Molecular Soil Biology 2025, Vol.16, No.2, 91-102 http://bioscipublisher.com/index.php/msb 97 make a trade-off between high yield and high NUE (Van Bueren and Struik, 2017; Neuweiler et al., 2021). Gene or QTL expression often varies between nitrogen environments, with some being condition-specific, which complicates breeding across multiple environments (Garoma et al., 2021; Chen et al., 2022). 6.2 Long breeding cycles and high resource requirements Breeding under low-nitrogen conditions demands multi-location, multi-year trials across varying nitrogen levels to accurately compare genotypes for yield and NUE performance (Zhao et al., 2019; Ertiro et al., 2020). These efforts require significant investment and organizational capacity, particularly in terms of funding and infrastructure (Chen et al., 2022). Molecular breeding methods such as genomic selection and marker-assisted selection also depend on high-throughput genotyping and phenotyping platforms, which are often burdensome for institutions in developing countries (Badu-Apraku and Fakorede, 2017). Establishing and maintaining low-nitrogen screening fields adds to ongoing costs (Van Bueren and Struik, 2017). Most NUE-related traits are quantitative and strongly influenced by the environment. Introducing these traits into elite cultivars often requires multiple generations of selection and backcrossing (Kamara et al., 2024). Traits like root structure or nitrogen remobilization are difficult to observe in early generations, and selection often occurs only in later stages, further prolonging breeding cycles (Ertiro et al., 2020; Ranjan and Yadav, 2020). 6.3 Difficulty in adapting to diverse ecological zones Genotype-by-environment interactions (G×E) are a persistent challenge in breeding and are especially significant in low-nitrogen breeding (Van Bueren and Struik, 2017). A variety may perform well under specific soil, climate, and management conditions but not in others (Ertiro et al., 2020; Kimutai et al., 2021). Systematic evaluation across multiple locations and years is necessary to identify genotypes with either broad stability or strong adaptation to specific environments (Bänziger et al., 1997). This requires extensive environmental testing at every breeding stage, which increases both time and cost. Certain NUE-related QTLs or genes may only be effective under specific nitrogen levels, and their functionality may be lost in other settings (Garoma et al., 2021). This limits gene selection and utilization and adds uncertainty to variety development (Neuweiler et al., 2021; Chen et al., 2022). Some genotypes that perform well under low nitrogen may underperform under high nitrogen or in response to other stresses such as drought or disease, making them unsuitable for broad deployment (Makinde et al., 2023). Therefore, breeding efforts must consider not only NUE but also stress resistance and yield stability (Ertiro et al., 2020; Kimutai et al., 2021). 7 Future Development Directions and New Opportunities 7.1 Application of CRISPR and genome editing in rice NUE gene improvement CRISPR/Cas9 and other tools have been widely used in rice breeding in recent years, especially in improving nitrogen use efficiency (NUE). Researchers have found many genes related to nitrogen absorption and utilization, such as nitrogen transporters, nitrogen metabolism-related enzymes, and regulatory factors (Fiaz et al., 2021; Hu et al., 2022; Wang et al., 2022). Knocking out, inserting or precisely editing these genes with CRISPR can change gene expression and thus improve nitrogen absorption capacity. For example, editing genes such as OsNRT1.1B and OsNLP4 has significantly enhanced the nitrogen uptake and utilization efficiency of rice (Yu et al., 2020; Fiaz et al., 2021). This technology can not only modify one gene, but also manipulate multiple genes at the same time, breaking through the previous limitation of only being able to improve a single trait and speeding up the breeding process (Fiaz et al., 2021; Hu et al., 2022). There are also newer tools, such as base editing and in situ editing, which can regulate DNA more finely and have higher operating efficiency, and are suitable for dealing with complex traits involving multiple genes such as NUE (Fiaz et al., 2021). However, this technology has not yet been promoted on
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