MPB2025v16n3

Molecular Plant Breeding 2025, Vol.16 http://genbreedpublisher.com/index.php/mpb © 2025 GenBreed Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Publisher

Molecular Plant Breeding 2025, Vol.16 http://genbreedpublisher.com/index.php/mpb © 2025 GenBreed Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. GenBreed Publisher is an international Open Access publisher specializing in molecular genetics, plant genes or traits, and plant breeding registered at the publishing platform that is operated by Sophia Publishing Group (SPG), founded in British Columbia of Canada. Publisher GenBreed Publisher Edited by Editorial Team of Molecular Plant Breeding Email: edit@mpb.genbreedpublisher.com Website: http://genbreedpublisher.com/index.php/mpb Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada Molecular Plant Breeding (ISSN 1923-8266) is an international, open access, peer reviewed journal published online by GenBreed Publisher. The journal publishes all the latest and outstanding research articles, letters and reviews in all areas of transgene, molecular genetics, crop QTL analysis, germplasm genetic diversity, and advanced breeding technologies. Molecular Plant Breeding is archived in LAC (Library and Archives Canada) and deposited in CrossRef. The Journal has been indexed by ProQuest as well. The Journal is expected to be indexed by PubMed and other databases in near future. All the articles published in Molecular Plant Breeding are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. GenBreed Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.

Molecular Plant Breeding (online), 2025, Vol. 16, No.3 ISSN 1923-8266 http://genbreedpublisher.com/index.php/mpb © 2025 GenBreed Publisher, an online publishing platform of Sophia Publishing Group. All Rights Reserved. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher Latest Content Application of Genome Editing in Sugarcane for Sugar Production: CRISPR/Cas9-Based Precision Improvement of Sugar Accumulation and Stress Tolerance Jianli Zhong Molecular Plant Breeding, 2025, Vol. 16, No. 3, 156-164 Molecular Mechanisms of Rice Drought Resistance Genes and Their Prospects in Breeding Nant Nyein Zar Ni Naing, Chunli Wang, Xiaoli Zhou, Cui Zhang, Junjie Li, Juan Li, Qian Zhu, Dongsun Lee, Lijuan Chen Molecular Plant Breeding, 2025, Vol. 16, No. 3, 165-179 Advances in Wheat Flour Processing: Strategies for Enhancing Nutritional Quality, Functional Properties, and Industrial Application Jianqiang Xu, Wei Hua, Min Fan, Weidong Wang, Jinghuan Zhu Molecular Plant Breeding, 2025, Vol. 16, No. 3, 180-190 Enhancing Postharvest Characteristics in Durian via Genome Editing: Regulation of Pericarp Softening and Shelf-Life Extension Dandan Huang, Haimei Wang Molecular Plant Breeding, 2025, Vol. 16, No. 3, 191-201 Improving the Fruit Flavor of Golden Pitaya through Gene Editing: Applications of CRISPR/Cas9 in Metabolic Pathways Zhen Li, Zhonggang Li Molecular Plant Breeding, 2025, Vol. 16, No. 3, 202-210

Molecular Plant Breeding 2025, Vol.16, No.3, 156-164 http://genbreedpublisher.com/index.php/mpb 156 Review Article Open Access Application of Genome Editing in Sugarcane for Sugar Production: CRISPR/Cas9-Based Precision Improvement of Sugar Accumulation and Stress Tolerance Jianli Zhong Hainan Provincial Key Laboratory of Crop Molecular Breeding, Sanya, 572025, Hainan, China Corresponding email: jianli.zhong@hibio.org Molecular Plant Breeding, 2025, Vol.16, No.3 doi: 10.5376/mpb.2025.16.0016 Received: 10 Apr., 2025 Accepted: 15 May, 2025 Published: 25 May, 2025 Copyright © 2025 Zhong, This is an open access article published under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Zhong J.L., 2025, Application of genome editing in sugarcane for sugar production: CRISPR/Cas9-based precision improvement of sugar accumulation and stress tolerance, Molecular Plant Breeding, 16(3): 156-164 (doi: 10.5376/mpb.2025.16.0016) Abstract This study explored the application of CRISPR/Cas9 genome editing technology in sugarcane improvement, which increased the sugar content and stress recovery ability of sugarcane. Sugarcane is an important crop for sugar production and bioenergy production. Its productivity and quality are limited by environmental challenges and the complexity of its genetic structure. The polyploid characteristics of sugarcane are facing huge challenges. However, the emergence of CRISPR/Cas9 genome editing technology provides precise tools to address these limitations, improving sugar metabolism and enhancing resistance to various stresses. The integration of multi-omics technologies and the pursuit of transgenic editing techniques have expanded the application scope of genome editing in sugarcane. Integrate multi-omics analysis and emerging gene editing tools in the future precise improvement research of sugarcane, a sugar crop, to promote the improvement of sugarcane varieties and the sustainable development of the industry. Keywords Sugarcane; CRISPR/Cas9; Genome editing; Sugar accumulation; Stress tolerance 1 Introduction As one of the major crops worldwide, sugarcane can provide bioenergy and sugar. The wide application of sugarcane in the food and processing industries supports the agricultural economy (Hussin et al., 2022; Krishna et al., 2023). As the global