Vincetoxicum spirale, known locally in Rajasthan as Aakari Bel or Doodhli, is far from an obscure botanical curiosity. In the Little Rann of Kachchh, a unique salt-marsh landscape in Gujarat, it is regarded as one of the region’s life-supporting plants, valued as food for both people and cattle. The plant carries a substantial chemical arsenal, including an acyclic diterpene ester, bergenin, ferulic acid, quercetin derivatives, squalene and triterpenes. Traditional medicine has exploited these constituents for their alterative, astringent, cooling, emetic, purgative, anti-inflammatory, antimicrobial and antifungal properties, deploying the plant against gonorrhoea, fever, indigestion, dysentery and muscular troubles. Native to Eritrea and Yemen and distributed across the dry shrublands of Africa, Australia and Asia, the species is already listed as threatened in the Karachi region of Pakistan, and ecological niche modelling predicts a high risk of habitat destruction in southeastern Sindh by 2050.

The obstacles to natural regeneration are formidable. The plant propagates by small, brown, winged seeds, but seed availability, germination and seedling establishment are all unreliable, and drought stress, overgrazing and mechanized agriculture compound the losses. Against this backdrop, the research team, led by Ashok Kumar Patel with Deepika Lodha, Sumitra Kumari Choudhary and N. S. Shekhawat, set out to build a regeneration system from scratch. Their strategy relied on indirect organogenesis, the process by which differentiated plant cells first dedifferentiate into an unorganized callus and then re-differentiate into shoots through newly formed meristematic centres. The process unfolds in three recognized stages: dedifferentiation, in which the explant responds to organogenetic signals; induction, in which cells commit to producing shoots or roots; and realization, when visible organs emerge.

The team began by comparing four types of explants: leaf, node, internode and root segments, all collected from a wild source plant near the village of Mogra in Jodhpur district. After surface sterilization with the systemic fungicide Bavistin followed by mercuric chloride, the explants were placed on Murashige and Skoog medium fortified with sucrose and varying concentrations of the synthetic auxins NAA or 2,4-dichlorophenoxyacetic acid. The verdict was unambiguous. Leaf explants cultured on medium containing 2.0 milligrams per litre of 2,4-D produced the highest callus induction rate, 76.7 percent, and the greatest fresh weight, about 747 milligrams after five weeks. Internodal explants performed respectably at 63.3 percent, while node and root explants lagged far behind, and root cultures suffered the heaviest contamination. The authors note that the midrib and veins of a leaf are extensions of the stem’s vascular bundles, surrounded by unspecialized parenchyma cells that are particularly competent to respond to growth regulators.

The choice of auxin proved decisive. While 2,4-D at 2.0 milligrams per litre produced cottony-white, proliferative and partially organized callus, the same concentration of NAA yielded brown, slow-growing, compact callus with a response of only 24.6 percent, and higher NAA levels pushed the tissue toward hard, rhizogenic growth. The researchers attribute 2,4-D’s potency to its role in reprogramming cells and driving cell division, effects that involve the control of endogenous auxin metabolism, DNA methylation and the induction of specific proteins. Yet callus induced on 2,4-D would not proliferate indefinitely on the same medium, because the synthetic auxin is metabolized slowly and can accumulate to toxic levels. The solution was to transfer the tissue to medium containing a reduced dose of 1.0 milligram per litre of 2,4-D combined with 0.5 milligrams per litre of the cytokinin Kinetin. This combination produced morphogenic, competent, light yellow-green, friable callus with a callus proliferation coefficient of 7.49, meaning the tissue nearly seven-and-a-half-folded in mass over five weeks, compared with just 3.01 on auxin alone.

With proliferating callus in hand, the team turned to the heart of the protocol: coaxing shoots to form. Proliferated callus was moved to media containing the cytokinins BAP or Kinetin, alone or with low levels of the auxins NAA or IAA. BAP outperformed Kinetin, and the two cytokinins together worked better than either alone, with a trace of NAA further boosting differentiation. The winning formula combined 0.5 milligrams per litre of BAP, 0.25 milligrams per litre of Kinetin and 0.1 milligrams per litre of NAA, supplemented with adenine sulphate, L-arginine and citric acid at 25 milligrams per litre each, ascorbic acid at 50 milligrams per litre, and 100 milligrams per litre of activated charcoal. This cocktail yielded an average of 30.17 shoots per culture, each about 6.26 centimetres long, after six weeks. Each additive earns its place: adenine sulphate is a purine precursor in cytokinin biosynthesis, L-arginine supplies extra reduced nitrogen, citric acid chelates and suppresses the phenolic browning that plagues many cultures, ascorbic acid acts as an enzyme cofactor and antioxidant, and activated charcoal adsorbs inhibitory metabolites while releasing growth-favouring substances.

