Legume Genomics and Genetics 2024, Vol.15, No.4, 199-209 http://cropscipublisher.com/index.php/lgg 203 4 Case Study: CRISPR/Cas9-Driven Improvement of Disease Resistance in Chickpeas 4.1 Identification of major disease resistance genes The identification of major disease resistance genes in chickpeas is a critical step towards enhancing disease resistance through genome editing. Recent advancements in CRISPR/Cas9 technology have enabled the precise targeting and modification of specific genes associated with disease resistance. For instance, the 4-coumarate ligase (4CL) and Reveille 7 (RVE7) genes have been identified as key players in drought tolerance and stress response in chickpeas. The 4CL gene is involved in the phenylpropanoid metabolism pathway, which regulates lignin accumulation under stress conditions, while the RVE7 gene is a MYB transcription factor that regulates circadian rhythm and stress responses (Badhan et al., 2021; Zhang et al., 2022). 4.2 CRISPR/Cas9-mediated targeted mutagenesis CRISPR/Cas9-mediated targeted mutagenesis has been successfully employed to edit the 4CL and RVE7 genes in chickpea protoplasts. This approach utilizes ribonucleoproteins (RNPs) composed of the Cas9 enzyme and a synthetically designed single guide RNA (sgRNA) to achieve high-efficiency editing. The DNA-free CRISPR/Cas9 system has shown promising results, with high-efficiency editing achieved for the RVE7 gene in vivo compared to the 4CLgene. This targeted mutagenesis represents a novel approach for achieving precise gene modifications in chickpeas, paving the way for improved disease resistance and stress tolerance (Figure 2) (Badhan et al., 2021). Figure 2 Flow chart of the steps performed during the protoplast isolation from leaf tissue of chickpea plants (Adopted from Badhan et al., 2021) Image caption: The leaves from the 4 to 5 weeks old plants were collected and surface sterilized. The leaves were cut into small pieces. The enzyme solution was prepared and cut leaf sections were kept in the dark for 4 to 5 h digestion resulting in protoplast formation. After 2 h, samples for protoplast formation were collected to check the protoplast formation stage, and once the protoplast were isolated, they were used for the PEG mediated transfections (Adopted from Badhan et al., 2021) 4.3 Field trials and performance evaluation Following successful gene editing, field trials are essential to evaluate the performance of the edited chickpea lines under natural conditions. Similar studies in other crops, such as soybean, have demonstrated that CRISPR/Cas9-mediated gene editing can enhance disease resistance without compromising agronomic traits. For
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