demand for sugar and biofuels continues to grow, the cultivation of sugarcane is becoming increasingly common, and increasing sugar production is also crucial (Krishna et al., 2023). Farmers are facing huge challenges. Pests, diseases and harsh environmental conditions, such as drought, saline-alkali soil or extreme weather, may significantly affect the yield of sugarcane (Li et al., 2022; Krishna et al., 2023). Furthermore, climate change and new stress factors have exacerbated these problems, leading to a decline in the stability of crops (Li et al., 2022; Krishna et al., 2023). Although traditional breeding methods are helpful, their progress is slow and the improvement results are usually small (Abdelrahman et al., 2018; Muha-Ud-Din et al., 2023). This study will explore the precise modification of sugarcane genes by CRISPR/Cas9 gene editing technology to cultivate new sugarcane germplasm that stores more sugar and has better resistance. Relying on the high-precision and high-efficiency features of CRISPR/Cas9 technology, it can promote the sustainable development of agricultural production and also solve the regulatory issues related to genetically modified crops. This study will also explore this method that combines modern gene editing technology with traditional breeding. The application of CRISPR/Cas9 gene editing technology is equivalent to injecting “technological impetus” into traditional breeding, accelerating the breeding speed of sugarcane resistant varieties, and improving the breeding efficiency and success rate. 2 Sugar Metabolism and Genetic Characteristics of Sugarcane 2.1 Key mechanisms of sugar accumulation Sugarcane is one of the main sources of sugar and bioenergy. The core of the economic value of sugarcane lies in the accumulation process of sugar. The accumulation of sugar in sugarcane involves complex metabolic pathways.

Molecular Plant Breeding 2025, Vol.16, No.3, 156-164 http://genbreedpublisher.com/index.php/mpb 157 Through photosynthesis, the sugar produced by sugarcane is transformed and stored in the stems of sugarcane. The regulatory network of enzymes and transport proteins can promote the transport and storage of sucrose in plants (Oz et al., 2021; Hussin et al., 2022). These mechanisms are highly efficient and can influence the final output of sugar. A key goal of growing sugarcane is to optimize the sugar accumulation process of sugarcane and increase its yield. The CRISPR/Cas9 gene editing technology can precisely adjust the pathways related to sugar metabolism. By targeting genes related to sucrose production and storage, in sugarcane breeding, researchers utilized CRISPR/Cas9 to enhance the sugar accumulation efficiency in sugarcane, thereby significantly increasing the overall sugar yield and improving other metabolic functions of sugarcane. CRISPR/Cas9 is an important means for improving sugarcane varieties (Hussin et al., 2022; Krishna et al., 2023). 2.2 Major metabolic and regulatory genes Sugarcane relies on a complex genetic network to control sugar metabolism, with many metabolic and metabolic genes. Some genes produce regulatory proteins that affect the activity levels of metabolic enzymes. Other genes can directly encode metabolic enzymes and participate in the production and decomposition of sucrose. Among these enzymes, sucrose phosphate synthase, sucrose synthase and invertase are particularly important in controlling the sugar content in plants (Zafar et al., 2020; Kumar et al., 2023). By using the CRISPR/Cas9 technology, researchers can edit the genes of sugarcane more accurately to increase the sugar content. The CRISPR/Cas9 technology alters the levels of regulatory genes and enzymes, making it easier for sugar in sugarcane to deposit. This gene editing method can improve sugarcane varieties and enable sugarcane to produce more sugar under different environmental conditions (Haque et al., 2018; Kumar et al., 2024). 2.3 Challenges of the polyploid genome In the genome editing of sugarcane, the polyploid characteristics of the genome are a challenge. The genome of sugarcane is composed of multiple sets of chromosomes, and genetic manipulation will be more complex. In traditional breeding and genetic engineering techniques, targeting multiple alleles simultaneously to achieve the desired traits is also a major challenge (Mohan, 2016; Oz et al., 2021). During the process of sugarcane breeding, the advancement of CRISPR/Cas9 technology has provided more opportunities for the study of sugarcane polyploid genomes. Enhancing the ability of precise gene editing of multiple alleles can improve the genetic traits of sugarcane. To stimulate the potential of genome editing in sugarcane, efficient transformation and screening technologies still need to be developed (Mohan, 2016; Tanveer et al., 2024). 3 The application of CRISPR/Cas9 in Sugarcane 3.1 Gene editing increases sugar production Researchers used CRISPR/Cas9 to adjust the genes of sugarcane that produce and store sugar. Sugarcane has a tricky genome, and CRISPR/Cas9 technology can perform small and precise edits. By altering key sugar-making genes, plants can produce and retain more sugar in their stems. Some edited types of sugarcane now store additional sugar (Augustine, 2017; Hussin et al., 2022). This proves that gene editing can help farmers cultivate new varieties of sugarcane that are sweeter and stronger. The CRISPR/Cas9 technology replaces “superior” genes with “inferior” ones to optimize the sugar metabolism process in sugarcane. By using the method of “homologous directed repair (HDR)”, researchers can carry out targeted nucleotide substitution, enabling sugarcane to better utilize its own genes to produce more sugar and improve sugar production efficiency (Oz et al., 2021). Since each gene in sugarcane has many copies, precise editing is of great significance. CRISPR/Cas9 helps improve the genetic combination of sugarcane and increase its sugar yield. 3.2 Edit stress resistance genes

