The initiation stage of micropropagation could be enhanced by the application of nanoparticles such as silver and copper oxide nanoparticles, which, as a consequence, not only improve explant viability but also reduce contamination and enhance morphogenic potential to develop successful plant tissue culture ( Table 1 ). Microbial contamination is widely perceived as the main bottleneck of plant tissue culture, which limits the efficacy of the micropropagation method. Common sterilization practices and techniques used in plant tissue culture can also damage plant tissues and/or produce toxic waste when not performed properly through the use of sodium hypochlorite, ethanol, or mercury chloride. However, nanoparticles, particularly silver nanoparticles, have gained attention as eco-friendly and effective alternatives for sterilization in tissue culture (Salisu et al., 2014).

Silver nanoparticles exhibit potent antimicrobial activity against a wide variety of bacteria, fungi and viruses. Their principal mechanisms result from the loss of structural integrity in microbial cell membranes and the production of reactive oxygen species. Other mechanisms of action include the disruption of microbial cell membrane structures, impairment of the electron transport chain, impairment of metabolic pathways for microbial growth, and induction of DNA replication interference, which ultimately leads to cell death (Dakal et al., 2016). These nanoparticles are different from traditional sterilization agents in that they work at low concentrations and do not damage plant tissues (Tung et al., 2021). In several reports, AgNPs have previously achieved high percentages of culture media sterilization and a reduction in microbial contamination (Munkager et al., 2020; Piecková et al., 2021; Wen et al., 2022; Alavi and Ashengroph, 2023).

Nanoparticles such as zinc oxide and titanium dioxide also have antimicrobial activity, which provides more options for sterilization (Abd-elsalam, 2013; Younis et al., 2022). Copper oxide nanoparticle usage also resulted in a 15–25% reduction in the explant infection rate during the initiation phase (Evlakov et al., 2020). Silver nanoparticles had a positive influence on SE and improved plantlet growth, which resulted in improved survival rates under greenhouse conditions (Cuong et al., 2021). There was improved branching of root systems in birch microplants with the introduction of CuO nanoparticles, demonstrating improved adaptive potential. However, some studies have also indicated possible negative effects associated with the introduction of nanoparticles during later stages, such as reduced viability and poor shoot and leaf development (Evlakov et al., 2020). Callus induction is the first step in the regeneration of a plant. This cell calling depends on the availability of important nutrients and growth regulators in the medium. NPs have been shown to increase callus induction via increased nutrient assimilation, altered hormonal activity and reduced oxidative stress (Irum et al., 2020; Malik et al., 2021).

Zinc oxide nanoparticles are essential for stimulating callus formation. These nanoparticles enhance cell division and proliferation by providing more macronutrients and micronutrients to the culture medium (Mousavi Kouhi and Lahouti, 2018). These nanoparticles are also treated as signaling molecules that regulate the synthesis of auxins and cytokinins, which are the two major hormones needed for callus initiation (Khan et al., 2022; Lee et al., 2023). ZnO nanoparticles have been demonstrated to improve callus induction in wheat (Triticum aestivum L.) by assisting in the uptake of important nutrients and facilitating cell activities that are important for plant growth and development Czyz˙owska and Barbasz, 2019).

NPs also strongly influence the morphogenic capabilities of explants during the initiation stage of micropropagation because of their enhanced growth characteristics, the fate of cell differentiation pathways, and increased pathogen resistance. Their unique properties allow for tailored applications that can optimize in vitro conditions for various plant species. In some cases, the presence of specific concentrations of nanoparticles in the nutrient medium, compared with controls, can yield enhanced morphogenic responses, and different plant species have been shown to have a range of optimal nutrient media concentrations to allow enhanced growth (Sichanova et al., 2023).

Gold nanoparticles affect SE by not allowing explant cells to follow a typical differentiation pathway and instead induce the formation of organ-like structures instead of somatic embryos. When the explants are exposed to nanoparticles, the tinkering of their chemical composition of the cell wall occurs, which further affects the developmental pathway (Godel-Je˛drychowska et al., 2023). The advent of nanoparticles into the culture media has decreased the occurrence of infections compared with that of control explants, increasing the survival rates of explants and thereby increasing their adaptability to stressful conditions during the micropropagation process (Evlakov et al., 2020; Pathak et al., 2023). While patterns that emerge from the emergence of nanoparticles may increase morphogenic potential in plants, notable concerns with their level of toxicity and whether nanoparticles have adverse long-term effects on plant health should remain a focus of debate (Pathak et al., 2023).

The shoot proliferation stage of micropropagation is a crucial phase that occurs simultaneously with shoot formation in cultured explants. The shoot proliferation stage is critical for calculating the overall multiplication rate and productivity of the process. The use of nanoparticles at the shoot proliferation stage of micropropagation has received much research attention, given their potential to facilitate plant growth and development ( Table 2 ).

Silver nanoparticles have been shown to increase the mean number of fresh shoots per explant and the number of explants that produce shoots due to an ethylene blocking mechanism (Aghdaei et al., 2012). Copper oxide and silver nanoparticles reduce the infection rate of explants and increase their morphogenic ability after explants are added to the culture medium (Evlakov et al., 2020). Potassium nanoparticles (K-NPs) working at optimal concentrations significantly increase the number of microtubers formed in potato cultivars (Farrag et al., 2024).

