Journal of Energy Bioscience 2025, Vol.16, No.5, 238-247 http://bioscipublisher.com/index.php/jeb 242 Figure 2 Challenges in sugarcane breeding and their biomass improvement. This figure illustrates the diverse challenges in sugarcane breeding and biomass improvement. Key areas include developing pest-resistant varieties to combat issues like Pyrilla perpusilla and Ceratovacoa lanigera and enhancing disease resistance against rust and smut. Environmental factors such as temperature, humidity, sunshine, and CO2 levels affect plant health, necessitating climate change resilience and soil adaptation. Yield and quality improvement focus on balancing high biomass yield and enhancing sucrose content. Technological limitations highlight the integration of biotech advances with conventional breeding, particularly through RNA sequencing. Genetic complexity, including polyploidy and a narrow genetic base, poses significant challenges (Adopted from Ahmad and Ming, 2024) 5 Emerging Technologies in C4 Engineering 5.1 CRISPR/Cas genome editing for precise modification of photosynthesis-related genes The CRISPR/Cas system has become an important tool for modifying the photosynthesis genes of C4 crops. It can achieve gene knockout, knock-in and base replacement, thereby significantly enhancing the efficiency of site-specific modification of key photosynthetic enzymes (such as PEPC, Rubisco, PPDK, etc.) and regulatory factors. CRISPR/Cas9 and its improved forms (such as base editing and guide editing) not only accelerate the trait improvement of C4 crops, but also can simultaneously regulate multiple photosynthetic or metabolic genes through multi-site editing (MGE), thereby promoting the cultivation of multiple traits such as high yield and stress resistance (Abdelrahman et al., 2021). In addition, CRISPR/Cas technology is also making continuous progress in reducing off-target effects, improving editing efficiency and obtaining transgenic offspring (Bao et al., 2019; Chen et al., 2019; Wada et al., 2020).
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