Perhaps the most commercially significant step is what the researchers did next. Instead of rooting the microshoots in sterile laboratory medium, they pulse-treated the cut bases with concentrated auxin solutions and planted them directly into solid substrates, a one-step strategy known as ex vitro rooting and concurrent acclimatization, or EVRCA. Dipping shoots in 300 milligrams per litre of NAA for exactly five minutes produced rooting in more than 75 percent of shoots, with an average of 4.45 roots per shoot reaching 5.58 centimetres within three weeks. Timing mattered: five minutes beat seven, three and nine minute treatments. Among the substrates tested, the porous, water-retentive Soilrite outperformed cocopeat and ordinary soil, both of which impede the delicate juvenile roots. Because EVRCA eliminates the entire in vitro rooting stage, it cuts chemical use, labour and time, reduces microbial contamination, and produces root systems with lateral root hairs and good vascular connectivity that resemble those of mother plants. About 70 percent of the rooted plantlets survived transplantation into polybags, and the cultures themselves were maintained for two years of subculturing without any decline in vigour.

The study’s second strand reaches into molecular territory. To understand why some callus regenerates and some does not, the team compared the total soluble protein profiles of non-regenerative callus, harvested from the induction medium, and regenerative callus, harvested from the shoot differentiation medium, using sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining. Total protein content rose from 34 micrograms per milligram fresh weight in non-regenerative callus to 42 in regenerative callus. The gels resolved 16 bands in the non-regenerative sample and 19 in the regenerative one, with significant differences at eleven polypeptide positions unique to the regenerative callus and three unique to the non-regenerative one, plus intensity variations at four further bands. Notably, the low molecular weight polypeptides labelled P17, P18 and P19 appeared only in regenerative tissue, suggesting they may underpin the callus’s capacity to build shoots. Protein expression is known to shift with developmental phase as structural and regulatory genes switch on and off, influenced by media composition, explant type and culture conditions, echoing earlier findings in sweet orange, where 2,4-D-responsive proteins revealed extensive gene reprogramming during somatic embryogenesis.

The implications extend well beyond a single species. Because the callus originates from mature leaf tissue, the system opens a door to secondary metabolite production in bioreactors, sidestepping the need to harvest wild plants. Indirect organogenesis through callus is also the gateway for genetic transformation, making the protocol a prerequisite for future CRISPR-Cas genome editing and molecular breeding aimed at improving the plant’s therapeutic traits. The authors are careful to frame the protein work as a first step: identifying and characterizing the specific regeneration-linked proteins will require further study, but the differential profiles provide the initial molecular map. In the broader picture, the study aligns with United Nations Sustainable Development Goal 15, which calls for the conservation of terrestrial ecosystems and the protection of threatened species. For a climber that has sustained desert communities for generations, science has now supplied a way to ensure it survives them.

Subject of Research: Indirect organogenesis and protein profiling in callus cultures of the threatened medicinal climber Vincetoxicum spirale

Article Title: Indirect organogenesis, ex vitro rooting, and protein profiling of callus cultures of Vincetoxicum spirale: a threatened climber

Article References: Indirect organogenesis, ex vitro rooting, and protein profiling of callus cultures of Vincetoxicum spirale: a threatened climber. (n.d.). https://doi.org/10.1007/s44340-025-00009-y

Image Credits: AI Generated

DOI: 10.1007/s44340-025-00009-y

Keywords: Vincetoxicum spirale, indirect organogenesis, plant tissue culture, callus, 2,4-D, BAP, ex vitro rooting, SDS-PAGE, protein profiling, plant conservation, Asclepiadoideae, micropropagation

The technique at the heart of the work is called layer-by-layer assembly, a method that builds films one molecular layer at a time by alternately dipping a charged substrate into solutions of positively and negatively charged polymers. In this case, the substrate was a commercially available nylon microfiltration membrane with a positively charged surface. Each dipping cycle deposits a nanometer-thin pairing, or bilayer, of negatively charged humic acid followed by positively charged chitosan, and repeating the cycle builds up a controlled nano-composite coating whose thickness and composition can be tuned with molecular precision.

