Pigeon pea is an important legume infested by a plethora of insect pests amongst which gram pod borer Helicoverpa armigera is very prominent. Imparting resistance to this insect herbivore is of global importance in attaining food security. Expression of insecticidal crystal proteins (ICP) in diverse crops has led to increased resistance to several pests. We report in this paper, expression of Cry2Aa in transgenic pigeon pea and its effectiveness towards H. armigera by employing Agrobacterium-mediated in planta transformation approach. Approximately 0.8% of T1 generation plants were identified as putative transformants based on screening in the presence of 70 ppm kanamycin as the selection agent. Promising events were further recognized in advanced generations based on integration, expression and bioefficacy of the transgenes. Seven T3 lines (11.8% of the selected T1 events) were categorized as superior as these events demonstrated 80–100% mortality of the challenged larvae and improved ability to prevent damage caused by the larvae. The selected transgenic plants accumulated Cry2Aa in the range of 25–80 µg/g FW. The transgenic events developed in the study can be used in pigeon pea improvement programmes for pod borer resistance.
Weeds burden plant growth as they compete for space, sunlight, and soil nutrients leading to 25-80% yield losses. Glyphosate [N-(phosphonomethyl)glycine] is a widely used broad spectrum non-selective herbicide that controls weeds by inhibiting 5enolpyruvylshikimate-3-phosphate synthase (EPSPS) enzyme and interfering with the shikimate biosynthesis pathway. Cotton (Gossypium hirsutum L.) is one of the most important commercial crops grown worldwide for its fiber. We have developed herbicide tolerant transgenic cotton (cv. P8-6) by introgression of a codon-optimized and modified EPSPS gene (CP4-EPSPS) possessing an N-terminal chloroplast targeting peptide from Petunia hybrida. Because of the recalcitrant nature of cotton, a genotype-independent non-tissue culture-based apical meristem-targeted in planta transformation approach was used to develop transformants. Although in planta transformation methodologies are advantageous in developing a large number of transgenic plants, effective screening strategies are essential for initial identification of transformants. In the present study, the use of a two-level rigorous screening strategy identified 2.27% of T1 generation plants as tolerant to 800 and 1,500 mg/L of commercially available glyphosate (Roundup). Precise molecular characterization revealed stable integration, expression, and inheritance of CP4-EPSPS in advanced generations of the promising transgenic events. Further, superiority of selected transgenic plants in tolerating increasing levels of glyphosate (500-4,000 mg/L) was ascertained through reduced accumulation of shikimate. This report is the first of its kind where cotton transformants tolerating high levels of glyphosate (up to 4,000 mg/L) and accumulating low levels of shikimate have been identified. This study not only reiterated the genotype-independent nature of the transformation strategy but also reiterated the translational utility of the CP4-EPSPS gene in management of weeds.
Plants are challenged incessantly by several biotic and abiotic stresses during their entire growth period. As with other biotic stress factors, insect pests have also posed serious concerns related to yield losses due to which agricultural productivity is at stake. In plants, trait modification for crop improvement was initiated with breeding approaches followed by genetic engineering. However, stringent regulatory policies for risk assessment and lack of social acceptance for genetically modified crops worldwide have incited researchers toward alternate strategies. Genome engineering or genome editing has emerged as a new breeding technique with the ability to edit the genomes of plants, animals, microbes, and human beings. Several gene editing strategies are being executed with continuous emergence of variants. The scientific community has unraveled the utility of various editing tools from endonucleases to CRISPR/Cas in several aspects related to plant growth, development, and mitigation of stresses. The categorical focus on the development of tools and techniques including designing of binary vectors to facilitate ease in genome engineering are being pursued. Through this Review, we embark upon the conglomeration of various genome editing strategies that can be and are being used to design insect pest resistance in plants. Case studies and novel crop-based approaches that reiterate the successful use of these tools in insects as well as in plants are highlighted. Further, the Review also provides implications for the requirement of a specific regulatory framework and risk assessment of the edited crops. Genome editing toward insect pest management is here to stay, provided uncompromising efforts are made toward the identification of amiable target genes.
Cotton (Gossypium hirsutum L.), a mercantile crop plant, is grown worldwide for fiber and seed oil. As with other economically important crops, cotton is bogged down with many biotic and abiotic stress factors. Towards this, genetic engineering offers numerous protocols to engineer plants for better resilience. However, recalcitrance of cotton to plant tissue culture has been the major constraint for successful in vitro regeneration. Hence, alternate methods that evade tissue culture regeneration have been envisaged. Non tissue culture-based in planta transformation strategies are in vogue due to amenability and ease in the generation of transgenic plants. In the present study, we demonstrate the utility of an in planta transformation protocol and establishment of a stringent selection agent-based screening for the identification of transgenics. The genotype independent nature of the protocol was validated in cotton cv. Pusa 8-6 using GFP. Preliminary transformation efficiency of 28% was achieved with a screening efficiency of 20% in the presence of hygromycin. The proof of T-DNA integration by various molecular and expression analysis in T1 and T2 generations proved that this technique can be employed to generate transgenic cotton.
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