Legume Genomics and Genetics 2025, Vol.16, No.5, 215-224 http://cropscipublisher.com/index.php/lgg 220 6.2 Gene editing process and validation Where does this resistance come from? The key still lies in the ALS gene. As the target of imidazolicotinic acid, as long as this gene becomes "able to see through the moves", it can withstand the attack of herbicides. In the past, people mostly relied on traditional mutagenesis and screening, which had some effects, but the accuracy was not high enough. Now, researchers in Canada have also begun to test the water with CRISPR/Cas9, attempting to perform "surgery" on ALS, introducing precise mutations, and pursuing more stable and heritable resistance (Figure 2). During the process, not only the genetic data was examined, but also field experiments were conducted: could the edited plants survive after being sprayed with pesticides? How do you look? Will there be a harvest in the end? At the same time, molecular testing was also carried out to confirm that the mutation did occur at the target site (Wall, 1996). Only when both ends are passed can it be truly considered a success. Figure 2 Schematic diagram of the targeted sequence in OsALS and CRISRP/Cas9-ALS vector (Adopted from Wang et al., 2020) Image caption: (A) A schematic description of the OsALS gene. The target site is located at the 3′ end of OsALS and indicated by a yellow arrowhead antisense to OsALS. The sgRNA and protospacer-adjacent motif (PAM) together with their target sequences are shown. (B) Linearized CRISPR/Cas9-ALS construct showing genes encoding hygromycin B phosphotransferase, Cas9 protein, and the sgRNA targeting OsALS (Adopted from Wang et al., 2020) 6.3 Impact and future applications After the emergence of resistant lentils, field management has become less tense. Originally, it was necessary to rush to spray pesticides before the weeds grew. Now, it's better to wait a little longer-to act when the crops and weeds start competing for territory. The effect will be better and the yield can be increased by about 30% (Fedoruk and Shirtliffe, 2011). This resistance strategy not only makes the timing of herbicide application more flexible but also avoids early damage to seedlings. Overall, it is more in line with the concept of sustainable agriculture. For now, this case in Canada serves as a reminder to other countries that it is not necessary to rely on genetic modification. By making good use of gene editing, lentils can also be "armed". In the future, in addition to continuing to build up the resistance to multiple herbicides, some agronomic traits can also be improved incidentally, bringing us one step closer to practical application (Shivani et al., 2023). 7 Future Prospects and Research Directions for Herbicide-Resistant Lentils 7.1 Expansion to other herbicide classes At present, most of the research on the herbicide resistance of lentils focuses on ALS and PSII inhibitors, with an emphasis on the two types of herbicides, imidazolicotinic acid and metochlor. But one should not just focus on these two. Herbicides with different mechanisms of action, such as oxyfluoxane, fluoxazuron, triazuron and methomylimate, have long been widely used in other crops. In fact, some screening experiments and mutagenesis studies have found that there are indeed genotypes in lentils that can tolerate these herbicides. This indicates that
RkJQdWJsaXNoZXIy MjQ4ODYzNA==