Humic acid, a major component of the natural organic matter found in soils and waterways, is rich in oxygen-containing functional groups such as carboxyl, carbonyl, and hydroxyl moieties that can complex with dissolved solutes. Chitosan, a linear polysaccharide derived from chitin, carries protonated amino groups under acidic conditions that form strong electrostatic salt bridges with the carboxylate groups of humic acid. Together, the two biopolymers create a dense, interactive separation skin on top of a membrane whose pores would otherwise be far too large to catch small organic molecules like herbicides.

Characterization of the coatings confirmed the assembly proceeded as designed. Ultraviolet-visible spectroscopy showed the absorbance at 256 nanometers rising linearly with each deposited bilayer, indicating uniform growth. Infrared spectroscopy revealed peak shifts consistent with electrostatic bonding between the carboxylate groups of humic acid and the ammonium groups of chitosan. Spectroscopic ellipsometry measured bilayer stacks growing from about 7 nanometers at three bilayers to roughly 19 nanometers at nine bilayers. Atomic force microscopy showed surface roughness increasing from 163 to 321 nanometers after modification, while the effective pore diameter shrank from 0.448 micrometers to 0.2 micrometers, and thermogravimetric analysis confirmed the modified membranes remained thermally stable up to around 450 degrees Celsius.

The filtration tests focused on four herbicides representing distinct chemical classes: the chlorophenoxy compounds 2,4-D and 2,4,5-T, the phenyl urea herbicide buturon, and the neutral amide herbicide diphenamid. All were tested at concentrations of 10 to the minus 4 moles per liter in a dead-end ultrafiltration cell operating at just 20 pounds per square inch and 500 revolutions per minute. The bare nylon membrane barely rejected any of the compounds, with removal rates between roughly 7 and 16 percent. Once coated, performance improved steadily with each added bilayer, and the nine-bilayer membrane delivered the best results of all.

The standout result came from 2,4-D, one of the most widely used weed killers in the world and a suspected human carcinogen, which was rejected at approximately 99 percent. Buturon followed at around 97 percent, 2,4,5-T at about 85 percent, and diphenamid at roughly 33 percent. The differences among these compounds reveal the physics of the separation. The two chlorophenoxy acids are anionic at neutral pH and hydrophobic, so they are retained through a combination of electrostatic repulsion from like charges in the polyelectrolyte matrix, hydrophobic adsorption, and steric blocking. The more polar 2,4-D experienced stronger repulsion than its less polar cousin, explaining its superior rejection.

Buturon, though non-ionic, carries a high dipole moment of 5.44 debyes and a log octanol-water partition coefficient near 3, indicating substantial hydrophobicity. Its rejection appears to arise mainly from hydrophobic adsorption onto humic acid sites combined with steric hindrance from the highly charged bilayer stack. Diphenamid fared worst because this neutral molecule has low polarizability, a modest dipole moment of 3.60 debyes, and limited hydrophobicity, leaving steric effects as its only barrier. Infrared spectra taken after filtration showed a new carbonyl peak at 1717 wavenumbers on the used membranes, direct evidence that herbicide molecules had been adsorbed within the bilayer architecture rather than simply screened by pore size.

The study also mapped how preparation and operating conditions shape performance. The pH of the chitosan deposition bath proved critical: at pH 1.7, chitosan is fully protonated and forms well-fabricated bilayers densely populated with solute-accessible interactive sites, delivering maximum rejection, while higher deposition pH values produced weaker coatings and lower efficiency. Adding salt to the deposition medium screened the charges on the polyelectrolytes, causing them to coil and thicken the multilayer while weakening electrostatic rejection of anionic herbicides. Similarly, anions such as phosphate, sulfate, nitrate, chloride, and acetate in the feed water reduced the rejection of the negatively charged herbicides but left the non-ionic compounds largely unaffected. Flipping the membrane so humic acid formed the exposed outer layer instead of chitosan slightly altered performance for several compounds, underscoring that solute interactions with the outermost layer matter.

