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    Establishment of highly efficient and reproducible Agrobacterium-mediated transformation system for tomato (Solanum lycopersicum L.)
    (Springer, 2022) Hashmi, Muneeb Hassan; Saeed, Faisal; Demirel, Ufuk; Bakhsh, Allah
    A simple and improved Agrobacterium-mediated transformation protocol of tomato (Solanum lycopersicum) cultivar Rio Grande was developed to inspect the potential of producing transgenic tomatoes. In this study, regeneration and transformation efficiency as assessed in response to the seedling age, explant type, co-infection, co-cultivation duration, selection pressure (kanamycin), and the optimal concentration of plant growth regulators (PGR) 6-benzylaminopurine (BAP), gibberellic acid (GA3), and indolebutyric acid (IBA) in a Murashige and Skoog (MS) basal medium. To accomplish this goal, 2- to 4-wk-old tomato explant cotyledons, hypocotyl, and cotyledonary nodes were excised and transformed with the EHA105 Agrobacterium tumefaciens strain harboring pBIN19 binary vector containing uidA reporter gene and nptII as a selectable marker. Results revealed that 14-d-old cotyledonary nodes and leaves inoculated for 15 min with A. tumefaciens strain EHA105 following 48-h co-cultivation were optimal for the highest percent transformation efficiency 27.31. Antibiotic kanamycin (Kan) at 25 mg L-1 in the regeneration selection medium was found to be effective. MS medium containing optimal concentrations of 1.5 mg L-1 of BAP, 0.2 mg L-1 GA(3), and IBA 1.5 mg L-1 showed a significant level of percent regeneration, shoot elongation, and rooting efficiency of transformed plantlets. Molecular analysis of T-0 transgenic tomato plants showed integration and a higher relative expression level of the uidA gene. The optimized A. tumefaciens-mediated transformation method for tomato cultivar Rio Grande showed the highest percent transformation efficiency (TE) and regeneration efficiency (RE) and is likely to give consistent results with different tomato cultivars.
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    Harnessing plant-mediated RNAi for effective management of Phthorimaea absoluta by targeting AChE1 and SEC23 genes
    (Elsevier, 2024) Hashmi, Muneeb Hassan; Tariq, Haneef; Saeed, Faisal; Demirel, Ufuk; Gokce, Ayhan; Merzendorfer, Hans; Aksoy, Emre
    Tomato production on a global scale is under persistent pressure due to the devastating impact of Phthorimaea absoluta Meyrick (Lepidoptera: Gelechiidae), the South American tomato leaf miner. To combat this devastating pest, we explored the potential of plant-mediated RNA interference (RNAi) as a novel strategy for its management. Using transgenic techniques, we developed RNAi constructs (p35S
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    Identification and characterization of RNA polymerase II (RNAP) C-Terminal domain phosphatase-like 3 (SlCPL3) in tomato under biotic stress
    (Springer, 2023) Saeed, Faisal; Hashmi, Muneeb Hassan; Aksoy, Emre; Demirel, Ufuk; Bakhsh, Allah
    BackgroundBacterial diseases are a huge threat to the production of tomatoes. During infection intervals, pathogens affect biochemical, oxidant and molecular properties of tomato. Therefore, it is necessary to study the antioxidant enzymes, oxidation state and genes involved during bacterial infection in tomato.Methods and resultsDifferent bioinformatic analyses were performed to conduct homology, gene promoter analysis and determined protein structure. Antioxidant, MDA and H2O2 response was measured in Falcon, Rio grande and Sazlica tomato cultivars. In this study, RNA Polymerase II (RNAP) C-Terminal Domain Phosphatase-like 3 (SlCPL-3) gene was identified and characterized. It contained 11 exons, and encoded for two protein domains i.e., CPDCs and BRCT. SOPMA and Phyre2, online bioinformatic tools were used to predict secondary structure. For the identification of protein pockets CASTp web-based tool was used. Netphos and Pondr was used for prediction of phosphorylation sites and protein disordered regions. Promoter analysis revealed that the SlCPL-3 is involved in defense-related mechanisms. We further amplified two different regions of SlCPL-3 and sequenced them. It showed homology respective to the reference tomato genome. Our results showed that SlCPL-3 gene was triggered during bacterial stress. SlCPL-3 expression was upregulated in response to bacterial stress during different time intervals. Rio grande showed a high level of SICPL-3 gene expression after 72 hpi. Biochemical and gene expression analysis showed that under biotic stress Rio grande cultivar is more sensitive to Pst DC 3000 bacteria.ConclusionThis study lays a solid foundation for the functional characterization of SlCPL-3 gene in tomato cultivars. All these findings would be beneficial for further analysis of SlCPL-3 gene and may be helpful for the development of resilient tomato cultivars.
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    Role of genetic engineering in improving potato production
    (Elsevier, 2022) Saeed, Faisal; Dangol, Sarbesh Das; Hashmi, Muneeb Hassan; Hossain, Md Jakir; Bakhsh, Allah
    Potato is believed to be one of the most significant noncereal food crops to address the food security concerns in future for the increasing population worldwide. Potatoes are rich in vitamins, minerals, and antioxidants that make them of paramount significance for impressive health benefits besides. Numerous improvements have been introduced in potatoes for yield and yield contributing traits; however, limitation still exist especially when it comes to abiotic and biotic stresses. Recently, developments in genetic engineering have revolutionized potato breeding remarkably by addressing bottlenecks encountered in conventional potato breeding successfully. The new breeding techniques have allowed the researchers to add, remove, alter, and manipulate gene(s) to encode tolerance/resistance against abiotic and biotic stresses. The improvements in cell biology to regenerate plants from single cells or organized tissues provide a prerequisite for the practical use of genetic engineering in crop improvement. In addition to that, the quality of the potato has also been improved significantly for industrial purposes. This chapter provides a comprehensive understanding of how genetic engineering has contributed to potato improvement in terms of quality, insect pests, and diseases. It also discusses how these modern techniques can shape potato breeding programs for the development of climate-resilient cultivars in future. © 2023 Elsevier Inc. All rights reserved.
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    Transgenic technologies for efficient insect pest management in crop plants
    (Elsevier, 2020) Saeed, Faisal; Hashmi, Muneeb Hassan; Hossain, Md. Jakir; Ali, Muhammad Amjad; Bakhsh, Allah
    Insect and pests impose significant yield losses in crops despite the use of pesticidal and nonchemical controls. These increasing yield losses have provided impetus for the development of new management strategies against various pests. In this regard, transgenic technologies have revolutionized agriculture remarkably with the development of pest-resistant crops that have been outstanding in terms of crop productivity and highly advantageous to the farming community worldwide. With the passage of time, these technologies have enabled the scientists to modify and manipulate crop plants and to provide new solutions to solve conventional barriers for the improvement of economic traits of crops. The advancements in cell biology to regenerate plants from single cells or organized tissues provide prerequisite for the practical use of genetic engineering in crops improvement. The use of insect-resistant endotoxins from Bacillus thuringiensis in commercialized crops has resulted in increased crop yield since their introduction. The plant chloroplastic genome has also been engineered to encode resistance against insects pests. Several transgenic approaches have been employed to enhance resistance in plants against plant parasitic nematode as well. More recently, new and robust genetic engineering techniques like RNAi and Crispr-Cas9 have also proved their usefulness as a potential strategy in crop improvement for the control of pests. This chapter provides comprehensive insights and discussion of use of modern transgenic technologies in today’s agriculture and integration to efficient integrated pest management. © 2020 Elsevier Inc. All rights reserved.