Nanoparticles, which primarily include gold (Au), silicon (Si), and carbon-based nanomaterials, have been shown to significantly increase shoot proliferation because of their ability to increase phytohormone levels and increase nutrient transport (Kim et al., 2017; Talebi, 2018). Gold nanoparticles have been shown to stimulate the activity of cytokinin, a hormone that facilitates shoot development. Research has shown that when these nanoparticles are introduced into a culture medium, the quantity and length of shoots increase significantly (Jadczak et al., 2019; Joshi et al., 2022). In addition, silicon nanoparticles can reduce oxidative stress by instigating antioxidant enzymes, which can help promote an environment for shoot proliferation (Mukarram et al., 2022). Carbon-based nanoparticles, such as graphene oxide (GO) and carbon nanotubes (CNTs), have shown potential as agents of stimulation for shoots. These factors are known to improve water and nutrient absorption, cell wall elasticity, and the transport of growth regulators, thus allowing for increased shoot multiplication (Mathew et al., 2021).

High doses of nanoparticles may inhibit plant viability and growth, such as in shoots and leaves, which limits the number of conversions (Evlakov et al., 2020). Additionally, a high dose of nanoparticles could stimulate auxin-influenced branching and root development despite negatively affecting health (Tymoszuk et al., 2024).

NPs can enhance shoot proliferation and overall plant quality; however, it is necessary to recognize that the effects of NPs are concentration-specific and that their overuse can lead to detrimental side effects. This duality emphasizes that optimization is necessary in micropropagation protocols. However, even after initial optimization, there are potential nanotoxicity risks and biosafety concerns associated with the use of nanoparticles in agricultural applications (Pathak et al., 2023).

Rooting is another significant step in micropropagation. An established root system leads to good plantlets. This phase often requires auxin-rich media; however, roots can be formed more efficiently via nanoparticles (Talebi, 2018). The authors of studies on various species have shown that nanoparticles can greatly increase plant growth and rooting efficiency during the rooting stage of micropropagation ( Table 3 ). The use of silver nanoparticles at the optimal concentration (12 mg/L), significantly increased both root count and length during banana micropropagation experiments (Tamimi and Othman, 2023). In strawberry micropropagation, the use of AgNPs decreased the level of ethylene accumulation, suggesting an improvement in plantlet quality that would facilitate root development and improve survival (Tung et al., 2021). Silver nanoparticles additionally improved the disinfection of strawberry explants along with the reduction of pathogen infestation and disease, and therefore, importantly, contributed to the establishment of a healthy and robust root system for woody species such as gray poplar (Vasyukova et al., 2021). Zinc oxide nanoparticles in combination with auxins greatly increased rooting percentages in seedling apple microcuttings and resulted in increased root length and decreased callus formation (Alizadeh and Dumanoglu, 2022). Iron oxide nanoparticles, silicon nanoparticles and graphene oxide have all joined the ranks of previously mentioned nanoparticles that have been widely studied for root growth promotion (Triphati et al., 2021; Zhao et al., 2022), whereas iron oxide nanoparticles are particularly influential on root induction and improve nutrient availability, auxin perception and signaling, and activate antioxidant defense pathways leading to increased root formation (Rai et al., 2022). Silicon nanoparticles have a complementary function in root development. They improve phosphorus and potassium assimilation, two nutrients that are directly related to root development, and improve root tissue structural integrity (El-Kady et al., 2017). More recently, silicon nanoparticles have been shown to protect from abiotic stress related to root formation. These NPs have been shown to effectively increase the growth and biomass of strawberry roots (Sener et al., 2023). Graphene oxide is a carbon-based nanomaterial that may promote rooting by increasing the ability of plants to take up water and transport nutrients. It interacts with auxins, facilitating cell elongation and division and allowing rooting for many different types of plant species (Cheng et al., 2016; Zhao et al., 2022).

Even though nanoparticles provide favorable advantages for micropropagation, it is important to assess the potential for toxicity and the environmental concerns associated with the use of materials at the molecular level. However, once again, given the potential outcomes of improved rooting, we need to consider any ecological concerns when utilizing this new form of plant biotechnology in the form of nanotechnology.

The last micropropagation step is acclimatization, in which the plantlets are cultured in vitro and then moved to natural conditions. Acclimatization is a step where there is a risk to the plantlets for mortality due to desiccation, pathogen infection, and stressful environmental conditions. The acclimatization process, where a plant survives, can be improved with nanoparticles by increasing stress tolerance and antimicrobial protection from pathogens (El-Saadony et al., 2022). The use of nanoparticles influences the acclimatization and hardening steps in micropropagation ( Table 4 ). The inclusion of AgNPs has been shown to improve the plant growth rate, increase the survival rate, and decrease microbial contamination, facilitating the transition from in vitro to ex vitro. These nanoparticles have been shown to improve somatic embryo formation and the overall number of somatic embryos in Panax vietnamensis (Cuong et al., 2021).

For blueberry micropropagation, the addition of AgNPs improved shoot propagation rates, which suggests that there is potential for improving growth during acclimatization (Tejada-Alvarado et al., 2022). Compared with those of the control plants (44.44%), the survival of the plants exposed to silver nanoparticles was much greater (93.65%) (Cuong et al., 2021). AgNPs are functional sterilants that minimize microbial contamination of growth media, which is crucial for successful acclimatization. The use of these nanoparticles in chrysanthemum micropropagation resulted in better acclimatization under greenhouse conditions, leading to earlier developmental stages (Tung et al., 2021). More intensely, AgNPs act to protect plants during this acclimatization process from fungi and bacterial pathogens. These nanoparticles serve to limit microbial infections and diminish each plant’s risk of loss following transplantation (Gautam et al., 2020; Tariq et al., 2020).

In general, silicon nanoparticles enhance acclimatization by improving water use efficiency, the strength of plant cell wall composition and photosynthetic efficiency, allowing plants to acclimatize better post-transplantation to environmental stresses (Moraes and Lacava, 2022). The advantages of the use of nanoparticles in micropropagation are remarkable however, some studies have noted that excessive amounts of nanoparticles can also have negative effects on plant morphology and development therefore, the precise fine-tuning of nanoparticles throughout various acclimatization processes could be highly important (Evlakov et al., 2020).

