Bare Plant Cells Offer a Fast Lane to Better Carrots and a First for Bitter Gourd

Researchers in India have reported a set of tissue culture advances that could quietly reshape how two staple vegetable crops of the subcontinent are bred. In a study published in Discover Biotechnology, a team from the Institute of Chemical Technology in Jalna and Mumbai, together with the seed company Kalash Seeds, optimized the enzymatic isolation of protoplasts from two varieties of carrot (Daucus carota) and, for the first time in any variety or tissue, from bitter gourd (Momordica charantia). The work also demonstrates that carrot protoplasts can be stored in a simple refrigerator for five months and still regenerate into healthy, harvestable carrots, a result with real consequences for how breeding programs manage their genetic material.

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