Practical durability is where the results become genuinely compelling. Nine-bilayer membranes stored for six months retained nearly all of their original rejection efficiency, and repeated filtration cycles over the same membrane showed only a slight, gradual decline attributed to a reversible fouling layer and the progressive occupation of active sites rather than any mechanical failure of the coating. Because the system operates at low pressure, it consumes far less energy than reverse osmosis or nanofiltration, produces less waste brine, and avoids the aggressive chemical cleaning cycles that shorten the life of high-pressure membranes. The entire separating layer is made from natural, biodegradable materials, giving the approach an environmental profile that synthetic polyelectrolyte coatings struggle to match.

The implications reach well beyond the four herbicides tested. Layer-by-layer coatings of humic acid and chitosan have previously been adapted to capture pesticides as diverse as atrazine, picloram, and metolachlor, and the present work extends that toolbox to chlorophenoxy, phenyl urea, and amide chemistries under a single platform. Because the assembly process works on substrates of varying geometry and can be scaled with straightforward dipping procedures, the author suggests the system could inform the design of pilot plants for membrane-based removal of chemical contaminants from drinking water, bringing affordable, low-energy herbicide filtration closer to real-world deployment for communities whose water supplies carry agricultural residues.

Subject of Research: Nano-composite biopolymer membranes for herbicide removal from water via layer-by-layer assembly under ultrafiltration

Article Title: Low pressure nano-composite biopolymer membranes for the removal of herbicides from water under ultrafiltration conditions

Article References: P., N. C. (2026). Low pressure nano-composite biopolymer membranes for the removal of herbicides from water under ultrafiltration conditions. Journal of Materials Science: Polymers, 1(1), Article 7. https://doi.org/10.1007/s44493-026-00007-4

Image Credits: AI Generated

DOI: 10.1007/s44493-026-00007-4

Keywords: ultrafiltration, layer-by-layer assembly, chitosan, humic acid, herbicides, water purification, nanocomposite membranes, biopolymers, 2,4-D, membrane filtration, micropollutants, low-pressure membranes

A research team led by Shruti Khanna of the California Department of Fish and Wildlife and the University of California, Davis, together with Jereme Gaeta, Krista Hoffmann, and remote sensing pioneer Susan Ustin, analyzed the Delta’s aquatic invasive plant control program across two five-year windows, 2004 to 2008 and 2014 to 2018. Their study, published in the journal Environmental Management, combined more than six hundred treated and reference sites with multi-year airborne hyperspectral imagery capable of mapping floating aquatic vegetation at the genus level. By fusing these vegetation maps with detailed herbicide application logs and boat path records, the team could ask questions that no small-scale field trial could answer: does the type of herbicide matter, does the dose matter, does the number of visits matter, and does any of it last?

The stakes are enormous. California spends more than 13 million US dollars each year treating invasive aquatic plants in the Delta, a 2,220-square-kilometer estuary formed by the confluence of the Sacramento and San Joaquin rivers. The main villains are water hyacinth (Pontederia crassipes), a Brazilian native that has plagued the Delta since 1904, and water primrose (Ludwigia hexapetala), present since 1949 and increasingly dominant since 2014. Two newer arrivals, spongeplant detected in 2008 and alligator weed in 2017, add to the threat. These plants form dense floating mats that darken water, deplete oxygen, block fish movement, and displace native communities, while their drifting fragments seed new infestations throughout the tidal system.

The control program itself has a long institutional history. Funded after 1982 legislation, it began as the Water Hyacinth Control Program and was renamed the Aquatic Invasive Plant Control Program in 2019, administered by California State Parks’ Division of Boating and Waterways. For most of its history it has relied on two systemic herbicides with remarkably long pedigrees: 2,4-dichlorophenoxyacetic acid, patented in 1945, which disrupts plant growth hormones and kills broadleaf dicots while sparing monocots, and glyphosate, patented by Monsanto in 1974, which blocks the shikimate pathway enzyme EPSPS essential to plant survival. The program straddled a major transition, relying mainly on 2,4-D in the early period and shifting to glyphosate in the later years, a natural experiment the researchers exploited to evaluate each chemical independently.

The analytical approach was rigorous. The team fitted mixed-effects logistic regression models, treating the probability of floating vegetation presence at each site as a function of herbicide application rate, spray frequency within a season, and consecutive years of treatment, with random intercepts for site and year to account for repeated observations. Separate models were built for each of the Delta’s three hydrologically distinct habitat types: fast-flowing tidal channels, slow shallow waters between remnant wetland patches, and flooded islands, lake-like expanses created by levee failures where water lingers and tidal action is weak.