Microbial contamination, mostly by fungi and bacteria, is a continued barrier in plant tissue culture and results in large financial losses in the sector, while also ending with somatic origins that would later become genetically regulated plants. Traditional chemical disinfectants are normally effective, but they can cause phytotoxicity as well as residual toxicity. Hence, the use of inorganic nanoparticles with antimicrobial properties has presented itself as a superior alternative to chemical sterility agents. Because of their multidimensional characteristics, metals and metal oxides (AgNPs, ZnO NPs, CuO NPs, and TiO₂ NPs) have inherently potent broad-spectrum antimicrobial activity via different mechanisms (Ranjan and Ramalingam, 2016; Kim et al., 2017; Fatima et al., 2021; Rahmawati et al., 2022; Kalra et al., 2022). Metallic nanoparticles, particularly silver and copper nanoparticles, have some effective antimicrobial properties that may reduce disease incidence by 50% (Francis et al., 2024).

The fundamental antimicrobial action of these nanoparticles is based on ROS (e.g., superoxide anions (O₂ ^-), hydroxyl radicals (•OH) and hydrogen peroxide (H₂O₂)). ROS can disturb microbial membranes, denature cellular proteins, and interfere with nucleic acids, leading to cell death (Kaur et al., 2023). Although silver nanoparticles are mostly composed of elemental silver (Ag), their surfaces may be functionalized with chemicals, which may also shed light on antimicrobial mechanisms. The interaction of AgNPs with thiol groups and phosphorus-containing compounds can lead to conformational changes and functional inactivity of these structural molecules. Importantly, these nanoparticles also permeabilize bacterial membranes, which also triggers cytoplasmic leakage and apoptosis (Bondarenko et al., 2018).

Both ZnO and TiO₂ NPs have photocatalytic properties related to the induction of light-excited electrons into holes that can catalyze ROS. The antimicrobial capabilities of all types of nanoparticles, including ZnO and TiO₂, are driven by their size at the nanoscale, surface area, and ability to interact intimately with microbial cells. The impressive capability of these phytocompatible drug nanotherapies for use in media sterilization means a stronger reliance on such methods than harsh disinfectants characterized by high toxicity (Zakharova et al., 2019).

Tissue culture media contain a balanced mix of macronutrients and micronutrients; however, the nutrient bioavailability is often still limited by precipitation processes, pH swings, and chemical interactions. Nanoparticles offer a novel approach to nutrient delivery, with potential advantages in terms of greater solubility and bioavailability (Panpatte et al., 2016; Jampilek and Králová, 2017; Tariq et al., Singh et al., 2024c). Novel engineered nanofertilizers can entrap essential nutrients or chelate them and deliver them directly to plant cells, bypassing barriers and enhancing uptake efficiency (Gohar et al.; Ghazaryan et al., 2024, simultaneously improving nutrient uptake in both the apoplastic and symplastic pathways (Khlebnikova et al., 2023; Djanaguiraman et al., 2024).

In in vitro propagation, iron oxide nanoparticles (Fe₃₄ NPs) have been successfully utilized to alleviate iron deficiency. Iron oxide nanoparticles can deliver Fe³⁺ ions in a controlled and sustained manner. This is important since Fe is critical for the biosynthesis of chlorophyll, electron transport, and a myriad of enzymatic functions (Feng et al., 2022). In addition, silicon nanoparticles positively contribute to the mechanical strength of cell walls, fortify cell walls, and increase stress tolerance, particularly under drought conditions, through regulating water retention and enzyme activities (Dhakte et al., 2022).

Researchers have also recently reported nanocarriers for macronutrients such as phosphorus (P) or potassium (K), which may be useful because of their limited ability in soil. Phosphorus in hydroxyapatite or other phosphate-bound nanocarriers assists with ATP synthesis and the metabolism of nucleic acids. Potassium-containing nanoparticles have been reported to regulate osmotic potential and activate enzymes. Overall, these advances improved nutrient uptake, promoting enhanced development of shoots and roots through various aspects of micropropagation (Ditta and Arshad, 2016).

Throughout micropropagation, phytohormones function to coordinate cellular differentiation, organogenesis, and plant morphogenesis. NPs alter phytohormone interactions by modifying biosynthesis, transport, sensitivity, and hormone-associated signaling pathways. Hormonal signaling modulation by NPs can take place either by direct contact with the hormone itself or indirectly through the transcriptional regulation of hormone-responsive genes.

In auxin (indole-3-acetic acid, IAA) signaling, which is critical for root induction, the addition of Fe₃₄ NPs and graphene oxide NPs increases the level of auxin signaling itself. In both cases, it not only enables the polar transport of auxins but also increases the expression of auxin efflux carrier genes to improve meristematic activity and root organogenesis (Cheng et al., 2016; Yan et al., 2020, Yan et al., 2020; Yadav et al., 2022). As another example of an increase in hormone response and activity, AuNPs were found to have a synergistic effect on cytokinin, promote shoot proliferation, translocate cytokinins by stabilizing, and increase multiplication and nodal development via signals from cytokinins. The combination of nanoparticles and hormones in the micropropagation of plants is encouraging, especially because of the interaction with fewer hormones, where some nanoparticles can exert hormone-like effects, while some can work synergistically with exogenous hormone applications and generally have the prospect of better adjustment of hormone balance without the risk of adding more chemical burden to the media (Vinkovic et al., 2017).

Notably, NPs interact directly with abscisic acid (ABA) and gibberellins (GA) and can regulate responses to stress as well as elongation growth (Quesada, 2022). Other nanoparticles may even mimic hormonal action alone or be additive to exogenous hormone treatments and provide new methods to manipulate hormonal balance without increasing the chemical load (Kandhol et al., 2022).