Molecular Plant Breeding 2025, Vol.16, No.3, 156-164 http://genbreedpublisher.com/index.php/mpb 158 Sugarcane is confronted with numerous troubles such as drought, saline-alkali land, high temperature and pests and diseases. The CRISPR/Cas9 technology can address these challenges by editing genes related to these stresses. Editing genes related to drought resistance helps sugarcane grow better in arid environments, which means more stable sugarcane yields and reduced sugarcane losses (Krishna et al., 2023; Tanveer et al., 2024). Technology is constantly advancing, and the genetic modification system is also constantly being optimized. Sugarcane can also be grown without transgenic genome editing. Through transient expression systems and precise screening processes, researchers can cultivate gene-edited plants without involving genetically modified organisms. This solved the regulatory problem and laid the foundation for the commercial application of CRISPR/Cas9 technology in sugarcane improvement (Krishna et al., 2023). Integrating genome editing technology into sugarcane breeding is a transformative innovation in this transformation method, which is of great significance and importance. 3.3 Optimization of sugarcane conversion system The application effect of CRISPR/Cas9 in sugarcane depends on the efficiency of the transformation system. Due to the complexity of the sugarcane genome, it is of great significance to optimize the transformation methods and obtain reliable genome editing results. Strengthening the transformation methods (including agrobacterium-mediated technology and bio-particle delivery methods) to improve the binding of CRISPR/Cas9 components in sugarcane cells (Eid et al., 2021; Laksana et al., 2024). These improvements have increased the success rate of genome editing in sugarcane and cultivated varieties with excellent genes. With the advancement of technology, the transformation system is constantly being optimized. Sugarcane plants can be grown without transgenic genome editing. Through transient expression systems and precise screening processes, researchers can cultivate gene-edited plants without involving genetically modified organisms. This resolves regulatory issues and lays a solid foundation for the commercial application of CRISPR/Cas9 technology in sugarcane improvement (Krishna et al., 2023). Integrating genome editing technology into sugarcane breeding is a transformative innovation in this transformation method and is of great significance. 4 Gene editing strategies to increase sucrose accumulation in sugarcane 4.1 Editing genes related to sucrose-synthesizing enzymes The coding gene of sucrose synthase was precisely modified, which increased the sugar accumulation in sugarcane. Using the CRISPR/Cas9 system, researchers can target key genes (sucrose phosphate synthase (SPS); sucrose synthase (SuSy). The CRISPR/Cas9 system regulates the expression of these genes, which can enhance the efficiency of sucrose production and thereby increase sugar output. The successful application on other crops further proves that this method can improve sugarcane varieties (Arora and Narula, 2017; Hussin et al., 2022). CRISPR/Cas9 simultaneously edits multiple alleles, which can significantly enhance the trait of sugar accumulation. At the same time, for multiple loci, sugarcane varieties with high sucrose content can be cultivated. The accuracy and efficiency of CRISPR/Cas9 technology lay a solid foundation for complex genetic modification and is a sustainable way to increase sugar production (Oz et al., 2021; Tanveer et al., 2024). 4.2 Movement and storage of sucrose in sugarcane In sugarcane plants, the key to increasing the sugar content of sugarcane lies in the transportation of sugar. The sugar transport process involves the synthesis of sugar in the leaves of sugarcane and its transportation to the stems for storage. The CRISPR/Cas9 technology can optimize the sugar production process by editing the genes that control this process. By modifying these genes with CRISPR/Cas9, sugar can be transported more efficiently, helping sugarcane store more sugar and thereby increasing the total sugar yield (Chen et al., 2019; Krishna et al., 2023). Gene editing can also change the distribution pattern of sugar in sugarcane. Some genes in sugarcane can determine whether sugar is stored or used for other functions in sugarcane growth. Editing these genes can promote more sugar storage rather than consumption in other parts to enhance the sugar accumulation efficiency in sugarcane (Zafar et al., 2020; Ahmar et al., 2023).

