The review, led by Francesca Cristiana Piritore and Maria Teresa Valenti of the University of Verona together with colleagues in Portugal, synthesizes a rapidly expanding body of evidence showing that plant cells actively secrete extracellular vesicles enriched in defense proteins, structural lipids, and regulatory RNAs. These vesicles, typically ranging from 30 to 300 nanometers in diameter, are now recognized as genuine mediators of intercellular communication rather than byproducts of cell wall remodeling. Crucially, vesicle release has been documented not only in intact plant tissues but also in callus and suspension cell cultures maintained in the laboratory, demonstrating that vesicle secretion is an intrinsic cellular process rather than one dependent on organized tissue architecture.

The functional repertoire of plant extracellular vesicles is striking. Early landmark work showed that sunflower vesicles, loaded with defense proteins and cell-wall-modifying enzymes, can bind to and kill cells of the fungal pathogen Sclerotinia sclerotiorum, causing morphological defects, growth retardation, and death in treated spores. More recent studies in Arabidopsis revealed that vesicles released during systemic acquired resistance are packed with immune signaling components and pattern-recognition molecules; recipient plant cells take up these vesicles and mount stronger defenses, reducing the virulence of bacterial and fungal pathogens in both infection assays and neutralization tests. Perhaps most remarkably, plant vesicles carry small interfering RNAs and microRNAs capable of silencing genes in interacting pathogens—direct evidence of cross-kingdom gene regulation mediated by vesicle traffic.

The molecular machinery behind this RNA trafficking is beginning to come into focus. Researchers identified several RNA-binding proteins in Arabidopsis that are secreted via exosome-like vesicles, including argonaute 1, RNA helicases RH11 and RH37, and the annexins ANN1 and ANN2. The helicases selectively associate with vesicle-enriched small RNAs, while the annexins bind them non-specifically; mutants lacking these proteins show reduced small RNA secretion, indicating that they are essential for loading or stabilizing RNA cargo within vesicles. This selectivity suggests a highly evolved mechanism by which plants can directly interfere with gene expression in their antagonists—a capability with obvious biotechnological appeal, though the authors caution that the relationship between cargo selection and specific vesicle biogenesis pathways remains poorly defined.

This is precisely where in vitro plant cell cultures enter the picture. The review highlights that callus and suspension cultures offer something whole plants cannot: a controlled, homogeneous, and potentially scalable environment for studying how vesicles are made, what they carry, and how their cargo can be modulated. Work on Arabidopsis cell cultures showed that vesicle biogenesis depends on the ESCRT trafficking pathway and on the physiological state of the cells, while studies of tobacco demonstrated that vesicles can be reproducibly generated and purified from calli and suspension cultures with morphological features comparable to those derived from whole tissues—although their composition differs, reflecting distinct cellular origins.

The practical advantages of culture-based production are considerable. Plant cell cultures can be maintained in simple, animal-component-free media without serum, reducing upstream costs, ethical concerns, and the risk of contamination by mammalian pathogens. They provide renewable biomass and are compatible with contained bioreactor production, including gentler airlift and temporary immersion systems that permit non-destructive collection of vesicles from the surrounding medium. Because vesicles can be harvested directly from conditioned culture media rather than from disrupted tissue, cultures also reduce interference from intracellular contents released during homogenization—a distinction the authors argue is critical for reproducibility. They draw a sharp line between bona fide extracellular vesicles secreted by living cells and the heterogeneous nanovesicle preparations generated by grinding or blending plant material, which contain membrane fragments, organelle-derived particles, and soluble contaminants that can confound both characterization and functional studies.