During the micropropagation process, oxidative stress commonly occurs due to high levels of ROS. The sources of oxidative stress include wounding, the application of exogenous hormones, and the artificial environmental components of in vitro cultivation (Hancock et al., 2001). In addition to acting as signaling molecules at lower concentrations, ROS can denature proteins, lipids, and nucleic acids at higher concentrations, leading to potentially nonviable plant tissue (Bhattacharjee, 2019). NPs can elicit these two types of ROS. They can produce ROS (especially at very high doses), but for the most part, they can increase the level of oxidative stress, which increases the activity of antioxidant defense systems (Kumar et al., 2018; Sachdev and Ahmad, 2021).

Silicon nanoparticles increase SOD, CAT, and POD enzyme activity to reduce oxidative damage and restore redox homeostasis (for example, during shoot elongation and root induction phases) (Mukarram et al., 2022). ZnO NPs and TiO₂ NPs also effectively scavenge ROS, thereby protecting tissue as viable and reducing pentane (Singh and Chaudhary, 2020; Faizan et al., 2021).

When applied to stress-responsive genes, nanoparticles have been shown to provide upstream signaling for salicylic acid, jasmonate (JA), and ethylene for establishing systemic plant protection. This elicitor-like activity not only enhances the poor defense of plants but also further promotes their survival in stressful plant tissue cultures (Khlebnikova et al., 2023; Francis et al., 2024; Djanaguiraman et al., 2024).

Plant metabolism includes three common processes: photosynthesis, respiration and secondary metabolism; together, these processes serve as the basis for biomass accumulation, as well as adaptive responses to the environment. NPs affect these processes at the molecular and physiological levels, although the stage of the in vitro to ex vitro transition (i.e., the acclimation/hardening phase) is critical (Selvakesavan et al., 2023; Sharma and Chauhan, 2023). The application of Si NPs, TiO₂ NPs and CeO₂ NPs has been shown to improve photosynthetic efficiency, enhance chlorophyll contents and improve nutrient use efficiency (Gohar et al., 2024).

Photosynthetic systems are affected by TiO₂ nanoparticles, as they increase photocatalytic activity in chloroplasts, promoting improved light absorption and better coupling to electron transport, which leads to an improved photosynthetic rate and increased stomatal conductance (Didaran et al., 2023). Similarly, Si NPs play seminal roles as osmotic buffers and induce structural changes in cell walls; these roles enhance stress resistance and ‘water retention’. These effects improve the overall ‘vigor’ and capacity for uniform hardening and acclimation of the plant (Siddiqui et al., 2020).

Furthermore, nanoparticles act as elicitors of secondary metabolite pathways, stimulating the production of phenolics, flavonoids, alkaloids and terpenoids, which are important for various facets of plant defenses and medicinal activities in plants (Sreelekshmi et al., 2022; Savanur, 2022; Inam et al., 2023).

While the use of nanoparticles as enhancers for micropropagation offers increased capacity to bioenhance multiple factors, including plant growth, re-establishment and rooting into natural growth media, it highlights the necessity for thorough attention to practical use in terms of nanoparticle dose-response relationships, phytotoxicity, and ecological cascading effects. Continued expansion of knowledge on plant–nanoparticle interactions are integral for understanding the molecular-level mechanisms that improve plant growth systems (Sharma et al., 2022; Khlebnikova et al., 2023).

An integral component of this framework is the standardization of nanomaterial characterization. Characterization ensures that the different physicochemical properties of NPs, which impact their biological performance, are relatively well defined and consistently reproduced. The key characterization parameters include the particle size distribution, morphology, surface area, zeta potential, chemical composition, and surface functionalization. Tools commonly employed for NP characterization include transmission electron microscopy (TEM), scanning electron microscopy (SEM), dynamic light scattering (DLS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and the Brunauer–Emmett–Teller (BET) surface area (Ramachandran et al., 2021).

A standardized characterization protocol should begin by measuring the particle size and distribution, which influences cellular uptake and reactivity. While DLS is a common technique because of its ability to analyze dispersed particles, it should always be followed up with TEM or SEM to obtain morphological confirmation. The next important measure is the zeta potential. The zeta potential helps characterize the coalescence of nanoparticles and their possible interactions with the plant cell membrane, providing further characterization of the nanoparticles. A relatively large absolute zeta potential (typically above ± 30 mV) reflects good coalescent particle stability, which is essential for determining the consistency of bioavailability in suspension culture media (Varenne et al., 2015a). The chemical composition and crystallinity of a nanoparticle is also an important part of its characterization because these parameters determine the rate of dissolution, the ion release profile and the redox potential of metal-based nanoparticles. These parameters may be characterized via XRD and energy-dispersive X-ray spectroscopy (EDX) (Reed-Gore et al., 2018; Deepty et al., 2019). The surface functional groups present on the nanoparticles matter because they contribute to the overall biocompatibility and plant hormone-mimicking capability of the nanoparticles to influence cellular signaling. The surface chemistry must also be characterized, which can be accomplished via FTIR or X-ray photoelectron spectroscopy (XPS). All these parameters must be reported by the researchers in detail. These guidelines must be followed when publishing and are recommended by the OECD Working Party on Manufactured Nanomaterials (WPMN), or ISO/TC 229 (Rasmussen et al., 2016).