Molecular Plant Breeding 2025, Vol.16, No.3, 156-164 http://genbreedpublisher.com/index.php/mpb 159 4.3 Altering key control genes (transcription factors) Edit the upstream transcription factors of gene expression related to sugar metabolism to increase sugar accumulation in sugarcane. Researchers used CRISPR/Cas9 technology to precisely edit these genes, improve the functions of multiple genes related to sugar, enhance the overall function of related metabolic pathways, and increase the sugar yield of sugarcane (Abdelrahman et al., 2018; Kumar et al., 2023). Precise regulation of the expression of transcription factors can optimize the glucose metabolism of sugarcane. Under the influence of climate change or some harsh conditions, sugarcane can still maintain a good yield. The application of CRISPR/Cas9 technology enables researchers to edit these transcription factors more easily and effectively, thereby promoting sugar accumulation in sugarcane and increasing sugarcane yield (Hussin et al., 2022; Tanveer et al., 2024). 5 Improvement of the stress resistance of sugarcane 5.1 Drought-resistant salt gene editing The CRISPR/Cas9 technology edits the key genes in sugarcane that help resist drought and salinization. Editing the genes that control the number of stomata (small holes on the surface of plants) can help sugarcane retain more water during drought and reduce water loss through transpiration (Kumar et al., 2020; Hussin et al., 2022). CRISPR/Cas9 can also target genes related to salt tolerance, those involved in molecular transfer or stress response signal transmission, enhancing the survival ability of sugarcane in saline-alkali environments (Farhat et al., 2019; Kumar et al., 2023). CRISPR/Cas9 can knock out harmful genes in sugarcane and precisely edit the genes related to some beneficial traits in sugarcane. For instance, a substance that helps plants cope with environmental stress, controlling osmotic protectants and editing the genes of this substance, can help sugarcane better maintain cellular balance and enhance stress resistance in harsh environments. CRISPR/Cas9 plays a significant role in crops such as rice and can enhance the drought resistance and salt tolerance of these crops (Figure 1) (Kumar et al., 2020; Kumar et al., 2024). Using CRISPR/Cas9 technology, researchers can cultivate sugarcane with greater stress resistance, and this variety of sugarcane can produce sugar stably for a long time. Figure 1 Types of biotic stresses that affect growth, yield, and productivity of sugarcane (Adopted from Kumar et al., 2024)

Molecular Plant Breeding 2025, Vol.16, No.3, 156-164 http://genbreedpublisher.com/index.php/mpb 160 5.2 Targeting disease resistance genes Through CRISPR/Cas9 editing, genes that play a key role in pathogen recognition and defense responses can enhance the disease resistance of sugarcane. Editing the resistance (R) gene can enhance the recognition ability of sugarcane against the effector of pathogenic bacteria, thereby strengthening its immune response ability. This method. CRISPR/Cas9 can edit the R gene and is also applicable to other crops, which can endow crops with resistance to various pathogens (Arora and Narula, 2017; Krishna et al., 2023). Researchers have utilized CRISPR/Cas9 to target the specific gene, the R gene, in sugarcane that is involved in pathogen recognition and defense responses, in order to enhance the susceptibility of sugarcane to diseases. As a result, the frequency of sugarcane being infected with diseases will be reduced, allowing farmers to use less spray and grow healthier sugarcane. This gene editing technology helps control other crops such as beans and rice, enhancing the resistance of these plants to bacteria, fungi and viruses (Wang et al., 2021; Kumar et al., 2024). The precise editing of these genes has made the new sugarcane varieties more stress-resistant and have higher yields. 5.3 Edit the stress response pathways CRISPR/Cas9 can help sugarcane cope with environmental stress during growth by modifying the genes that control the stress response. These genes, transcription factors and kinases play an important role in the process by which plants cope with adverse conditions. For example, by editing the genes in the ABA signaling pathway, CRISPR/Cas9 can enhance the water-saving capacity of sugarcane, thereby enhancing the drought resistance of sugarcane (Farhat et al., 2019; Ahmar et al., 2023). Similarly, the genes in the salicylic acid (SA) and jasmonic acid (JA) pathways were edited to enhance the disease and pest resistance of sugarcane (Kumar et al., 2023). CRISPR/Cas9 can simultaneously edit multiple genes and is also more likely to regulate complex stress response systems. Sugarcane is polyploid, and each gene in sugarcane can have multiple copies. CRISPR can target multiple genes simultaneously, which is particularly beneficial for sugarcane. CRISPR/Cas9 modifying different genes of the same pathway can more effectively enhance the stress resistance of sugarcane, enabling it to grow stronger and have a higher yield under harsh conditions, and also laying the foundation for cultivating better sugarcane varieties (Hussin et al., 2022; Mir et al., 2022). 6 Challenges and Future Perspectives 6.1 Complexity and off-target issues of the sugarcane genome The genome of sugarcane is very complex because it is polyploid. Polyploid leads to multiple copies of genes, so it is difficult to achieve targeted modifications without affecting the copies of other genes, and it may accidentally change the wrong part of the DNA. When using CRISPR/Cas9, it may also affect areas that should not be changed. These unwanted changes may bring about new problems or reduce the ability of plants to adapt to the environment (Mohan, 2016; Oz et al., 2021; Hussin et al., 2022). Researchers are also striving to improve the CRISPR/Cas9 system, studying a newer and more precise version of the Cas9 enzyme and designing better guide RNA to help the system locate the correct position (Oz et al., 2021; Hussin et al., 2022). Combining multi-omics tools such as genomics, transcriptomics and proteomics can help researchers better understand the genome of pitaya, which is conducive to selecting better target genes and conducting more precise gene editing (Figure 2) (Oz et al., 2021; Hussin et al., 2022). 6.2 Management challenges and public acceptance Sugarcane is one of the genome-edited crops. The main challenge in genome-edited crops is the relevant laws and regulations. Many countries have strict management regulations on genetically modified organisms (Gmos), and genome-edited plants usually also face scrutiny. The presence of genome editing in plants has raised concerns among regulatory agencies, leading to restrictions or delays in the application of some technologies in agriculture (Krishna et al., 2023; Kumar et al., 2024). Some genome editing methods, such as ribonucleoprotein technology, do not introduce exogenous DNA into plants, which can reduce some regulatory challenges (Arora and Narula, 2017; Krishna et al., 2023).

