Legume Genomics and Genetics 2025, Vol.16, No.3, 143-152 http://cropscipublisher.com/index.php/lgg 149 6.3 Integration of scRNA-seq with spatial and temporal resolution approaches The spatial information gets lost when using scRNA-seq as a standalone method which makes it hard to determine where specific genes are expressed in the tissue. The integration of scRNA-seq with spatial transcriptomics and lineage tracing and time-course experiments enables scientists to construct developmental pathways while observing cell-cell interactions in their natural tissue environment. The integration of scRNA-seq with ATAC-seq and proteomics enables researchers to better understand gene regulatory networks and epigenetic landscapes (Denyer et al., 2019; Shaw et al., 2020; Zheng et al., 2021). 6.4 Prospects for translating findings into crop improvement and breeding strategies The large dataset from scRNA-seq allows researchers to discover essential regulators of root development and stress adaptation and symbiotic interactions which scientists can use to develop genetic engineering and breeding targets. Single-cell methods will establish themselves as essential tools for improving legume crops through improved protocols and reduced costs which will allow scientists to create new crop varieties with superior nutrient absorption and stress resistance and symbiotic relationships (Shaw et al., 2020; Bawa et al., 2022; Zheng et al.,2021). 7 Challenges and Future Directions in scRNA-Seq for Legume Root Development 7.1 Technical limitations: protoplasting, cell viability, and biases in cell capture The main technical hurdle in plant scRNA-seq analysis requires protoplasting to remove cell walls but this process triggers cellular stress reactions and cell death and generates artificial gene expression modifications. Cell dissociation methods fail to effectively break down cells with thick or lignified walls which leads to unbalanced cell collection and incomplete cellular mapping. The study of single-cell RNA sequencing data faces two major challenges which include technical noise and batch effects that need proper experimental design and data normalization methods (Shaw et al., 2020; Sun et al., 2024). 7.2 Need for cross-species root cell atlas development in legumes The root atlases of Arabidopsis and several crop species exist in detail but most legumes do not have complete cross-species cell-type maps. The creation of these atlases serves three main purposes which include comparative research and marker gene transfer and identification of shared and distinct root biology features in legumes. Scientists can discover common regulatory patterns and unique control systems of each plant species through studying single cells across different legume species (Shaw et al., 2020; Bawa et al., 2022). 7.3 Integration of scRNA-seq with spatial and temporal resolution approaches The main challenge of scRNA-seq data analysis arises from its inability to maintain spatial data which prevents researchers from identifying specific tissue locations for gene expression. Researchers can create developmental pathways and observe cell-cell interactions in their native tissue locations through the combination of scRNA-seq with spatial transcriptomics and lineage tracing and time-course experiments. The integration of scRNA-seq with ATAC-seq and proteomics enables researchers to study gene regulatory networks and epigenetic landscapes better (Denyer et al., 2019; Shaw et al., 2020; Zheng et al., 2021). 7.4 Prospects for translating findings into crop improvement and breeding strategies Scientists can identify essential regulators of root development and stress responses and symbiotic relationships through scRNA-seq data which serves as a basis for genetic modification and plant breeding programs. Single-cell methods will drive forward legume crop development through improved methods and reduced costs to produce new varieties that show better nutrient absorption and stress resistance and symbiotic relationships (Shaw et al., 2020; Zheng et al., 2021; Bawa et al., 2022). 8 Conclusion Single-cell RNA sequencing (scRNA-seq) has transformed the study of legume root development because it allows scientists to analyze cell diversity and detect infrequent cell populations while building comprehensive models of developmental progression. The technology has delivered complete information about cell fate determination and gene regulatory mechanisms and root and nodule development processes in legumes. The
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