In addition to valuable characterization, dose–response determination approaches are integral to understanding the concentration-dependent responses exerted by NPs during micropropagation. Low dosages of NPs such as silver, zinc oxide, or graphene oxide may promote callus induction, root formation, or shoot elongation, but high dosages often react to oxidative stress, genotoxicity, and loss of growth. As a means to define practical application limits, reliable replications of dose-response curves must be developed. Thus, a tiered experimental design needs to be developed that includes several concentrations of the NPs (0.1 to 100 mg/L), multiple time points (24 h to weeks), and separation of the culture stages (initiation, proliferation, rooting, and acclimatization). The biological endpoints should consist of quantitative parameters such as callus mass, shoot number, root length, chlorophyll content, and antioxidant enzyme activities (SOD, CAT, POD) and stress markers (MDA, H₂O₂ levels). Additionally, transcriptomic and proteomic profiling can provide mechanistic details related to the dose-dependent modulation of signaling and metabolic pathways (Claeys et al., 2014; Kumar et al., 2016).

Aquiring clearance projections from the start, as well as acute and chronic exposure models, for both acute and chronic dose-response analyses. For example, some acute responses may only be seen as immediate toxicity, whereas chronic low doses could very easily lead to discrete epigenetic or physiological responses that occur cumulatively over successive subcultures. Advanced statistical models such as nonlinear regression, NOAEL (No Observed Adverse Effect Level) estimates, and benchmark dose (BMD) analysis can provide insight into safe and non-harmful nanoparticle concentrations, particularly when combined with in vitro–in vivo correlation studies, where plantlets initially treated in vitro are subsequently followed following acclimatization, and further bolster the ecological relevance of dose to indications (Nikinmaa, 2014; Savage et al, 2019).

Safety assessment protocols represent another critical element of the framework. It is much broader, as it demands attention beyond phytotoxicity to consider shifts in the microbial community, the expression of genes, and long-term ecology. For plant tissue culture, safety assessment should ultimately begin with cytotoxicity tests using a cell viability assay, such as 2,3,5-triphenyl tetrazolium chloride (TTC) or MTT, with suspension cultures or calli (Karakas et al., 2017). Genotoxicity tests, including the comet assay, micronucleus assay, and RAPD-PCR, may reveal DNA damage due to exposure to nanoparticles and, as such, can serve as a guide for the risk of chromosomal instability or unintended changes that can occur under somaclonal variation (Verdon et al., 2022). In addition, oxidative stress assays, which are used to determine the levels of ROS and lipid peroxidation, and assays of antioxidant enzymes produce a biochemical safety profile (Shukla et al., 2012; Gašparović et al., 2013; Agarwal et al., 2014).

At the microbial level, the unforeseen actions of various nanoparticles could also affect positive endophytes or rhizosphere consortia, either in the process or after hardening. Thus, culture-dependent and culture-independent techniques (e.g., qPCR and 16S rRNA sequencing) are useful for assessing microbial diversity in treated and control samples (Chhipa, 2023). Transgenerational studies, although infrequently published, are equally important for determining latent or heritable effects of NP exposure on seed germination, plant vigor, and reproductive outcomes. Future toxicology assessments will benefit from these multigenerational safety data to obtain regulatory clearance and general acceptance (Poma et al., 2014).

QC measures must be taken during the entire process of NP production and applied to end-use studies. QC starts with the NP synthesis stage and must ensure consistency between different batches, and along a line, the particle size, charge and composition must be ensured. For green-synthesized NPs, extract variability from plants is a barrier to consistency; hence, standardization of the source material is needed (e.g., at the minimum age of the plant, the plant part used, or the solvent system). However, for all NPs, the sterility, endotoxin (most importantly for bioderived NPs) and stability must be assessed, including storage assessments. Where possible, certificates of analysis (CoAs) should be reported listing, at a minimum, all critical physicochemical properties (Maity et al., 2021; Saleh and Fadillah, 2023).

During application in micropropagation systems, media preparation must follow sterile, reproducible conditions, with uniform NP dispersion achieved via sonication or mechanical stirring. To avoid aggregation or precipitation, NP suspensions should be freshly prepared or stabilized via biocompatible agents (e.g., citrate and polyethylene glycol). The risk of cross-contamination between treatment groups should be minimized through spatial or temporal separation and rigorous tool sterilization. Regular checks for NP leaching, sedimentation, and degradation in culture media are essential to ensure consistent bioavailability (Kim et al., 2017; Gupta et al., 2020b).

Internal QC audits should involve routine calibration of instruments (e.g., pH meters, laminar flow cabinets), validation of assay protocols (e.g., controls in antioxidant enzyme tests), and replication of experiments across different labs or technicians (Kinns et al., 2013; Zhuravleva, 2014). Documentation standards, including detailed lab notebooks, digital records, and standardized reporting templates, support transparency and traceability. Furthermore, collaboration with accredited nanotechnology or toxicology laboratories for independent verification of findings adds robustness to the dataset and aids in regulatory compliance (Elberskirch et al., 2022).

As the field matures, there is a growing call for harmonized guidelines and databases for nanoparticle use in plant biotechnology. This includes establishing repositories for NP physicochemical and biological data, similar to the eNanoMapper or OECD NanoStat database. Such initiatives can help in the predictive modeling of NP behavior, reduce redundancy, and promote responsible innovation. The methodological framework described here aims to lay a foundation for such harmonization, ensuring that nanotechnology in plant tissue culture evolves along safe, effective, and scientifically rigorous lines (Kokina and Plaksenkova, 2022; Naidoo, 2022).

The biological efficacy and safety of nanoparticles in micropropagation are related to their dose-related response, which constitutes one of the greatest challenges in nanobiotechnology. NPs possess certain unique properties depending on their application in micropropagation, as they have a much higher surface area-to-volume ratio, are reactive and contain properties based on quantum mechanics, unlike traditional agrochemicals, which alter their association with plant tissues. These characteristics can promote or prevent the micropropagation process, as they have the potential to encourage growth in plants or raise concerns regarding phytotoxicity (Ramkumar et al., 2022). Whereas low concentrations of some NPs, such as AgNPs, ZnO NPs and Fe₃₄ NPs, can stimulate growth or differentiation processes in the form of shoot proliferation, rooting and somatic embryogenesis, all of the above NPs select for cellular damage after certain thresholds involving oxidative stress, membrane leakage and genotoxicity (Irum et al., 2020; Tymoszuk et al., 2022).