Molecular Plant Breeding 2025, Vol.16, No.3, 156-164 http://genbreedpublisher.com/index.php/mpb 161 Public understanding and acceptance of genome-edited crops are equally important. Due to concerns over safety and ethical issues, the public is skeptical of genetically modified crops. It is of great significance for regulatory agencies to explain to the public the safety and benefits of genome editing technology and gain public trust (Arora and Narula, 2017; Kumar et al., 2024). Involving farmers, consumers and policymakers in discussions, the public can understand the potential of genome editing, which can increase sugarcane yields and thus make these genome-edited crops more socially acceptable (Arora and Narula, 2017; Kumar et al., 2024). Figure 2 Identification of gene targeting in sugarcane by restriction endonuclease assays and herbicide application (Adopted from Oz et al., 2021) Image caption: (A) Repair template compared to sugarcane acetolactate synthase (ALS) gene. The 1 833 bp template carries four nucleotide substitutions, one at the codon positions W574L (introducing the MmeI recognition site) and S653I (eliminating BfaI recognition site), as well as two modified PAM sites (PAM1 and PAM2) (preventing template cleavage by Cas9 complexed with sgRNA1 or sgRNA2). Amino acid residue numbering in the sugarcane ALS gene follows the Arabidopsis nomenclature. (B) Schematic representation of primer annealing positions. Primers DO1 and UP6 were used to amplify a 1,913 bp fragment. Using primer UP6 prevented amplification of randomly inserted template by annealing outside the repair template. Primers F1 and R1 were used to generate a 455 bp nested PCR amplicon for analyses by restriction enzyme digestion. (C) Restriction-digestion pattern of nested PCR amplicons from wild-type (WT) and edited lines after digestion with MmeI or BfaI. + indicates positive control that includes the MmeI site and lacks the BfaI site. (D) Vegetatively propagated edited line L1 with multi allele conversion of W574L and S653I was tolerant to application of the herbicide nicosulfuron (Accent® DuPont) at 95 g ha-1, in contrast to non-edited WT plants (Adopted from Oz et al., 2021) 6.3 Emerging tools and multi-omics integration Genome editing technology is developing rapidly. New tools such as base editing and start editing are integrated with CRISPR technology. These new technologies can directly modify genes without cutting the DNA strand, thereby enhancing the safety and accuracy of crop gene editing. Because the genome of sugarcane is very complex, the advancements of these technologies are of great significance to sugarcane. Using these new tools, researchers can make smaller and more precise alterations to the DNA of plants (Arora and Narula, 2017; Haque et al., 2018).

Molecular Plant Breeding 2025, Vol.16, No.3, 156-164 http://genbreedpublisher.com/index.php/mpb 162 By bringing in multi-omics data- including gene sequences, RNA activity, and protein levels- researchers can better understand how sugarcane works. This helps them choose the best targets for editing. Combining these tools may lead to better sugarcane varieties that grow well and produce more under different conditions. 7 Concluding Remarks CRISPR/Cas9 is an important tool in sugarcane breeding. The genome of sugarcane is extremely large and complex. CRISPR/Cas9 can precisely and on a small scale edit specific genes of sugarcane or edit these genes in a controllable way, thereby helping to cultivate new sugarcane varieties more quickly. These newly cultivated varieties usually have a higher sugar content, while reducing unnecessary genetic changes and risks in sugarcane. They can better adapt to extreme climates or poor soil, making the breeding process of sugarcane more efficient and stable. The use of CRISPR/Cas9 helps sugarcane grow better and achieve good yields even under harsh conditions. By editing genes related to yield and tolerance with CRISPR/Cas9, researchers can cultivate sugarcane varieties that still grow well in drought, pest attacks or saline-alkali land, which is important for maintaining stable sugar production and helping to produce biofuels. CRISPR/Cas9 endows sugarcane with stronger natural defense capabilities and is the key to coping with the constantly changing environment. In sugarcane breeding, the use of CRISPR/Cas9 can ensure better development of sustainable agriculture, reduce the demand for chemical fertilizers and pesticides, make the plants themselves stronger, and also be beneficial to environmental protection. The new gene editing method does not add exogenous DNA. These crops may not be classified as genetically modified organisms, meaning that these genetically modified organisms can be approved more quickly and be more easily sold and accepted in markets around the world. Acknowledgments I would like to thank the two peer reviewers, Rudi Mai and Qixue Liang, for their feedback on this study. Their evaluations and suggestions have greatly contributed to the improvement of the manuscript. Funding This study was funded by the Hainan Tropical Agricultural Resources Research Institute Research Fund (Project No. H2025-01). Conflict of Interest Disclosure The author affirms that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. Reference Abdelrahman M., Al-Sadi A., Pour-Aboughadareh A., Burritt D., and Tran L., 2018, Genome editing using CRISPR/Cas9-targeted mutagenesis: an opportunity for yield improvements of crop plants grown under environmental stresses, Plant Physiology and Biochemistry, 131: 31-36. https://doi.org/10.1016/j.plaphy.2018.03.012 Ahmar S., Hensel G., and Gruszka D., 