The research team, led by Clara U. Nwankwo and corresponding author Michael E. Ikehi, set out to answer a practical question that has grown more urgent in recent years. Global disruptions to fertilizer supply chains have sent prices soaring and left many farmers in developing regions unable to afford synthetic nutrients at the recommended rates. At the same time, demand for carrot foliage as livestock feed has been rising, turning what was once agricultural waste into a marketable secondary product. The authors frame this as a dual-market challenge: optimize nutrients for the taproot, optimize them for the leaves, or find a middle path. Their experiment was designed to test all three possibilities under identical conditions so the outcomes could be compared directly.

The methodology was deliberately straightforward. The researchers used a completely randomized design with three replicates, growing the Kuroda carrot variety over a 12-week period in four treatment groups. One group served as an unamended control, receiving no nutrient additions at all. A second received poultry manure, representing the organic option. A third received NPK 15:15:15, a balanced synthetic fertilizer supplying nitrogen, phosphorus, and potassium in equal proportions, representing the inorganic option. The fourth received a combination of poultry manure and NPK, representing integrated nutrient management. Data were analyzed using means, and analysis of variance was applied to test the null hypotheses at the 0.05 significance level, the conventional threshold in agricultural science for judging whether differences between treatments are likely to be real rather than the product of random variation.

When it came to what happens above the ground, the integrated treatment dominated. Carrots grown with the combined poultry manure and NPK treatment reached an average height of 34.39 millimeters, taller than those grown with inorganic fertilizer alone at 28.54 millimeters, organic manure alone at 32.35 millimeters, and the untreated control at 26.39 millimeters. Leaf biomass followed the same pattern, with the integrated treatment producing a value of 12.23, compared with 11.37 for organic manure, 9.10 for inorganic fertilizer, and 8.44 for the control. The pattern makes agronomic sense: organic manures release nutrients slowly as they mineralize, while synthetic fertilizers deliver an immediate pulse, and combining the two gives plants both a steady background supply and readily available nitrogen during peak vegetative growth.

Below the ground, however, the ranking flipped. Carrots grown with inorganic NPK fertilizer alone developed the longest taproots, averaging 17.86 millimeters, and the heaviest taproots, averaging 59.52 grams. The integrated treatment came in second at 14.84 millimeters and 54.15 grams, followed by organic manure at 13.35 millimeters and 42.48 grams, and the control at 11.55 millimeters and 31.25 grams. In other words, the synthetic fertilizer delivered roughly 90 percent more root weight than doing nothing at all, while the organic treatment delivered about 36 percent more. For a farmer whose income depends on selling graded taproots at market, those numbers carry real economic weight, and they explain why synthetic fertilizers have remained attractive even as their cost and availability have become less predictable.

The statistical analysis added an important layer of nuance to these numbers. While the integrated treatment produced numerically higher values for leaf biomass, the differences among treatments for that parameter did not reach statistical significance at the 0.05 level. Root tuber performance, by contrast, showed a statistically significant difference among treatments, meaning the advantage of inorganic fertilizer for taproot length and weight is unlikely to be a fluke of this particular experiment. The authors are careful to acknowledge this distinction, and it matters for how the findings should be used. A farmer choosing a fertilizer strategy to boost foliage would be making a decision on weaker statistical ground than one choosing a strategy to boost roots, and future replication studies could help firm up the biomass conclusions.

One of the more intriguing results concerns the organic treatment. Although integrated nutrient management produced the tallest plants and the highest leaf biomass score, the authors note that organic manure application alone was associated with a higher optimum harvestable root yield in their interpretation of the results, positioning it as a viable option for growers who want to avoid synthetic inputs altogether. Poultry manure is widely available and inexpensive in many parts of the global South, often generated as a byproduct of the region’s rapidly expanding poultry industry. Recycling it onto vegetable fields closes a nutrient loop, reduces waste disposal problems, and improves soil organic matter over time, benefits that extend beyond a single season’s yield even if they are harder to capture in a 12-week trial.

The broader context of the study is what gives it its urgency. The authors write that amid global fertilizer supply chain disruptions and the emerging dual-market demand for carrot taproots and leafy biomass, optimizing nutrient management is now essential for sustainability. Imported synthetic fertilizer exposes farmers in the global South to currency fluctuations, shipping delays, and geopolitical shocks that they have little power to influence. Integrated nutrient management offers a partial hedge, because a farmer who can meet part of the crop’s nutrient demand from local manure needs to purchase less synthetic product, reducing both cost and risk. The study’s results suggest that this hedge does not necessarily cost much in yield terms, since the integrated treatment was statistically comparable or superior in most measured parameters.

The dual-market angle is equally significant for the economics of smallholder carrot production. If the goal is cultivating carrots primarily for leafy biomass destined for livestock feed, the integrated treatment emerges as the optimal strategy in this experiment, maximizing the above-ground vegetative parameters that define that harvest. If the goal is the traditional taproot market, inorganic fertilizer remains the top performer, though organic manure offers a respectable and more affordable alternative. This goal-oriented framing is a departure from the usual framing of the organic-versus-conventional debate, which tends to ask which approach is universally better. The Nigerian team’s data suggest the more useful question is which approach is better for a specific product, market, and risk profile.

The study, conducted by researchers in the Department of Agricultural Education at the University of Nigeria, Nsukka, the Department of Environmental Management and Toxicology at the University of Delta, Agbor, and the Department of Home Economics and Hospitality Management at the University of Nigeria, received no specific financial support and was published as open access research. The authors conclude that adopting targeted organic and integrated nutrient management practices could provide sustainable, goal-oriented pathways to optimize agricultural production while buffering domestic farming in the global South against global challenges in food production. For a crop as familiar and widely grown as the carrot, that is a quietly radical proposition: the future of sustainable vegetable farming may depend less on choosing sides in the fertilizer debate than on matching the nutrient strategy to the harvest a farmer actually intends to bring to market.

Subject of Research: Comparative effects of organic, inorganic, and integrated nutrient management on carrot growth and yield

Article Title: A comparative analysis of organic, inorganic manures, and integrated nutrient management in carrot growth and yield in the global south

Article References: Nwankwo, C. U., Ikehi, M. E., Ashikodi, M. O., Ugwu, E. I., Nosakhare, D. O., & Ifeanyieze, F. O. (2026). A comparative analysis of organic, inorganic manures, and integrated nutrient management in carrot growth and yield in the global south. Discover Sustainability. https://doi.org/10.1007/s43621-026-04876-1

Image Credits: AI Generated

DOI: 10.1007/s43621-026-04876-1

Keywords: carrot, Daucus carota, integrated nutrient management, poultry manure, NPK fertilizer, leafy biomass, root yield, sustainable agriculture, global South, soil fertility, livestock feed, fertilizer supply chain

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