A major complication in the analysis of dose-response relationships is related to the plant species and their developmental stages and the physical and chemical properties of the nanoparticles (Rawat et al., 2021). The size, shape, surface charge, and functionalization of nanoparticles can change their bioavailability and uptake (Ma et al., 2013). Smaller nanoparticles are more likely to simply traverse cell membranes and quickly be sequestered in organelles, whereas larger nanoparticles can end-up outside of the cell, with the potential to interfere with metabolic and signaling pathways (Mosquera et al., 2018). However, nanomaterials can generate ROS concentrations that are high-enough to potentially lead to oxidative stress, which may have a deleterious effect on cell viability, leading to lipid peroxidation, protein denaturation, and DNA damage (Manke et al., 2013).

The absence of a therapeutic index for the use of nanoparticles in plant systems has encouraged their use in micropropagation protocols. An index of the therapeutic window for dosage in pharmaceutical therapies has been established, but we do not have a similar range of dosages that are considered safe and efficacious from nanoparticles in plant systems (Kumari et al., 2022). Moreover, the interaction of nanoparticles with plant growth regulators can further complicate inference regarding optimal dosages. Therefore, specific treatments in a systematic manner for any one or a range of species are needed to assess potential concentrations that are intended to maximize efficacy with minimal cytotoxic effects. Longer-term studies may also be beneficial for identifying whether repeat doses through subcultures, or across generations, result in the collection of toxic effects or stress-related adaptations of the plantlets (Kandhol et al., 2022).

The inconsistency in experimental designs reported in the literature, including everything from the composition of culture media utilized to the environments imposed during our experiments, adds another layer of complexity to the establishment of dose-response curves (Cvrčková et al., 2015). Without standardized protocols, cross comparison of the impact of nanoparticles is an unreliable process. We need to standardize bioassay protocols that have measurable endpoints (i.e., chlorophyll concentration, root/shoot biomass, electrolyte leakage and enzyme activity). Advances in molecular diagnostics and imaging, such as transcriptomics and proteomics, could reveal valuable information on the sublethal effects of nanoparticles, which could help appropriately define toxicity thresholds and limits of exposure (Kang et al., 2023).

The environmental impacts of the use of nanoparticles in plant micropropagation are becoming more significant, particularly with respect to their persistence, mobility and bioavailability through bioaccumulation in ecosystems. If nanoparticles are added to plantlets in the in vitro phase, once they are assembled into the ex vitro environment, they can be transferred to soils or aquatic environments, including the direct transfer of plantlets, disposal of culture media, or leaching from ex vitro additions. After being released into the environment, nanoparticles may undergo physical/chemical changes, such as aggregation, dissolution, biomolecular and macromolecular association and involvement, all of which have a compound effect on their fate and ecological toxicity (Batley et al., 2013; Wojcieszek and Ruzik, 2022).

Bioaccumulation in trophic chains may be one of the most serious hazards. For example, silver, copper, and zinc oxide nanoparticles, among others, may persist in soil matrices, become associated with, and be taken up by, plant roots and microbial communities and subsequently translocated into edible flora/plant tissues, thus entering the food chain. Numerous studies have shown the bioavailability of engineered nanoparticles with agricultural crops, which raises the possibility of engineered nanoparticles accumulating in higher organisms, potentially including humans. Many of the studies have focused primarily on those crops grown in soils, but the same hazards may also exist for crops derived from tissue cultures after acclimatization and following transplantation despite having undergone, in addition, planting medium recognition/realization (Murali et al., 2022).

The ecotoxicological effects of nanoparticles are not limited to only plants but also to soil microorganisms, fungi, nematodes, and aquatic organisms (Chhipa, 2021). For example, silver nanoparticles are used as in vitro antimicrobial agents but can inhibit nitrogen-fixing bacteria and interfere with the mycorrhizal relationships necessary for plant uptake of nutrients (Cao et al., 2017; Yu et al., 2021) in addition to causing unplanned collateral damage. These effects may negatively impact soil fertility and crop yield over time. Furthermore, nanoparticles that enter water can have a negative impact on the aquatic environment. Once in aquatic systems, they reduce photosynthetic productivity in algae, promote bioaccumulation in fish, and impair the coral cycle in invertebrates.

Methodologically, environmental monitoring of nanoparticles is an additive challenge because of their nanoscale size; they are uniquely variable in chemical composition and often interact with disparate environmental matrices. The environmental risk assessment tools are designed primarily for bulk chemicals. When attempting to add the exposure and effects of nanomaterials, this approach is not straightforward, and the use of complex compounds is unlikely to yield the same results as those of bulk chemical assessment methods. We need more advances in detection methodologies, such as isotopically labeled nanoparticles or synchrotron spectroscopy electronics, single particle inductively coupled plasma mass spectrometry (spICP-MS), and other more sophisticated methodologies to track the compartmental distribution and quantify the accumulation of nanoparticles within biological tissues and environmental compartments (Cornelis et al., 2021; Singh et al., 2024d).

There are at least two main methods for addressing environmental risks related to the use of nanoparticles in micropropagation. First, synthesis methods that are deemed more environmentally friendly and that use plant-based or microbial principles to make biodegradable or less persistent nanoparticles should be used whenever possible. Second, pre-acclimatization procedures should be able to degrade or eliminate any nanoparticles from plant tissues prior to field transplantation. Government regulatory agencies need to have some type of framework for the disposal of nanoparticles (more research related to this topic is needed). This is particularly important for laboratories and greenhouses where researchers use nanoparticles, and there are risks of unintended release into the environment (Kim et al., 2017).

