Protoplasts are plant cells that have been stripped of their rigid cell walls, leaving behind a fragile, membrane-bound sphere of living cytoplasm. Because the wall is removed, protoplasts readily take up foreign DNA, making them a workhorse system for plant research. In a typical protoplast experiment, scientists can introduce reporter constructs to watch gene expression unfold in real time, map protein localization inside the cell, dissect gene regulatory networks, test gene-editing reagents, and probe protein function, all within a matter of days and without the months required to generate a stable transgenic plant. The technique has been refined for major crops and model species such as Arabidopsis, tobacco, and rice, where leaves provide an abundant and convenient source of cells. But the standard recipe assumes a plant with expanded leaves and roots, and dodder has neither.

That anatomical peculiarity posed the central challenge for the research team at Virginia Tech, led by Hope Gruszewski and Matilda Cashman, who contributed equally to the work, alongside Joseph S. Taylor, Bastiaan O. R. Bargmann, and James H. Westwood. As an obligate parasite, C. campestris has evolutionarily abandoned most of the structures that define a conventional plant. It produces slender, leafless, yellow-orange stems that twine around hosts, and it lacks true roots, relying entirely on haustoria for sustenance. The team therefore had to rethink the very first step of the protocol: which tissue to use as the starting material. The options available were limited. They tested shoots harvested from plants that were actively growing on a host, young seedlings, and undifferentiated callus tissue grown in culture, systematically evaluating how well each source yielded intact, viable protoplasts after enzymatic digestion of the cell walls.

The answer, somewhat unexpectedly, came from the tissue least connected to the parasite’s natural lifestyle. Shoots that had developed from callus culture, essentially laboratory-grown stem tissue coaxed into existence on nutrient media, proved to be the best starting material, outperforming both host-grown shoots and seedlings. The researchers grew the callus on modified Murashige and Skoog media supplemented with the plant hormones 6-benzylaminopurine and naphthalene acetic acid, which promote the proliferation of the soft, actively dividing tissue that digests cleanly into single cells. Once the optimal tissue source was identified, the team turned to optimizing the remaining parameters of the isolation procedure, including the enzyme cocktail, digestion conditions, and purification steps, which involve filtering and washing the fragile protoplasts in solutions buffered with MES and protected with bovine serum albumin to preserve membrane integrity.

The optimized protocol delivers numbers that make real experiments possible. From just three grams of starting material, the researchers can routinely isolate approximately one million protoplasts, with more than 85 percent of them remaining viable after the isolation process. That viability figure is critical, because protoplasts are notoriously delicate; even in well-established systems, the stresses of enzymatic digestion and purification can rupture a large fraction of the cells. Achieving consistently high yields and viability from a parasitic plant with no conventional leaves means that downstream applications, from fluorescence microscopy to flow cytometry, now have enough material to work with. The method transforms what was once a bespoke, uncertain exercise into a repeatable routine that any competent plant molecular biology lab could adopt.

Isolation, however, was only half the battle. A protoplast is only useful if foreign DNA can be delivered into it and expressed. The team tackled this with polyethylene glycol-mediated transformation, the most widely used chemical method for shuttling DNA across protoplast membranes. In this approach, protoplasts are suspended in a mannitol-magnesium transformation buffer, mixed with plasmid DNA, and exposed to PEG, which transiently disrupts the membrane and permits DNA uptake before the membrane reseals. The researchers transformed the C. campestris protoplasts with multiple different constructs, and the technique reliably produced an approximate transformation rate of 10 percent. That robustness across constructs matters, because it demonstrates that the method is not a one-off success tuned to a single plasmid but a general platform for genetic manipulation of the parasite’s cells.

To showcase the research value of the new system, the team put the transformed protoplasts to work on two fronts. First, they expressed subcellular-targeted fluorescent proteins, confirming that the parasite’s cells can correctly sort tagged proteins to their intended destinations, a prerequisite for studying protein localization and trafficking in Cuscuta. Second, they used flow cytometry to quantify the activation of an exogenous auxin-responsive reporter gene, treating the protoplasts with the plant hormone auxin, indole-3-acetic acid, and measuring the fluorescent output across large cell populations. Auxin signaling is a master regulator of plant growth and development, and being able to measure its response quantitatively in dodder cells opens the door to comparing hormone signaling between the parasite and its autotrophic relatives at single-cell resolution.

The significance of the work extends well beyond the technical achievement. Parasitic plants such as Cuscuta, Striga, and Orobanche inflict enormous crop losses worldwide, and understanding how their cells sense hosts, form haustoria, and negotiate molecular exchanges with their victims is a major goal of plant science. Cuscuta is particularly intriguing because the haustorial connection allows the movement of RNAs and proteins between parasite and host, raising questions about cross-species signaling that cannot be answered with conventional model systems. Until now, however, progress has been hampered by the scarcity of genetic and cellular tools for these organisms. A reliable protoplast platform fills a crucial gap, offering a fast, flexible system for testing gene function without waiting for slow and difficult stable transformation protocols that have yet to be perfected for obligate parasites.

The Virginia Tech team, whose work was supported by the United States National Science Foundation and the United States Department of Agriculture, emphasizes that protoplasts are efficient systems for analyzing cell-level events, including gene expression, gene regulatory networks, gene editing, protein localization, and protein function. With the new protocol in hand, researchers can begin asking how the fundamental machinery of a parasitic plant cell differs from that of an autotrophic one, whether hormone signaling pathways have been rewired to serve the parasite’s needs, and which cellular programs underpin the formation of the haustorium. The method has the potential to accelerate research into the basic biology of parasitic plants, and in doing so, it may eventually inform new strategies for controlling one of agriculture’s most persistent enemies. For a plant that has spent millions of years perfecting the art of stealth, Cuscuta campestris has just lost a significant measure of its anonymity.

Subject of Research: Protoplast isolation and transient genetic transformation of the obligate parasitic plant Cuscuta campestris

Article Title: Isolation and transient transformation of protoplasts from Cuscuta campestris, an obligate parasitic plant

Article References: Gruszewski, H., Cashman, M., Taylor, J. S., Bargmann, B. O. R., & Westwood, J. H. (2026). Isolation and transient transformation of protoplasts from Cuscuta campestris, an obligate parasitic plant. Plant Methods. https://doi.org/10.1186/s13007-026-01600-y

Image Credits: AI Generated

DOI: 10.1186/s13007-026-01600-y

Keywords: Cuscuta campestris, protoplasts, parasitic plant, plant genetic transformation, haustoria, auxin signaling, flow cytometry, fluorescent proteins, plant cell biology, Plant Methods, callus culture, gene expression