Molecular Plant Breeding 2024, Vol.15, No.5, 209-219 http://genbreedpublisher.com/index.php/mpb 211 selection of genes that target the same pest species, ensuring synchronous expression in the same tissues, and avoiding cross-resistance among the stacked genes (Gressel et al., 2017). Figure 1 Schematic representation of Tranformation for gene stacking (Adopted from Shehryar et al., 2019) Image caption: A-C, Sequential transformation of two disease resistance genes. a and b Marker-free transgenic potato plants were produced by MAT vector system. c The Marker-free transgenic potato plants were re-transformed with wasabi defensin gene. d Dm-AMP1 and Rs-AFP2 genes were connected by linker peptide on same plasmid between leftand right borders. e Three genes, Rpi-sto1, Rpi-vnt1.1 and Rpiblb3 were transformed simultaneously in potato. f In GAANTRY system more than three genes could be transformed simultaneouslyin plants. GUS, beta-glucuronidase. hpt, hygromycin phophotransferase. nptII, neomycin phophotransferase. LP, linker peptideregion isolated from the seeds of Impatiens balsamina. Dm-AMP1, Antimicrobial proteins from Dahlia merckii. Rs-AFP2, Antimicrobial proteins fromRaphanus sativus. Rpi-sto1, Resistance gene for Phytophthora infestans from Solanum stoloniferum. Rpi-vnt1.1, Rpi fromS. venturii. Rpi-blb3, Rpi fromS. bulbocastanum. TBS transformation booster sequence, MYBCsMybA, Bar bialaphos resistance, GFPenhanced green fuorescent, Luc frefy luciferase, Sul1 sulfadiazine resistance (Adopted from Shehryar et al., 2019) 3.2 Methods and technologies for gene stacking Several advanced biotechnological methods are employed for gene stacking, including CRISPR/Cas9, TALENs, and zinc finger nucleases (ZFNs). These technologies enable precise editing and integration of multiple genes into the plant genome. CRISPR/Cas9: This technology allows for targeted gene editing by creating double-strand breaks at specific locations in the DNA, which can then be repaired by the cell's natural repair mechanisms, incorporating the desired genes. TALENs: Transcription activator-like effector nucleases (TALENs) are engineered proteins that can be designed to bind to specific DNA sequences, enabling targeted gene insertion or modification. ZFNs: Zinc finger nucleases are another class of engineered proteins that facilitate targeted genome editing. They have been used in combination with modular ‘trait landing pads’ (TLPs) to enable precise and efficient stacking of multiple traits in crops (Ainley et al., 2013). These technologies have revolutionized the field of genetic engineering by providing tools for precise and efficient gene stacking, thereby enhancing the durability and effectiveness of pest resistance in crops. 3.3 Selection of genes for stacking based on pest resistance mechanisms The selection of genes for stacking is critical and is based on understanding the pest resistance mechanisms. Genes are chosen to target different aspects of the pest's biology, such as metabolic pathways, detoxification processes, and reproductive mechanisms. For instance, genes encoding for proteins that interfere with the pest's digestive system or reproductive cycle can be stacked to provide a multi-faceted defense (Liu et al., 2011; Salim et al., 2020).
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