Governmental efforts to establish regulatory frameworks for the secure, uniform and ethical practice of nanoparticles in agriculture and biotechnology cannot seem to keep up with the acceptance of nanoparticles in micropropagation systems. The specific physicochemical traits that nanoparticles exhibit create complexities for regulatory frameworks that have relied on typical bulk assessment. Nanoparticles behave differently than typical agrochemicals because their size and reactivity are dependent, and the different interactions of nanoparticles depend on their performance, provider and biological system, so they do not assess nanoparticles in the same manner as traditional agrochemicals do. This gap in regulations has created a significant bottleneck to scaling up nanotechnology with plant tissue culture (Kumari et al., 2023).

Most regulatory authorities have not developed eligible and scientifically validated regulations specific to the use of nanoparticles in plant biotechnology. The few regulations that do exist, such as the EU REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) regulations or U.S. EPA documents that discuss the usage of this nanomaterial, focus on its industrial usage rather than its applications in agricultural biotechnology. Consequently, researchers and commercial end-users practice in a regulated or informal regulated state that creates inconsistencies in applications, safety and quality control, including labeling. Furthermore, there are exposed liability concerns for institutions that use nanoparticle-based micropropagation methods without completed risk assessments (Ngarize et al., 2013; Bajpai et al., 2020).

The lack of standardized testing protocols only adds to the regulatory issue. Even if laboratories follow unique procedures to produce, characterize, and test nanoparticles, they compile different and heterogeneous datasets that are not representative, reliable or comparable, or aggregable in meta-analysis. The heterogeneity and endpoint variation make it much more difficult for regulators to determine the maximum acceptable exposure or the bioequivalence of different formulations of nanoparticles. Although international organizations responsible for developing protocols, such as the International Organization for Standardization (ISO) or the Organization for Economic Cooperation and Development (OECD), have developed high-throughput screening protocols for characterizing and testing nanoparticles as they relate to toxicology, many in plant sciences are just emerging (Gomes et al., 2021; Bleeker et al., 2023).

Risk assessment pathways for nanoparticles used in micropropagation should also include assessments of acute and chronic toxicity, in conjunction with ecological information, and the consequences of any genotoxicity and lifecycle assessment data. If regulatory pathways are developed to assess the safety of nanoparticles, they must also allow for any interactions with and how nanoparticles will function as plant growth regulators or other components of the culture media, as well as any potential synergies or antagonistic consequences. We need to be able to determine the direct or indirect effects (or absence) of nanoparticles on gene expression and epigenetic stability in regenerated plants, particularly when regenerated plants are consumed as food (Ranjan et al., 2021).

Tackling these issues involves active and cross-disciplinary collaboration. Researchers, regulators, and industry will have to combine their efforts to produce a comprehensive regulatory regime that will cater to the unique properties of nanoparticles. This needs to include premarket approval processes, required testing for safety, post-market surveillance of use and market disclosure requirements. An expanded role for regulatory agencies will be in producing guidelines for required documentation on nanoparticle-based culture media and formulations; labeling, disclosure and transparency within the value chain; and traceability.

The economic viability and scale of nanoparticles for use in micropropagation are two of the most important but still underexplored dimensions for understanding why nanoparticles have not gained wider use and acceptance in commercial horticulture and plant biotechnologies (Wu and Li, 2022). The documented benefits of the use of nanoparticles, such as increased shoot multiplication, reduced contamination, increased rooting efficiency and stress tolerance, are well known under laboratory conditions (>500 papers), but determining how to obtain practical laboratory benefits or advantages for economically viable large-scale operations is challenging. This is primarily due, in part, to the costs incurred from producing, stabilizing, characterizing and integrating nanoparticles into tissue culture procedures (Abdelmajeed and Aboul-Nasr, 2013).

NPs are generally produced through energy-hungry physical or chemical processes that are generally costly due to expensive precursors, reducing agents and specialized instrumentation (ultrasonication, high-temperature furnaces, autoclaves, and reactors are just a few examples). While greener synthesis approaches using biological templates (such as plant extracts or microbes) are emerging, they have not achieved the consistency or scale of manufacture to be widely adopted in industrial applications. The functionalization of nanoparticles with appropriate ligands is fundamental to creating nanoparticles with stability, specificity, and controlled release, which also adds to production costs. In general, tissue culture laboratories (especially when they operate in low-resource situations or with non-commercial or low-margin crops such as trees) are unlikely to ever recoup the financial costs of adopting a consistently reliable nanoparticle-based protocol, regardless of any direct benefits such as production efficiency, improved quality (e.g., improved propagation accuracy), throughput or other secondary benefits from the normalization of nanoparticle use (Afonso et al., 2024).

When undertaking cost analyses, one must always consider how nanoparticle systems will fit with the current infrastructure. Most tissue culture laboratories have no existing experience with nanoparticle dispersion, characterization, or tracking, and the introduction of nanoparticles as a component in media preparation, sterilization and delivery will most likely require new methods, new equipment, new personnel training, and even significant changes to how waste is managed (e.g., disposal of residues of nanoparticles used in media preparation), so the associated costs to the operating budget, while likely not relevant when capital costs are considered, must be justified by justifiable measurable returns, such as propagation efficiencies, product quality (e.g., improved accuracy of propagation), throughput, or additional value for normalizing the use of nanoparticles (Tariq et al., 2020; Su et al., 2022).