2023, CRISPR/Cas9-mediated genome editing techniques and new breeding strategies in cereals- current status, improvements, and perspectives, Biotechnology Advances, 69: 108248. https://doi.org/10.1016/j.biotechadv.2023.108248 Arora L., and Narula A., 2017, Gene editing and crop improvement using CRISPR-Cas9 system, Frontiers in Plant Science, 8: 1932. https://doi.org/10.3389/fpls.2017.01932 Augustine S., 2017, CRISPR-Cas9 system as a genome editing tool in sugarcane, In: Mohan C. (eds.), Sugarcane biotechnology: challenges and prospects, Springer, Cham, Switzerland, pp.155-172. https://doi.org/10.1007/978-3-319-58946-6_11 Bao A., Burritt D., Chen H., Zhou X., Cao D., and Tran L., 2019, The CRISPR/Cas9 system and its applications in crop genome editing, Critical Reviews in Biotechnology, 39: 321-336. https://doi.org/10.1080/07388551.2018.1554621 Chen K., Wang Y., Zhang R., Zhang H., and Gao C., 2019, CRISPR/Cas genome editing and precision plant breeding in agriculture, Annual Review of Plant Biology, 70: 667-697. https://doi.org/10.1146/annurev-arplant-050718-100049 Demirci Y., Zhang B., and Unver T., 2018, CRISPR/Cas9: an RNA-guided highly precise synthetic tool for plant genome editing, Journal of Cellular Physiology, 233: 1844-1859. https://doi.org/10.1002/jcp.25970

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Molecular Plant Breeding 2025, Vol.16, No.3, 165-179 http://genbreedpublisher.com/index.php/mpb 165 Research Insight Open Access Molecular Mechanisms of Rice Drought Resistance Genes and Their Prospects in Breeding Nant Nyein Zar Ni Naing1,4, Chunli Wang1,3, Xiaoli Zhou1,5, Cui Zhang1,3, Junjie Li1,3, Juan Li1,2,3, Qian Zhu1,2,3, Dongsun Lee1,2,3, Lijuan Chen1,2,3 1 Rice Research Institute, Yunnan Agricultural University, Kunming, 650201, Yunnan, China 2 The Key Laboratory for Crop Production and Smart Agriculture of Yunnan Province, Yunnan Agricultural University, Kunming, 650201, Yunnan, China 3 State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan Agricultural University, Kunming, 650201, Yunnan, China 4 Department of Plant Breeding, Physiology and Ecology, Yezin Agricultural University (YAU), Nay Pyi Taw, 15013, Myanmar 5 College of Agricultural Science, Xichang University, Liangshan, 615013, Sichuan, China Corresponding email: chenlijuan@hotmail.com Molecular Plant Breeding, 2025, Vol.16, No.3 doi: 10.5376/mpb.2025.16.0017 Received: 19 Apr., 2025 Accepted: 21 May, 2025 Published: 30 May, 2025 Copyright © 2025 Naing et al., This is an open access article published under the terms of the creative commons attribution license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Naing N.N.Z.N., Wang C.L., Zhou X.L., Zhang C., Li J.J., Li J., Zhu Q., Lee D.S., and Chen L.J., 2025, Molecular mechanisms of rice drought resistance genes and their prospects in breeding, Molecular Plant Breeding, 16(3): 165-179 (doi: 10.5376/mpb.2025.16.0017) Abstract Drought resistance in rice is a critical trait for ensuring stable yields in the face of increasing water scarcity. This review explores the molecular mechanisms underlying drought resistance genes in rice and their potential applications in breeding programs. Drought tolerance in rice is a complex trait influenced by various genetic and physiological factors. Recent advancements in genetic engineering, marker-assisted selection (MAS), and genome-wide association studies (GWAS) have identified key genes and quantitative trait loci (QTLs) associated with drought resistance. Several key genes closely associated with drought resistance have been appraised for their appreciable potential in drought resistance breeding. For instance, the overexpression of OsERF71 in transgenic rice has been shown to enhance drought tolerance by modulating global gene expression and energy allocation. Additionally, the identification of drought-responsive genes through transcriptome analysis and gene co-expression networks has provided insights into the biological processes and metabolic pathways involved in drought tolerance. The integration of these molecular insights into breeding programs, such as the use of MAS and genetic transformation, has led to the development of rice varieties with improved drought resistance. This review highlights the importance of a multidisciplinary approach, combining molecular genetics, plant physiology, and advanced breeding techniques, to develop rice cultivars that can withstand drought conditions and ensure food security. Keywords Drought resistance; Rice breeding; Molecular genetics; Gene expression; Marker-assisted selection (MAS) 1 Introduction Rice (Oryza sativa L.) is a staple food for more than half of the world’s population, particularly in developing countries (Hadiarto and Tran, 2011; Chen et al., 2013). However, rice cultivation is highly susceptible to drought stress, which significantly affects its growth, development, and yield (Sandhu and Kumar, 2017; Oladosu et al., 2019). Drought is one of the most severe environmental stresses, leading to substantial crop yield losses and posing a major threat to food security (Hu and Xiong, 2014; Pant et al., 2022). The increasing global population and changing climatic conditions exacerbate the challenge of ensuring stable rice production under water-deficit conditions. Therefore, developing drought-resistant rice varieties is crucial for sustaining rice production and ensuring food security (Hadiarto and Tran, 2011; Chen et al., 2013; Swamy and Kumar, 2013). Drought resistance in rice is a complex trait governed by multiple genes and involves various physiological and molecular mechanisms (Chen et al., 2013; Selamat and Nadarajah, 2021). Key mechanisms include osmotic adjustment, scavenging of oxidative radicals, and regulation of endogenous hormones such as abscisic acid (ABA) and jasmonic acid (JA) (Chen et al., 2013; Pant et al., 2022). Advances in biotechnology have enabled the identification and manipulation of drought-responsive genes, including transcription factors (TFs), protein kinases, and other regulatory proteins (Hadiarto and Tran, 2011; Selamat and Nadarajah, 2021; Pant et al., 2022). QTL mapping and MAS have been instrumental in identifying genetic regions associated with drought tolerance and incorporating them into breeding programs (Swamy and Kumar, 2013; Sandhu and Kumar, 2017). Additionally,

