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

Protoplasts are plant cells stripped of their rigid cellulose walls, usually digested free from leaf tissue by cocktails of commercial enzymes. Freed from the wall, they behave like single cells in culture: they can be fused with cells of other species to sidestep sexual incompatibility, transformed with gene-editing machinery such as CRISPR components, and pushed to regenerate entire plants through somatic embryogenesis. Because protoplasts can develop into plantlets faster and at larger scale than conventional callus-based micropropagation, they have long been attractive to breeders of crops where open-pollinated cultivars produce inconsistent yields, as is the case for carrot and many other members of the Apiaceae family. Carrot itself is a historic model in this field; the first protoplast isolation from carrot root slices was reported in 1972, and the species has since served as a workhorse for studies of somatic hybridization, cytoplasmic male sterility, and transformation.

The new study compared two carrot varieties, the hybrid Champion F1 and the male-sterile Naigara, using leaves from aseptically grown seedlings as the starting material. Leaf tissue avoids the laborious cell suspension cultures traditionally used for carrot protoplasts, which can take three to eight months to establish and have historically delivered plating efficiencies of twenty percent or less. Leaves of three- to four-week-old seedlings were finely chopped, plasmolyzed in mannitol solution, and digested overnight with enzyme mixtures built around Cellulase Onozuka R-10, Macerozyme R-10, and Pectolyase Y-23. Released protoplasts, roughly 22 micrometers in diameter and green with randomly distributed chloroplasts, were sieved, pelleted by gentle centrifugation, and purified on a sucrose gradient before counting in a hemocytometer.

The enzyme recipes mattered, and in a variety-dependent way. For Naigara, the mixture of one percent Cellulase Onozuka R-10 with 0.1 percent Pectolyase Y-23 yielded 2.29 million protoplasts per gram of fresh leaf, while a stronger mixture of two percent cellulase with 0.2 percent Macerozyme R-10 raised the yield to 3.74 million per gram. Champion F1 showed the opposite trend: the pectolyase-based mixture delivered the best result, 4.9 million protoplasts per gram, nearly double the 2.42 million obtained with the macerozyme recipe. Viability, assessed by fluorescein diacetate staining under a fluorescence microscope, exceeded 70 percent in all cases, peaking at 87.5 percent for Champion F1 with the pectolyase mixture. The authors attribute the different responses of the two varieties primarily to variations in cell wall structure, a reminder that even within a single species, protoplast work remains a bespoke craft. The yields reported are comparable to those in prior Daucus studies, which have ranged from roughly 1.3 to 5.9 million protoplasts per gram across species and varieties.

The bitter gourd result is the study’s most novel contribution. Bitter gourd is valued across Asia for both nutrition and medicinal compounds, but its long juvenile phase, resistance to transformation, and variety-dependent regeneration protocols have hampered conventional breeding. The team tested ten different enzyme combinations on leaves of the open-pollinated variety KSP 1522, and only one recipe, combining 1.5 percent Cellulase Onozuka R-10 with 0.2 percent Macerozyme R-10, worked. The protoplasts proved highly sensitive: most other enzyme treatments either failed to liberate cells or produced protoplasts that burst during centrifugation. The successful combination yielded 1.18 million protoplasts per gram, about 24 micrometers in diameter, with viability of just over 80 percent that declined only modestly, by around eight percent, after 24 hours. Because viability above roughly 65 percent is generally considered sufficient for regeneration, the authors present this as a foundational protocol, while cautioning that it is optimized for this specific variety and explant, and that yields remain sub-optimal pending further refinement.

For carrot, the team went beyond isolation and demonstrated the full pipeline from naked cell to field-ready root. Protoplasts were embedded in thin layers of calcium alginate, a technique that protects the fragile cells, allows undisturbed cell walls to regenerate, prevents clumping, and improves mass transfer of nutrients because the matrix is thin. Within four to six weeks, visible macrocolonies emerged, developed into proembryonic masses, and then differentiated into globular, torpedo, and cotyledonary-stage somatic embryos over one to two months. Embryos were released from the alginate with a mannitol and sodium citrate solution and transferred to regeneration medium containing zeatin and naphthaleneacetic acid. Plantlets appeared within weeks, and germination frequency was so high that thousands of plantlets, too many to count individually, were produced. Of 332 plantlets hardened off in cocopeat under gradually reduced humidity, 319 survived acclimatization, a 96 percent success rate with no morphological abnormalities.

The cold storage experiments are arguably the most practically significant. Plant genetic resources are conventionally conserved as seeds, field collections, or in vitro cultures, with cryopreservation in liquid nitrogen as the gold standard for recalcitrant material. But cryopreservation is expensive, technically demanding, and, for carrot protoplasts, previously documented only once, with a colony-forming capability of just 19 percent after six months in liquid nitrogen. The Indian team instead stored Champion F1 protoplasts in carrot petiole protoplast medium at ordinary refrigerator temperatures of 2 to 8 degrees Celsius. After five months, the protoplasts were still viable by FDA staining, although exact counts were complicated by clumping, and they showed partial cell division upon rewarming. When embedded in alginate and cultured, the cold-stored protoplasts regenerated into plantlets within a week of transfer to regeneration medium.

The outcome of that regeneration pipeline was remarkable: from 253 plants hardened after regeneration from five-month-old cold-stored protoplasts, 216 survived acclimatization, an 85 percent efficiency. The regenerated plants completed a three-month growth cycle and produced healthy orange carrots, confirming that the entire pathway, from wall-less cell stored in a refrigerator to harvestable storage root, can function without cryogenic equipment. The authors are careful to note the limitations: the study rests on only two data points, and systematic work across longer storage durations with multiple time points will be needed to establish how far this simple approach can be pushed.

Taken together, the results offer breeding programs a practical toolkit. Reliable leaf-based protoplast isolation eliminates the need for long-lived suspension cultures; demonstrated cold storage at 2 to 8 degrees Celsius gives labs a low-cost buffer for scheduling fusions, transformations, and regeneration experiments weeks or months apart; and the first protoplast protocol for bitter gourd opens a route toward transgene-free genome editing in a crop whose medicinal value and breeding difficulties have long made it a difficult target. With CRISPR components already shown to be deliverable to protoplasts, and gene-edited carrots previously produced via protoplast transformation, the ability to isolate, bank, and regenerate these cells on demand brings somatic hybridization and precision editing for both crops a step closer to routine practice.

Subject of Research: Protoplast isolation, cold storage and plant regeneration in carrot and bitter gourd

Article Title: Protoplast isolation in Daucus carota and Momordica charantia: regeneration and cold storage studies in carrot

Article References: Ranaware, A. S., Kushwaha, S. B., Kunchge, N., Prakash, G., & Lele, S. S. (2025). Protoplast isolation in Daucus carota and Momordica charantia: regeneration and cold storage studies in carrot. Discover Biotechnology, 2(1), Article 24. https://doi.org/10.1007/s44340-025-00034-x

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00034-x

Keywords: protoplasts, carrot, bitter gourd, Daucus carota, Momordica charantia, plant regeneration, somatic embryogenesis, cold storage, tissue culture, cellulase, plant breeding, CRISPR