In addition, the viability of using nanoparticles in micropropagation via economies of scale is also not understood. While many small-scale studies demonstrate the efficacy of a few milligrams or micrograms of nanoparticles, the process of scaling up to kilograms for commercial propagation, with millions of plantlets, creates many challenges with sourcing nanoparticles in the same form, preventing agglomeration of the nanoparticles, and obtaining batch consistency. These challenges could ultimately result in inconsistent responses of the nanoparticles and negatively impact product quality, as well as the trust of stakeholders and end-users alike (Kokina and Plaksenkova, 2022).

The cost-benefit ratio is also related to the type of crop being produced. For high-value crops, including orchids, elite fruit cultivars, and medicinal plants, any increase in propagation efficiency or improved plant resistance to disease increases the costs associated with nanoparticles. Low-margin crops, including leafy vegetables and fodder plants, may not be able to provide sustainable economic viability for practices that rely on costly nanoparticle protocols. Crop economic modeling provides a precise number that will equate break-even and profitability under different crop production scenarios (Zaman and Maitra, 2018).

Things that might help reduce economic feasibility include reusable nanoparticle matrices, time-released formulations to reduce waste, or the use of nanocomposites added to culture vessels or media gels to minimize the need for repeated applications. The use of various precision delivery systems, such as microfluidics, nanofiber scaffolds or encapsulated growth matrices, may lead to a gain in the delivery efficiency of these nanoparticles, indicating that the delivery of fewer nanoparticles will result in a problematic dose. Relationships with nanomaterial suppliers and some investments in local manufacturing will increase the cost of nanoparticle usage and access (Delgado, 2009).

With respect to some costs, if they are deemed in the best interest of governments or funding bodies, regulation, partnerships with nanotechnology regarding public and private relations, and international collaboration might also increase the likelihood of making lower cost barriers for adoption and the transfer of technologies. As an example, government and funding bodies explicitly subsidizing green nanoparticle synthesis as examples being used for agricultural uses or adding nanotechnology to the scopes of innovation-related schemes in agricultural use signifies alternatives to support the engagement of facilitating technologies for utilization. Generally, a multidisciplinary approach involving nanotechnology, economics, plant science, and industrial engineering means that knowledge of nanoparticle-assisted micropropagation should grow to be a meaningful, scalable, and economically useful technology in common culture.

Importantly, standardization is a key component of scientific reproducibility and industrial scalability. Without standardization, we would be unable to scale up the production of nanoparticles or their integration into micropropagation. The inconsistencies in nanoparticle synthesis, characterization, and, ultimately, their application in horticulture are the primary impediments to integrating nanoparticles into the micropropagation process because they make it impossible to compare and establish best practices across studies. Furthermore, such inconsistencies mean that regulatory bodies are unable to regulate these substances or assess their risk and prevent the development of effective, crop specific, nanoparticle protocols (Zhang and Ge, 2018).

The range of nanoparticles used in plant tissue culture can vary significantly on the basis of core material (e.g., silver, gold, zinc oxide, iron oxide, silicon), morphology (e.g., spherical, rod, flower-like), size distributions, surface charges, and levels of functionalization. Small differences in synthesis conditions (e.g., pH, temperature, reactant concentration, reducing agents) can lead to large variations in the properties of nanoparticles and, consequently, in their biological effects. Small nanoparticles may penetrate cell walls more easily but could be more phytotoxic. However, without standardized protocols, researchers cannot reproduce research findings or transfer technology commercially (Mishra et al., 2022).

In addition to problems with synthesis variability, there is an absence of standard protocols for the characterization of nanoparticles. Techniques such as DLS, TEM, SEM, zeta potential measurements and UV-Vis spectroscopy have been used with varying degrees of application by researchers, including differing application parameters and reporting methods. These various methods lead to variable conclusions on the size, stability, and reactivity of nanoparticles, which makes it difficult to predict their biological outcomes. Most published studies report nominal concentrations (mg/L), without evaluation or reporting on the actual bioavailable fraction of nanoparticles in culture media. This would be informative in establishing their functional relevance (McNeil, 2011).

The application methods are also variable. Plant cultures can be exposed to nanoparticles in solid or liquid culture media via foliar spraying, seed priming, or coatings on the surfaces of culture vessels. The form of exposure, timing, time spent, frequency, etc., are also generally variable among the studies, leaving little to no common strategy for these variables across the literature. This inconsistency limits the generalizability of the results and complicates efforts to optimize nanoparticle use for specific micropropagation stages, such as callus induction, organogenesis, rooting, or acclimatization (Wohlmuth et al., 2022).

When these issues are considered, a priority is to create consensus standards regarding how particles are synthesized, characterized, and utilized in micropropagation. Creating a standard operating procedure (SOP) will require explicit descriptions of the physicochemical characteristics of the particles, protocols for the dispersion of particles, sterilization protocols and storage conditions. There is also potential for academic institutions, industry, and regulatory authorities in collaboration to develop certified reference materials (CRMs) and validated test methods related to agri-nanotechnology.

An additional important aspect of standardization includes incorporating nanotoxicology assessments into standard operating procedures. Considerations for standardized assays for cytotoxicity, genotoxicity and ecotoxicity should be included in the routine evaluation of nanoparticle formulations and could include high-throughput screening approaches and new omics-based technologies in search of elicited sub-lethal/long-lasting effects on plant physiological processes and genetic integrity for nanoparticles used in micropropagation (Koedrith et al., 2021; Pokhrel et al., 2021).

There should also be efforts toward digital standardization, and centralized databases or even metadata repositories where researchers can deposit and retrieve nanoparticle characterization, exposure conditions, and biological responses would be beneficial in considerably enabling meta-analyses, machine learning-based predictive modeling, and the evolution of evidence-based protocols (Elberskirch et al., 2022).