Title : The effect of fixation methods on gene expression from single-Cell RNA sequencing in somatic embryogenesis cells of orchid (Phalaenopsis aphrodite)
Abstract:
Single-cell RNA sequencing (scRNA-seq) advances transcriptomic analysis by enabling high-resolution gene expression profiling at the cellular level, thereby overcoming the limitations of bulk RNA sequencing in capturing heterogeneity. However, scRNA-seq for plant samples still faces a challenge in protoplast isolation due to the low cell wall enzymatic efficiency, which leads to subsequent stress responses and cell type capture bias. This study evaluates the effectiveness of a pre-enzymatic fixation method compared to the conventional fresh method in Phalaenopsis aphrodite, using Arabidopsis callus as a benchmark. The transcriptomic cell atlases were compared from both methods, then applied this fixation method to identify somatic cell type and molecular mechanisms initiating embryogenic competence in Phalaenopsis aphrodite. This study optimized protoplast isolation by fixing samples in Methacarn for 1-hour prior to 2-hour enzymatic digestion at 37°C, while fresh samples underwent 3-hour digestion at 25°C. The fixation method demonstrated superior performance, yielding higher number of protoplasts (10,000 cells) and improved sequencing quality (1,609 genes/cell and 2,590 UMI counts), while preserving cell integrity. Furthermore, fixed samples enabled higher-resolution cell-type annotation, identifying 12 distinct clusters, and reducing mixed identities. This approach revealed distinct identities including meristematic, procambium, and guard cell lineage stages. Key somatic embryogenesis (SE) trajectories were reconstructed using Monocle3, showing that procambium-like cells (Cluster 4) serve as the primary origin of embryonic competence, transitioning through auxin-mediated reprogramming (LAX2, MP) and embryonic commitment (SERK1, SERK2, Cyclin genes). Overall, these findings validate fixation-based scRNA-seq as a robust approach for plant single-cell transcriptomics, enhancing cell capture and developmental trajectory mapping. Significantly, this research provides a feasible solution for exploring the single-cell transcriptomic landscape of samples that protoplast isolation is otherwise challenging