The results were strikingly habitat-dependent. Both herbicides performed best in slow, shallow, open-water habitats and flooded islands, and worst in fast-flowing channels. Glyphosate was essentially ineffective in channels at any application rate, while 2,4-D showed significant impact there only at the highest rates used in the field. The likely explanation lies in the Delta’s relentless tidal dynamics. Previous work using GPS trackers showed that water hyacinth mats drift constantly in all directions with the tides, so treated plants in channels are quickly replaced by fresh propagules floating in from untreated areas. Flooded islands, isolated from these tidal conveyor belts, allow treatment effects to persist and become detectable months and even years later.

Perhaps the most actionable finding concerns dose and frequency. Across habitats and time periods, applying a higher concentration of herbicide in fewer visits consistently outperformed delivering the same total amount in smaller, more frequent doses. Low-dose applications were not merely ineffective; in some cases they were associated with a higher probability of floating vegetation than untreated reference sites. The authors point to two possible mechanisms. The first is hormesis, a well-documented phenomenon in which sublethal chemical stress actually stimulates plant growth and defense mechanisms, potentially triggering a post-treatment rebound. The second is herbicide resistance: sites treated with glyphosate for more than six consecutive years required noticeably higher application rates to achieve significant reductions than sites treated for only one or two years, a pattern consistent with resistance evolving under repeated sublethal selection pressure.

The legacy effects, meaning how long benefits persisted after spraying stopped, differed sharply between the two chemicals. Sites treated with 2,4-D showed no detectable reduction in floating vegetation one, two, or three years after treatment ended, in any habitat. Glyphosate, by contrast, left a measurable legacy in flooded islands, where the probability of floating vegetation remained significantly below reference levels even three years after the last application. In slow shallows, longer histories of glyphosate treatment, five to six consecutive years, produced stronger lasting suppression than shorter histories. In channels, neither herbicide left any lasting benefit, and longer treatment histories there appeared if anything counterproductive.

The study also revealed an unsettling background trend: floating vegetation cover and occurrence probability were higher across all habitats in 2014–2018 than in 2004–2008, suggesting the invasion problem has intensified even as treatment continued. Slow shallows consistently showed the highest occurrence, likely because their nutrient-rich, slow-moving waters combine continuous propagule input with ideal conditions for fragments and seedlings to take root.

For managers, the recommendations are concrete. Avoid sublethal doses that risk stimulating growth or breeding resistance; concentrate herbicide into fewer, higher-dose applications; match herbicide choice to habitat, favoring glyphosate’s longer-lasting action in stable flooded islands while recognizing that neither chemical offers much hope in fast channels; and rotate active ingredients, a strategy the Division of Boating and Waterways has begun testing with newer herbicides such as imazamox and penoxsulam in demonstration zones. Above all, the authors argue, chemical control must be embedded in a holistic program of monitoring, integrated methods, and native plant restoration. What this study demonstrates most powerfully is the value of scale: only by combining ten years of treatment records with a decade-spanning remote sensing archive could the true contours of herbicide efficacy in a tidal ecosystem come into focus, and only at that scale can the difference between money spent and money wasted finally be seen.

Subject of Research: Herbicide efficacy against invasive floating aquatic vegetation in the Sacramento–San Joaquin Delta

Article Title: Multi-Year Regional-Scale Efficacy Assessment of Two Herbicides for Treatment of Invasive Floating Aquatic Vegetation in a California Estuary

Article References: Khanna, S., Gaeta, J. W., Hoffmann, K., & Ustin, S. L. (2026). Multi-Year Regional-Scale Efficacy Assessment of Two Herbicides for Treatment of Invasive Floating Aquatic Vegetation in a California Estuary. Environmental Management, 76(10), Article 326. https://doi.org/10.1007/s00267-026-02625-8

Image Credits: AI Generated

DOI: 10.1007/s00267-026-02625-8

Keywords: glyphosate, 2,4-D, invasive species, water hyacinth, water primrose, Sacramento-San Joaquin Delta, remote sensing, estuary management, herbicide resistance, aquatic plants, tidal wetlands, environmental management

Plant tissue culture begins with a biological reset. Under carefully controlled conditions, specialized cells can lose aspects of their original identity and begin dividing as an undifferentiated tissue known as callus. With the right hormonal signals, some callus can later form roots, shoots or embryos and eventually develop into a complete plant. This ability, called cellular totipotency, underpins several crop technologies, including Agrobacterium-mediated transformation, particle bombardment, doubled-haploid production and CRISPR-based genome editing. Maize, however, is notably difficult to culture, particularly when researchers work with tropical and subtropical genotypes. Immature zygotic embryos have historically been favored because they can produce embryogenic callus, but they are available only during a narrow developmental window and generally require controlled pollination, seasonal planning and suitable greenhouse facilities. Seedling-derived explants could offer a more accessible alternative because they can be produced from mature seeds and prepared under laboratory conditions throughout the year.

