A new nanotube-based RNA delivery system boosts plant regeneration by switching off a molecule that blocks shoot growth, without changing the plant’s DNA.
(Nanowerk Spotlight) Plant biotechnology has reached an inflection point. Advances in genome editing have made it possible to design crops with precision traits — drought resistance, disease tolerance, higher nutritional value — but turning these edits into viable plants still hinges on a slow and unreliable process: regeneration from cultured cells. For many economically important crops, inducing plant cells to regenerate into full organisms in the lab remains a technical deadlock. The cells resist, the transformation fails, and the engineered traits never make it into the field.
This barrier isn’t due to poor lab technique. The problem lies deeper, in the plant’s own regulatory systems. Molecular pathways designed to maintain developmental stability often suppress the very genes needed for regeneration. One such regulator, microRNA396 (miR396), blocks a family of genes essential for forming new shoots. Attempts to override this suppression have been limited by the lack of a delivery system that can temporarily and precisely modulate gene expression without permanently altering the genome.
Conventional methods like Agrobacterium-mediated transformation and particle bombardment have helped introduce genetic material into plant cells, but these techniques either lack precision, cause permanent genomic changes, or are incompatible with regeneration-recalcitrant species. Nanomaterials have emerged as a promising alternative, offering the ability to carry RNA and other molecules into cells without integration. Yet most nanocarriers fail to deliver cargo where and when it’s needed, or they clump together before reaching their targets.
A study published in Advanced Functional Materials (“Oligohistidine‐Functionalized Single‐Walled Carbon Nanotube‐Guided RNA Delivery to Improve Shoot Regeneration Efficiency in Plant Calli”) offers a practical solution to this long-standing problem. The team developed a novel nanocarrier based on single-walled carbon nanotubes (SWNTs) functionalized with a six-histidine peptide. These modified nanotubes, called His₆-SWNTs, can transport synthetic RNA into plant callus tissue — undifferentiated cells cultured for regeneration — and release it inside the cytoplasm at just the right time. The RNA molecule, called STTM396, binds and inactivates miR396, relieving its repression of growth-regulating genes and allowing regeneration to proceed more efficiently.
This diagram shows how the new nanotube system helps plants grow shoots more efficiently. A special type of carbon nanotube was coated with a short chain of histidine molecules so it could carry a designed RNA (called STTM396) into plant cells. Once inside, the RNA is released and blocks another RNA (miR396) that normally slows down shoot growth. By removing this block, the genes that promote shoot formation become more active, leading to better regeneration of shoots in plant tissue. (Image: Reprinted from DOI:10.1002/adfm.202510105, CC BY) (click on image to enlarge)
The underlying biology of shoot regeneration is complex. In tissue culture, plant cells must first form a callus — a cluster of undifferentiated cells — before reorganizing into shoot-forming structures. This process is mediated by hormones like auxin and cytokinin and controlled by networks of transcription factors. Among them, the Growth-Regulating Factor (GRF) family plays a central role. GRFs are repressed by miR396, which prevents their mRNA from being translated into protein. Without GRF activity, shoot regeneration is limited or fails altogether.
To overcome this block, the authors designed STTM396, a synthetic, noncoding RNA that mimics the natural targets of miR396. When delivered into the cytoplasm, it sequesters miR396 and prevents it from binding to GRF transcripts. The challenge was getting STTM396 into plant cells efficiently and ensuring that it would be released in the right place. That’s where His₆-SWNTs come in.
The nanotubes were modified by attaching oligohistidine peptides using a pyrene linker. This functionalization creates a nanocarrier that binds RNA through electrostatic interactions in slightly acidic conditions, such as the plant cell wall and extracellular spaces. Once inside the cytosol — where the pH is closer to neutral — the histidine loses its positive charge, weakening the RNA–nanotube interaction and triggering release. This pH-sensitive mechanism ensures that the RNA payload is delivered where it can function.
The authors confirmed the structural integrity and functional properties of the His₆-SWNTs using spectroscopy and microscopy. Nanoparticle tracking analysis showed a consistent size distribution, and Raman spectroscopy confirmed that the optical properties of the nanotubes were preserved. Zeta potential measurements revealed that the surface charge of the complex decreased with increasing pH, supporting the release mechanism. Importantly, RNA loaded onto the nanotubes was protected from degradation by nucleases, ensuring it remained intact during delivery.
To test the biological effect, the researchers treated calli from Arabidopsis and tomato with the STTM396–SWNT complex. In Arabidopsis, a single 18-hour treatment led to a 1.7-fold increase in shoot regeneration compared to untreated controls. Quantitative PCR confirmed that miR396 levels were reduced and that GRF gene expression was elevated during the early stages of regeneration. In tomato, which maintains high miR396 levels for a longer period, repeated treatments every five days produced a 2.1-fold increase in regeneration efficiency. Similar results were observed in a second tomato cultivar, suggesting the method’s broad applicability.
The study also showed that the STTM396–SWNT complex successfully penetrated deep into plant calli. Confocal Raman microscopy detected nanotube signals as far as 300 micrometers from the surface. Fluorescence microscopy further confirmed that both the nanotubes and their RNA cargo were released into the cytoplasm. Controls using RNA alone or the His₆ peptide without nanotubes did not produce the same effect, indicating that the complete nanocarrier system was essential for delivery.
This approach addresses a key limitation in plant biotechnology: the inability to transiently manipulate gene expression in a targeted, non-integrative way. The His₆-SWNT system does not alter the genome and degrades over time, minimizing the risk of unintended developmental effects. By targeting a known repressor of regeneration, it activates an endogenous gene network that promotes shoot formation without needing permanent genetic modification.
While the authors tested their method in Arabidopsis and tomato, the underlying principles could apply more widely. Many crop species — including wheat, citrus, and grape — suffer from poor regeneration rates in tissue culture. These species often cannot be transformed using conventional methods. A delivery platform like His₆-SWNT could help circumvent these barriers, enabling more efficient genome editing and trait development in crops that have previously resisted improvement.
The study also contributes to the growing body of research on nanocarriers in plant systems. Most prior work focused on chloroplasts or nuclei, where delivery is more difficult. By optimizing release conditions for the cytosol and ensuring compatibility with the plant cellular environment, this platform offers a more generalizable tool for molecular delivery. The design allows for high RNA loading, precise tuning of surface properties, and biocompatibility with minimal stress response.
The researchers have demonstrated a delivery system that is both technically sophisticated and biologically effective. The His₆-SWNT platform provides a model for how nanotechnology can be applied to one of the most persistent bottlenecks in plant genetic engineering. By targeting regulatory RNAs at the right time and place, the method unlocks a new strategy for improving regeneration efficiency — one that could be extended to a wide range of plant species and applications.
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