for commercial cul-tivation in India. The technology was introduced into India by MAHYCO (Maharashtra Hybrid Seeds Company Ltd, Jalna, India) under license from Monsanto, USA. Five more Bt-cotton hybrids (Bollgard-RCH-2, Bollgard-RCH-20, Bollgard-RCH-134, Bollgard-RCH-138 and Bollgard-RCH-144 from RASI Seeds Pvt Ltd, Attur, Tamil Nadu) were approved by the Government of India, for large-scale field trials during the 2002 and 2003 crop-ping seasons, before being released for commercial culti-vation. All the Bt-cotton hybrids mentioned above were developed using Indian parent varieties into which the cry1Ac gene was introgressed from a transgenic Bt-cotton variety, Coker 312. All the current Bollgard cotton hybrids have descended from a common parent with a single genetic transformation event 1 'Monsanto-531', which was trans-formed with a vector containing a full-length cry1Ac coding sequence driven by an enhanced 35S promoter that enables the production of Cry1Ac protein in almost all parts of the plant. When injested by larvae, Cry1Ac binds to specific receptors in the midgut region. Toxin-binding in susceptible insects disrupts midgut epithelium, thereby causing overall toxic effects and ultimately resulting in death of the larvae. The novel transgenic technology was found to be highly beneficial in almost all parts of the world in terms of its capabilities to keep the target pests such as bollworms under check. However, for the Bt-transgenic technology to be sustainable, it is important that the toxin expression levels be expressed at adequate quantities in appropriate plant parts at the requisite time of the season to afford protec-tion against major target insect pests, which primarily in-clude the bollworms. Studies in Australia 2–5 and USA 6–11 showed that Cry1Ac toxin expression was variable among Bt-cotton plant parts and that the Cry1Ac toxin expres-sion and bollworm mortality levels decreased consistently as the plant aged. Our field experience with Bt-cotton hy-brids in India showed that the cotton bollworm Heli-coverpa armigera (Hübner) was able to survive more on some particular fruiting parts, and the pest infestation ex-ceeded economic threshold levels more readily on some specific commercial hybrids compared to others. The main objectives of this study were to determine a critical level of Cry1Ac expression in Bt-cotton plants that would be required for effective bollworm control; estimate the variability of Cry1Ac expression among plant parts and elu-cidate temporal changes in expression of Cry1Ac protein in eight commercial Bt-cotton hybrids using ELISA (enzyme linked immunosorbent assay).