Decoding grapevine somatic embryogenesis through 3D spatial transcriptomics

Abstract

Somatic embryogenesis refers to the process through which differentiated plant cells reacquire totipotency and regenerate into whole plants. In grapevine (Vitis vinifera L.), this developmental reset represents a crucial regeneration pathway supporting vegetativepropagation, genetic transformation and, increasingly, DNA-free genome editing approaches whose successful application dependson robust and predictable regeneration systems. Despite its relevance, embryogenic competence remains incompletely understood, asthe complex regulatory networks underlying totipotency are typically inferred from bulk transcriptomic analyses that lack cellular resolution and obscure spatial heterogeneity. Although single-cell RNAsequencing increases resolution, it disrupts tissue architectureand positional information that are central to plant developmental regulation. Spatial transcriptomics (ST) overcomes these limitationsby enabling in situ gene expression profiling while preserving spatial relationships between neighbouring cells. Recent technological advances allow the integration of serial sections into three-dimensional reconstructions, generating combinedmolecular and histological tissue atlases at subcellular resolution (Schott et al., 2024). However, 3D ST has not been implemented ingrapevine and remains largely unexplored in plant developmental reprogramming. Here, we propose the first adaptation of thisapproach to grapevine somatic embryogenesis to generate spatially resolved expression maps of embryogenic calli and developing embryos. This strategy will allow the identification of transcriptional domains, embryogenic niches and cellular microenvironmentsassociated with the transition from differentiated cells to totipotent embryogenic states, with a particular focus on the spatialcoordination of transcription factors and hormone-related pathways shaping embryogenic reprogramming. Preliminary optimizationof the workflow in grapevine embryogenic tissues provides encouraging evidence of technical feasibility and enabling subsequent spatial analyses.

More broadly, the implementation of 3D ST in grapevine represents a significant methodological advance for perennial crops, expanding the frontiers of spatial gene expression studies and strengthening the innovation capacity of viticulture research.

References

Carratore, C.; Amato, A.; Pezzotti, M.; Bellon, O.; Zenoni, S. Genome Editing and Integrative Breeding Strategies for Climate-Resilient Grapevines and Sustainable Viticulture. Horticulturae 2026, 12, 117. https://doi.org/10.3390/horticulturae12010117

Carra A, Wijerathna-Yapa A, Pathirana R, Carimi F. Development and Applications of Somatic Embryogenesis in Grapevine (Vitisspp.). Plants (Basel). 2024 Nov 7;13(22):3131. doi: 10.3390/plants13223131. PMID: 39599340; PMCID: PMC11597845.

Nuzzo F, Gambino G, Perrone I. Unlocking grapevine in vitro regeneration: Issues and perspectives for genetic improvement and functional genomic studies. Plant Physiol Biochem. 2022 Dec 15;193:99-109. doi: 10.1016/j.plaphy.2022.10.027. Epub 2022 Nov 1. PMID: 36343465.

Ikeuchi M, Sugimoto K, Iwase A. Plant callus: mechanisms of induction and repression. Plant Cell. 2013 Sep;25(9):3159-73. doi:10.1105/tpc.113.116053. Epub 2013 Sep 27. PMID: 24076977; PMCID: PMC3809525.

Ikeuchi M, Shibata M, Rymen B, Iwase A, Bågman AM, Watt L, Coleman D, Favero DS, Takahashi T, Ahnert SE, Brady SM, Sugimoto K. AGene Regulatory Network for Cellular Reprogramming in Plant Regeneration. Plant Cell Physiol. 2018 Apr 1;59(4):765-777. doi: 10.1093/pcp/pcy013. Erratum in: Plant Cell Physiol. 2018 Apr 1;59(4):876. doi: 10.1093/pcp/pcy075. PMID: 29462363; PMCID: PMC6018650.

Koska S, Leljak-Levanić D, Malenica N, Bigović Villi K, Futo M, Čorak N, Jagić M, Ivanić A, Tušar A, Kasalo N, Domazet-Lošo M, Vlahoviček K, Domazet-Lošo T. Developmental phylotranscriptomics in grapevine suggests an ancestral role of somatic embryogenesis. Commun Biol. 2025 Feb 20;8(1):265. doi: 10.1038/s42003-025-07712-w. PMID: 39972184; PMCID: PMC11839975.

Giacomello S. Anew era for plant science: spatial single-cell transcriptomics. Curr Opin Plant Biol. 2021 Apr;60:102041. doi: 10.1016/j.pbi.2021.102041. Epub 2021 Apr 26. PMID: 33915520.

Yu X, Liu Z, Sun X. Single-cell and spatial multi-omics in the plant sciences: Technical advances, applications, and perspectives. Plant Commun. 2023 May 8;4(3):100508. doi: 10.1016/j.xplc.2022.100508. Epub 2022 Dec 20. PMID: 36540021; PMCID: PMC10203395.

Schott M, León-Periñán D, Splendiani E, Strenger L, Licha JR, Pentimalli TM, Schallenberg S, Alles J, Samut Tagliaferro S, Boltengagen A, Ehrig S, Abbiati S, Dommerich S, Pagani M, Ferretti E, Macino G, Karaiskos N, Rajewsky N. Open-ST: High-resolution spatial transcriptomics in 3D. Cell. 2024 Jul 25;187(15):3953-3972.e26. doi: 10.1016/j.cell.2024.05.055. Epub 2024 Jun 24. PMID: 38917789.

Schott M, León-Periñán D, Splendiani E, Ferretti E, Macino G, Karaiskos N, Rajewsky N. Protocol for high-resolution 3D spatialtranscriptomics using Open-ST. STAR Protoc. 2025 Mar 21;6(1):103521. doi: 10.1016/j.xpro.2024.103521. Epub 2024 Dec 19. PMID: 39708325; PMCID: PMC11731217.

Giacomello S, Lundeberg J. Preparation of plant tissue to enable Spatial Transcriptomics profiling using barcoded microarrays. Nat Protoc. 2018 Nov;13(11):2425-2446. doi: 10.1038/s41596-018-0046-1. PMID: 30353173.

Giacomello S, Salmén F, Terebieniec BK, Vickovic S, Navarro JF, Alexeyenko A, Reimegård J, McKee LS, Mannapperuma C, Bulone V, Ståhl PL, Sundström JF, Street NR, Lundeberg J. Spatially resolved transcriptome profiling in model plant species. Nat Plants. 2017May 8;3:17061. doi: 10.1038/nplants.2017.61. PMID: 28481330.

Saarenpää S, Shalev O, Ashkenazy H, Carlos V, Lundberg DS, Weigel D, Giacomello S. Spatial metatranscriptomics resolves host-bacteria-fungi interactomes. Nat Biotechnol. 2024 Sep;42(9):1384-1393. doi: 10.1038/s41587-023-01979-2. Epub 2023 Nov 20. PMID: 37985875; PMCID: PMC11392817.

Ge X, Yu X, Liu Z, Yuan J, Qin A, Wang Y, Chen Y, Qin W, Liu Y, Liu X, Zhou Y, Wang P, Yang J, Liu H, Zhao Z, Hu M, Zhang Y, Sun S, Herrera-Estrella L, Tran LP, Sun X, Li F. Spatiotemporal transcriptome and metabolome landscapes of cotton somatic embryos. Nat Commun. 2025 Jan 20;16(1):859. doi: 10.1038/s41467-025-55870-6. Erratum in: Nat Commun. 2025 Mar 5;16(1):2213. doi: 10.1038/s41467-025-57675-z. PMID: 39833155; PMCID: PMC11747644.