Molecular Plant Breeding 2025, Vol.16, No.3, 165-179 http://genbreedpublisher.com/index.php/mpb 166 transgenic approaches have shown promise in enhancing drought tolerance by overexpressing specific genes or silencing negative regulators (Hu and Xiong, 2014; Pant et al., 2022). This review summarizes the molecular mechanisms underlying drought resistance in rice and explores their potential applications in breeding programs. Specifically, it seeks to identify and characterize key drought-responsive genes and their regulatory networks, investigate the physiological and biochemical pathways involved in drought tolerance and assess the effectiveness of genetic engineering and MAS in developing drought-resistant rice varieties. Additionally, the review will provide insights into integrating molecular and conventional breeding approaches to enhance drought resistance in rice. Ultimately, this review aims to support the development of rice varieties that can withstand drought stress, thereby ensuring stable rice production and food security in the face of increasing environmental challenges. 2 Drought Stress in Rice: Physiological and Molecular Responses 2.1 Impact of drought stress on rice growth and yield Drought stress is a major abiotic factor that severely affects rice production, leading to significant reductions in growth and yield. Under drought conditions, rice plants experience reduced water availability, which directly impacts various physiological processes, such as photosynthesis, transpiration, and nutrient uptake. The severity of drought stress can cause stunted growth, delayed flowering, and reduced grain filling, ultimately leading to lower grain yield and quality (Lafitte et al., 2006). During the vegetative stage, drought reduces plant height, biomass, and tiller numbers, and causes leaf rolling in rice. The stress results from reduced soil moisture, which limits nutrient absorption and inhibits cell division in meristem tissues. At the tillering stage, water stress significantly impacts growth by hindering food production. Drought during flowering is especially damaging, disrupting pollination, inducing flower abortion, and leading to higher rates of unfilled grains. Prolonged moisture stress during the panicle initiation stage can reduce rice yields by 53.7%~63.5%, a major loss for farmers. Drought also hampers grain development during the reproductive stage, leading to spikelet infertility, decreased tillering capacity, and reduced photosynthesis due to leaf shrinkage. Water stress during the grain-filling stage accelerates plant senescence, shortens the grain-filling period, and reduces yield (Patnaik et al., 2021). Studies indicate that drought conditions during the critical flowering and grain-filling periods can reduce rice yields by up to 50%. Drought stress also affects the root system, reducing root length and density, which further impairs the plant’s ability to absorb water and nutrients from the soil (Serraj et al., 2011). 2.2 Physiological responses to drought stress Rice plants have developed a range of physiological responses to cope with drought stress. Chlorophyll is a key element in the photosynthesis of green plants and is positively correlated with the photosynthetic rate. Under drought stress, chlorophyll pigments can degrade and oxidize, which are common indicators of oxidative stress, resulting in a reduction in chlorophyll content. Both chlorophyll a and chlorophyll b are impacted by drought conditions (Islam et al., 2021). One of the primary responses is the closure of stomata to reduce water loss through transpiration. This stomatal closure, however, also limits CO2 uptake, thereby reducing photosynthetic efficiency. Drought stress decreases the efficiency of photosystem II, impairing energy conversion in the chloroplasts (Flexas et al., 2006). Osmotic adjustment is another critical response, where plants accumulate compatible solutes such as proline, glycine betaine, and sugars to maintain cell turgor and protect cellular structures (Serraj and Sinclair, 2002). Relative water content (RWC) is an important indicator of a plant’s water status, reflecting the metabolic activity within tissues and playing a crucial role in evaluating dehydration tolerance. RWC is associated with both water absorption by roots and water loss through transpiration. Many studies have reported a decrease in RWC in response to drought stress in various plant species (Nayyar et al., 2006). Additionally, rice plants enhance the expression of antioxidant enzymes like superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) to mitigate oxidative damage caused by reactive oxygen species (ROS) generated during drought stress (Mittler, 2002). Drought stress often leads to an increase in root-to-shoot ratio as plants invest more in root growth to enhance water uptake. Deeper and more extensive root systems help rice plants access water from deeper soil layers, improving drought tolerance (Henry et al., 2011).