Danielle Christelle Tinak Ekom and Abba Haïcha Diko evaluated the approach using the local varieties ATP, CHABA, CHH and KASSAI. The seeds were surface-sterilized and germinated on Murashige and Skoog basal medium supplemented with sucrose and plant growth regulators. After two weeks, the researchers cut the seedlings into two types of explants. Internodes were split longitudinally to expose tissue near the shoot meristem, while young leaves were cut into pieces approximately half a centimeter long. The explants were then placed on five callus-induction media, each based on the same mineral salts and vitamins but containing different combinations of the synthetic auxin 2,4-dichlorophenoxyacetic acid, or 2,4-D, and the cytokinins benzylaminopurine, known as BAP, or kinetin. The formulations also included casein hydrolysate, silver nitrate and spermidine, compounds used to support culture performance and, in the case of silver nitrate, reduce ethylene accumulation that can inhibit maize callus growth.

The researchers maintained the cultures in complete darkness at approximately 25 degrees Celsius for six weeks, transferring them to fresh medium after 21 days. They recorded the percentage of explants that formed callus and calculated relative fresh weight growth rate, a measure based on the increase between initial and final tissue weight. Statistical analysis used a randomized complete block design covering four varieties, two explant types and five media, with two-way analysis of variance followed by Duncan’s multiple range test at a significance threshold of 0.05. Callus became visible after about one week on all media and from both types of explant. By the end of the culture period, the tissues displayed several forms, including soft, watery white or cream callus; cream-to-brown callus; and more friable, granular tissue that showed a tendency toward early root formation. The authors emphasize that these appearances are preliminary indicators, not proof that a callus is embryogenic.

The clearest pattern was the strong influence of genotype. Split internodes from CHABA produced the highest reported induction response, reaching 76.2 percent on one medium, while ATP and CHH generally performed better than KASSAI. Media designated M1 and M3 were broadly effective for internode-derived callus, with induction above 50 percent in several variety combinations. M2 produced the weakest responses, indicating that its balance of auxin and cytokinin was poorly suited to internode callogenesis in these materials. The leaf explants told a slightly different story. ATP reached the highest leaf-based induction rate, 80.25 percent on M3, followed by CHABA and KASSAI, whereas CHH was the least responsive leaf source. Even so, every variety formed callus from leaf pieces under the tested conditions, showing that the tissue could provide a useful secondary route when internodes are unsuitable.

Growth rate revealed another important distinction. Split internodes consistently generated more rapidly expanding callus than leaves. CHABA showed the strongest proliferation, with relative fresh weight growth rates exceeding 5,000 percent on M1 and M3. Leaf-derived callus from the same variety also grew vigorously, surpassing 3,000 percent on those media, but remained less proliferative overall. The researchers caution that such striking percentages should not be interpreted as equivalent to a five-thousand-fold increase in useful biological material or as evidence of superior regeneration potential. Fresh weight can rise sharply when callus absorbs water, becomes highly vacuolated and develops a loose, watery structure. In other words, rapid tissue expansion may reflect hydration as much as the production of dense, developmentally competent cells. This distinction is crucial for laboratories choosing material for transformation, because abundant callus is not necessarily embryogenic callus.

The hormone results fit the basic biology of plant regeneration. Auxins such as 2,4-D can promote dedifferentiation and stimulate the formation of early callus, while cytokinins help regulate cell division and influence whether tissue continues proliferating or begins differentiating. The most favorable responses generally came from media containing 2 to 2.5 milligrams per liter of 2,4-D together with BAP or kinetin, corresponding to the M1–M3 group. The outcome was not universal, however: the same medium could produce very different results in different varieties, and the interaction between genotype and medium was statistically significant. That variability reflects the fact that tissue culture is governed not only by the recipe in the vessel but also by the genetic and physiological state of the plant. Differences in hormone signaling, cell-cycle control, stress responses and tissue organization can determine whether an explant remains inactive, produces watery callus or enters a pathway capable of regeneration.