Isolation and purification remain the field’s most stubborn obstacles. Plant extracts are rich in pigments, phenolics, starch, polysaccharides, and secondary metabolites that coprecipitate with vesicles and interfere with downstream analysis. Differential ultracentrifugation remains the workhorse method, but high centrifugation forces co-sediment protein aggregates, prompting many laboratories to combine it with size-exclusion chromatography or density-gradient ultracentrifugation, which exploits the characteristic buoyant density of plant vesicles. Polymer-based precipitation offers a low-cost, scalable alternative at the price of contaminant carryover, while newer technologies—tangential flow filtration for pharmaceutical-grade scalability, the EXODUS system using oscillating membrane vibration, hydrophobic interaction chromatography on polymer fiber phases, and label-free microfluidic and acoustofluidic platforms—promise higher purity and throughput, though most still require validation on plant-derived material.

Characterization suffers from an equally fundamental gap: the absence of universal plant vesicle markers. Mammalian extracellular vesicle research relies on well-established markers such as CD9, CD63, and CD81, but no plant equivalents have been validated. Candidate proteins including TETRASPANIN8, PEN3, HSP70, HSP90, and PATL1 are frequently detected, yet their abundance varies with species, tissue, physiological state, and vesicle subpopulation, and antibodies raised against mammalian markers cannot be assumed to recognize plant homologs specifically. The review calls for plant-specific characterization guidelines combining multiple positive markers with negative markers for intracellular contaminants, aligned with the International Society for Extracellular Vesicles’ MISEV framework, alongside standardized reporting of cell-line origin, culture age, subculture number, and growth-regulator regime to guard against somaclonal variation during prolonged propagation.

On the translational side, the momentum is unmistakable. Plant-derived vesicles have shown antioxidant and anti-inflammatory activity, immune modulation, inhibition of tumor cell growth, and promotion of tissue repair in mammalian experimental systems. Cannabis-derived vesicles enriched in cannabidiol reduced the viability of hepatocellular carcinoma cell lines, with activity tracking CBD content across chemotypes. Strawberry and citrus vesicles carrying vitamin C and microRNAs protected human mesenchymal stromal cells from oxidative stress, with encapsulation enhancing the stability and bioavailability of the antioxidant cargo. Engineering strategies are advancing rapidly: LED irradiation exploits the intrinsic photosensitizing properties of vesicles to transiently increase membrane permeability, achieving roughly 80 percent loading efficiency under optimized conditions; electroporation loaded RNA into high-yield vesicles from poplar callus cultures, which were then internalized by Botrytis cinerea hyphae; and genetically modified tobacco plants produced vesicles selectively enriched with artificial microRNAs that induced sequence-specific gene silencing in recipient cells—the first proof of concept that plant vesicles can be programmed for targeted RNA delivery.

The authors are careful to temper enthusiasm with rigor. Most biomedical evidence comes from in vitro systems or acute animal models, with little known about biodistribution, pharmacokinetics, immune interactions, and long-term safety following systemic administration. Functional studies must exclude contributions from co-isolated free metabolites, and dose-response relationships, uptake mechanisms, and off-target effects remain largely unexplored. Yet the convergence of plant cell biology, vesicle research, and biotechnology that this review charts suggests a coherent path forward: controlled cultures as model systems for understanding vesicle-mediated communication, and as production platforms for biologically defined vesicles whose cargo, purity, and function can finally be specified, standardized, and translated.

Subject of Research: Extracellular vesicles released by in vitro plant cell cultures and their role in vesicle-mediated communication and biomedical applications

Article Title: Extracellular vesicles released by in vitro plant cell cultures: emerging systems for vesicle-mediated communication and biomedical translation

Article References: Extracellular vesicles released by in vitro plant cell cultures: emerging systems for vesicle-mediated communication and biomedical translation. (n.d.). https://doi.org/10.1007/s00299-026-03983-7

Image Credits: AI Generated

DOI: 10.1007/s00299-026-03983-7

Keywords: extracellular vesicles, plant cell cultures, plant-derived exosomes, intercellular communication, drug delivery, RNA cargo, vesicle biogenesis, plant immunity, nanocarriers, biotechnology, ESCRT pathway, cross-kingdom signaling