Molecular Plant Breeding 2025, Vol.16, No.3, 165-179 http://genbreedpublisher.com/index.php/mpb 167 2.3 Molecular responses and signaling pathways At the molecular level, rice plants activate a complex network of signaling pathways and gene expression changes in response to drought stress. Key signaling molecules such as ABA play a central role in drought response by regulating stomatal closure and inducing the expression of drought-responsive genes (Shinozaki and Yamaguchi-Shinozaki, 2006). Former research has identified two primary regulatory pathways that influence gene expression patterns related to drought resistance mechanisms: (1) ABA-dependent pathways and (2) ABA-independent pathways. The ABA-dependent pathway is driven by MYB, NAC, and AREB/ABF TFs, whereas the ABA-independent pathways are regulated by DREB TFs (Du et al., 2011; Fu et al., 2017). TFs like DREB (dehydration-responsive element-binding), AREB/ABF (ABA-responsive element-binding protein/ABRE-binding factor), and NAC (NAM, ATAF, and CUC) are crucial for the activation of downstream drought-responsive genes that encode for osmoprotectants, late embryogenesis abundant (LEA) proteins, and heat shock proteins (HSPs) (Nakashima et al., 2007). Additionally, rice plants utilize microRNAs (miRNAs) to fine-tune gene expression during drought stress. For example, miR169 and miR393 have been shown to regulate genes involved in stress responses and hormone signaling pathways (Zhao et al., 2007). In response to drought, rice plants generate ROS, which act as signaling molecules in stress adaptation. Low to moderate ROS levels trigger the activation of stress-responsive TFs and antioxidant defenses. ROS interact with other signaling pathways, such as ABA and MAPK, to mediate drought responses (Mittler et al., 2011). The integration of these physiological and molecular responses enables rice plants to survive and adapt to drought conditions. Understanding these mechanisms provides valuable insights for developing drought-resistant rice varieties through genetic engineering and molecular breeding approaches. 3 Identification of Drought Resistance Genes in Rice 3.1 Methods for identifying drought resistance genes Identifying drought resistance genes in rice involves a combination of traditional and modern molecular techniques. GWAS have been instrumental in identifying loci associated with drought resistance traits. A study utilized a non-destructive phenotyping facility to extract 51 image-based traits (i-traits) from 507 rice accessions, leading to the identification of 470 association loci, some containing known drought resistance (DR)-related genes (Guo et al., 2018). Additionally, genetic linkage maps and QTL mapping have been used to dissect the genetic basis of drought resistance. A comprehensive study mapped QTLs for osmotic adjustment and root traits in a doubled-haploid rice population, identifying 41 QTLs that explained 8%~38% of the phenotypic variance (Zhang et al., 2022). Transcriptomic analyses and gene co-expression networks also play a crucial role in identifying differentially expressed genes (DEGs) under drought conditions, as demonstrated by the identification of key modules and hub genes associated with drought sensitivity in rice (Yu et al., 2020). 3.2 Key drought resistance genes discovered in rice Several key drought resistance genes have been discovered in rice through various studies. The OsPP15 gene, identified through GWAS and confirmed by genetic transformation experiments, plays a significant role in drought resistance (Guo et al., 2018). Another study highlighted the role of the AP2/ERF TF family member OsERF71, which, when overexpressed, conferred a drought-resistant phenotype by modulating global gene expression to prioritize survival-critical mechanisms (Ahn et al., 2017). Additionally, a meta-analysis of QTLs identified stable QTLs across different genetic backgrounds and environments, pinpointing genes such as ABA-Insensitive Protein 5 (ABI5) and G-box binding factor 4 (GBF4) as crucial for drought response (Selamat and Nadarajah, 2021). DREB1 and DREB2 have been extensively studied for their role in drought tolerance. These genes bind to dehydration-responsive elements (DRE) in the promoter regions of stress-inducible genes and activate their expression, leading to improved drought tolerance (Lata and Prasad, 2011). Furthermore, genes like WRKYs and PR family proteins have been identified as differentially expressed hub genes involved in ROS scavenging, contributing to drought resistance (Yu et al., 2020). Successful drought-resistant advantages of upland rice have motivated researchers to explore related genetic mechanisms. Sun et al. (2022) found that the elite haplotype of DROUGHT1 (DROT1) exists in upland rice, and the key SNP variation in the promoter region results in higher expression of DROT1, thereby enhancing drought

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