Bioscience Methods 2024, Vol.15, No.6, 315-326 http://bioscipublisher.com/index.php/bm 318 Targeted editing strategies using CRISPR/Cas9 involve precise modifications of resistance genes to improve their efficacy. For example, the Lr34/Yr18 genes, which confer durable resistance to leaf rust and stripe rust, have been mapped to a specific locus on chromosome 7DS. By using CRISPR/Cas9 to introduce targeted mutations in these genes, researchers can enhance their resistance properties and potentially extend their effectiveness to other pathogens, such as powdery mildew (Spielmeyer et al., 2005). This approach not only improves resistance but also helps in understanding the genetic basis of disease resistance in wheat. 4 Wheat Stress Tolerance Improvement 4.1 Impact of abiotic stresses (e.g., drought, salinity, cold) on wheat production Abiotic stresses such as drought, salinity, and cold significantly impact wheat production, leading to substantial yield reductions globally. These environmental factors create hostile conditions that adversely affect plant growth and development, ultimately compromising overall productivity (Zafar et al., 2020; Bhat et al., 2021; Kumar et al., 2023). Drought stress, for instance, limits water availability, which is crucial for various physiological processes in plants. Salinity stress, on the other hand, disrupts ion homeostasis and water uptake, while cold stress affects membrane fluidity and enzyme activities, all of which are vital for plant survival and growth (Bhat et al., 2021; Nazir et al., 2022; Erdoğan et al., 2023). The economic implications of these stresses are profound, as they lead to decreased crop yields and increased production costs. Traditional breeding methods have been employed to develop stress-tolerant wheat varieties, but these approaches are often time-consuming and less effective in addressing the multifaceted nature of abiotic stresses (Jaganathan et al., 2018; Karunarathne et al., 2023). Therefore, there is a pressing need for innovative solutions to enhance wheat's resilience to these environmental challenges. 4.2 Application progress of CRISPR/Cas9 in regulating stress tolerance-related genes The advent of CRISPR/Cas9 technology has revolutionized the field of plant genetic engineering, offering a precise and efficient method for editing stress tolerance-related genes in wheat. This genome-editing tool allows for targeted modifications at specific genomic loci, enabling the development of wheat varieties with enhanced tolerance to abiotic stresses (Zafar et al., 2020; Bhat et al., 2021; Kumar et al., 2023). Researchers have successfully utilized CRISPR/Cas9 to edit genes involved in stress response pathways, such as those regulating osmotic balance, ion transport, and antioxidant defense mechanisms (Nazir et al., 2022; Erdoğan et al., 2023; Nascimento et al., 2023). One of the significant advancements in this area is the ability to create transgene-free plants, which are more likely to gain public acceptance and regulatory approval. By employing CRISPR/Cas9, scientists can introduce beneficial mutations without incorporating foreign DNA, thus addressing concerns related to genetically modified organisms (GMOs) (Jaganathan et al., 2018; Karunarathne et al., 2023). Additionally, the development of novel CRISPR/Cas9 variants and delivery methods has further improved the specificity and efficiency of gene editing, making it a robust tool for wheat genetic improvement (Biswas et al., 2021; Wang et al., 2022). 4.3 Successful examples of gene editing to enhance wheat stress tolerance Several successful examples highlight the potential of CRISPR/Cas9 in enhancing wheat stress tolerance. For instance, researchers have edited the TaERF3 gene, which plays a crucial role in the ethylene response pathway, to improve drought tolerance in wheat. The edited plants exhibited better water-use efficiency and maintained higher photosynthetic rates under drought conditions (Zafar et al., 2020; Kumar et al., 2023). Another notable example is the modification of the TaHKT1;5 gene, which is involved in sodium transport. By knocking out this gene, scientists have developed wheat varieties with improved salinity tolerance, as the plants were able to maintain ion homeostasis more effectively (Nazir et al., 2022; Erdoğan et al., 2023). In addition to these examples, CRISPR/Cas9 has been used to edit genes associated with cold tolerance. The TaCBF1 gene, which is part of the C-repeat binding factor (CBF) pathway, was targeted to enhance cold tolerance in wheat. The edited plants showed increased survival rates and better growth under low-temperature conditions (Jaganathan et al., 2018; Karunarathne et al., 2023). These successful applications demonstrate the versatility and
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