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

Collagen peptides are short chains of amino acids produced by enzymatically breaking down collagen from skin, bone, and cartilage. They have attracted attention for antioxidant, antihypertensive, and anti-aging properties, and previous work has shown that proline- and arginine-rich sequences at their C-terminal ends can inhibit dipeptidyl peptidase-IV (DPP-IV), an enzyme whose suppression prolongs the action of incretin hormones that stimulate insulin release. But the researchers argue that hitting DPP-IV alone is often not enough. Persistent hyperglycemia fuels overproduction of reactive oxygen species, and when antioxidant defenses falter, the resulting oxidative stress damages pancreatic beta cells, muddles insulin signaling, and derails glucose handling in the liver, accelerating the progression of type 2 diabetes.

To find a suitable partner for the peptides, the team screened four plant extracts, from blue plum, tremella, mulberry leaf, and bitter melon, alongside the cowhide collagen peptides themselves. In radical-scavenging assays, bitter melon extract stood out: its DPPH radical scavenging activity was nearly five times that of the collagen peptides, and its hydroxyl radical scavenging capacity exceeded the peptides by more than 85 percent. It also showed a stronger, though statistically not significant, tendency to inhibit alpha-amylase, the digestive enzyme that cleaves dietary starch into sugars. That dual profile, antioxidant power plus suppression of carbohydrate breakdown, made bitter melon the natural candidate for a combination formulation.

The interaction between the two components turned out to be a genuine molecular partnership rather than a simple mixture. Fluorescence spectroscopy showed that as bitter melon extract concentration rose, the peptides’ fluorescence was progressively quenched, and Stern-Volmer analysis indicated static quenching, meaning the two had formed non-fluorescent complexes. Thermodynamic calculations revealed positive enthalpy and entropy changes and negative free energy, signatures of a spontaneous, endothermic binding process driven primarily by hydrophobic interactions. Molecular docking of two representative collagen peptides, GPVGPPG and GPVG, against momordicine I, a key bioactive constituent of the extract, confirmed that hydrogen bonds and hydrophobic pi-alkane and pi-sigma contacts stabilize the assembly. Infrared spectroscopy and calorimetry backed this up: the amide I and amide II bands of the peptides shifted after binding, and the complex melted at higher temperatures, indicating enhanced thermal stability. Microscopy even showed the fragments reassembling into larger, flake-like structures.

What matters most for an oral supplement, however, is survival in the gut. The researchers subjected both the plain peptides and the complex to simulated gastric and intestinal digestion. Both withstood pepsin admirably, retaining over 80 percent of their activities. But trypsin in the intestinal phase proved far more destructive, significantly degrading the bioactivity of the plain peptides. The bitter melon complex, by contrast, retained significantly higher alpha-amylase inhibition and antioxidant capacity after intestinal digestion, suggesting the molecular partnership shields the peptides from enzymatic attack. Notably, DPP-IV inhibition was no different between the two after digestion, likely because continued hydrolysis of the proline-rich peptides keeps generating new DPP-IV-inhibiting fragments regardless.

Then came the animal trial. The team induced type 2 diabetes in mice with a high-sugar, high-fat diet followed by streptozocin injections, then treated the diabetic animals for four weeks with collagen peptides, the peptide-bitter melon combination, or metformin as a positive control. After four weeks, fasting blood glucose fell by nearly 38 percent in the peptide group and about 39 percent in the combination group, compared with roughly 52 percent for metformin. Glucose tolerance improved in both treatment groups, again with a slight edge for the combination, as measured by a standard oral glucose tolerance test.

The combination also tackled oxidative stress more effectively. Diabetic mice showed depressed total antioxidant capacity and reduced activities of glutathione peroxidase and superoxide dismutase in the liver, along with elevated malondialdehyde, a marker of lipid damage. Peptide supplementation partially reversed these changes, and the combination pushed them further toward normal, consistent with the in vitro radical-scavenging results. The liver told a similar story structurally: diabetic mice had depleted hepatic glycogen stores, swollen and vacuolated hepatocytes, and disrupted kidney tubules, all of which improved after treatment, with the combination group showing the clearest restoration of glycogen deposition and tissue architecture.

Mechanistically, the benefits appear to route through a well-known metabolic control axis. AMP-activated protein kinase, or AMPK, is a master sensor of cellular energy status whose activation suppresses hepatic gluconeogenesis, the liver’s internal production of new glucose. In the diabetic mice, the phosphorylation ratio of AMPK was significantly depressed. Both treatments restored it, with the combination showing the strongest effect, while simultaneously reducing the expression of FoxO1, a transcription factor that drives gluconeogenic genes, and PEPCK, the rate-limiting enzyme of that process. In other words, the supplement combination seems to flip the liver from a glucose-producing state toward a glucose-storing one.

The study also ventured into the gut microbiome, sequencing bacterial DNA from cecal contents and profiling cecal metabolites by untargeted metabolomics. Diabetic mice exhibited an elevated ratio of Firmicutes to Bacteroidetes, reduced microbial diversity, and shifts in genera such as Candidatus Arthromitus and Anaerostipes. The combination treatment partially reversed these dysbiotic patterns and shifted the overall community composition toward that of healthy controls. Metabolomics revealed 89 significantly altered metabolites, including stress-related steroid hormones like cortisol and aldosterone, which rose with diabetes and fell back toward normal with treatment, along with antioxidant compounds like ascorbate that were depleted in disease and partially restored. Association analysis linked these metabolic shifts to specific microbial phyla, hinting that the gut ecosystem participates in the formulation’s effects.

The authors are careful to frame the microbiota findings as correlational, offering mechanistic clues rather than proof of a causal host-microbe pathway, and the work remains at the mouse-model stage, with human efficacy, dosing, and safety still untested. Yet the study makes a compelling case that food-derived ingredients can be engineered into something greater than the sum of their parts. By pairing waste-stream collagen with a traditional medicinal plant, and letting noncovalent chemistry, digestive stability, AMPK signaling, and gut ecology all pull in the same direction, the researchers suggest that the future of glycemic management may partly lie not in a single magic molecule, but in thoughtfully assembled natural complexes. If subsequent studies confirm these effects in humans, cowhide, one of the meat industry’s most abundant leftovers, could find itself at the center of a new generation of functional foods for blood sugar control.

Subject of Research: Antihyperglycemic effects of cowhide collagen peptides combined with Momordica charantia extract in type 2 diabetes

Article Title: Enhanced antihyperglycemic effects of cowhide collagen peptides by the combination with Momordica charantia extract: AMPK–FoxO1 pathway engagement and gut microbiota remodeling

Article References: He, L., Wang, X., He, J., Gao, Y., Yu, Q., & Zhang, L. (2026). Enhanced antihyperglycemic effects of cowhide collagen peptides by the combination with Momordica charantia extract: AMPK–FoxO1 pathway engagement and gut microbiota remodeling. Journal of Agriculture and Food Research, 31, Article 103298. https://doi.org/10.1016/j.jafr.2026.103298

Image Credits: AI Generated

DOI: 10.1016/j.jafr.2026.103298

Keywords: collagen peptides, bitter melon, Momordica charantia, type 2 diabetes, AMPK, FoxO1, gut microbiota, antioxidant, DPP-IV, alpha-amylase, hepatic gluconeogenesis, functional foods