The study therefore represents a foundation rather than a finished transformation platform. The authors did not test whether the induced calli could produce shoots, roots and fertile plants, nor did they use histological or molecular markers to confirm embryogenic competence. Further experiments must identify which callus types can regenerate, determine whether the tissues remain genetically stable during prolonged culture and optimize the transition from induction media to regeneration media. Those steps will be especially important for CHABA and ATP, the varieties that displayed the most promising combinations of induction and proliferation. If subsequent work succeeds, seedling-derived split internodes and leaves could make tropical maize biotechnology less dependent on immature embryos and specialized facilities. That would give researchers a practical starting point for improving locally adapted varieties threatened by climate change, pests, diseases and declining soil quality, while preserving the genetic resources that farmers already rely on.

One practical strength of the protocol is that it begins with mature caryopses rather than relying on a precisely timed reproductive tissue. The seedlings were generated under defined laboratory conditions, and the explants were prepared at a common two-week developmental stage. That standardization can reduce one source of experimental variation: the physiological differences associated with embryo age. It does not eliminate variation altogether, because seed quality, germination behavior and the exact position of an internode or leaf segment may still affect the cells that respond. For this reason, a useful next step would be to define explant sampling landmarks and seedling size criteria in enough detail for independent laboratories to reproduce the comparison.

The treatment design also illustrates why tissue-culture optimization is usually empirical rather than transferable as a single universal recipe. The five media varied the relative influence of 2,4-D, BAP and kinetin, allowing the researchers to examine hormonal combinations rather than testing an auxin alone. Such comparisons can reveal a response window in which cells divide without immediately differentiating, but the best induction medium may not be the best medium for later plant development. Regeneration commonly requires a change in hormonal conditions, and the study’s results should therefore be used to select candidate combinations for subsequent experiments, not as a complete culture system.

Several components of the medium are particularly relevant to interpreting the results. Casein hydrolysate supplies a complex mixture of nitrogenous and other organic compounds, while spermidine is associated with processes involved in cell proliferation and stress responses. Silver nitrate was included because ethylene can accumulate in sealed culture vessels and suppress callus proliferation. These additives may have contributed to the observed responses, but their effects were not separated experimentally from those of the growth regulators. A future factorial comparison could determine whether each component is necessary for every genotype or whether some varieties would respond equally well to a simpler and less costly formulation.

The statistical structure provides a framework for identifying interactions that would be missed by comparing averages alone. With varieties, explant sources and media combined in a randomized complete block design, the researchers could assess whether a medium’s effect depended on genetic background or tissue type. Nevertheless, callus induction percentage and fresh-weight growth rate describe quantity more directly than developmental quality. Confirmation of embryogenic potential will require regeneration tests, characterization of shoot and root formation, and evaluation of plants recovered from culture. Testing regenerated plants for fertility and phenotypic or genetic stability would then determine whether the method can support breeding and transformation rather than merely produce proliferating tissue.

That distinction is important for tropical maize improvement. A protocol that works across several locally maintained varieties can serve as a screening platform, helping researchers compare transformation or editing conditions without first obtaining immature embryos from every genotype. It may also support experiments on varieties whose agronomic value is local but whose tissue-culture behavior has not been extensively documented. The immediate contribution of this work is thus methodological: it expands the set of accessible starting tissues and identifies genotype-specific responses that can guide the more demanding stages of regeneration and genetic improvement.

Subject of Research: Callus induction from seedling-derived explants in tropical maize

Article Title: Seedling-derived split internode and leaf explants as efficient alternatives for callus induction in tropical maize

Article References: Tinak Ekom, D. C., & Diko, A. H. (2026). Seedling-derived split internode and leaf explants as efficient alternatives for callus induction in tropical maize. BMC Agriculture, 2(1), Article 28. https://doi.org/10.1186/s44399-026-00051-z

Image Credits: AI Generated

DOI: 10.1186/s44399-026-00051-z

Keywords: Tropical maize, Callus induction, Plant tissue culture, Split internodes, Leaf explants, 2,4-D, Plant regeneration, Crop biotechnology, Seedling-derived, split, internode, leaf