Genetic Engineering Research Articles

Binary vector copy number engineering improves Agrobacterium-mediated transformation.

The copy number of a plasmid is linked to its functionality, yet there have been few attempts to optimize higher-copy-number mutants for use across diverse origins of replication in different hosts. We use a high-throughput growth-coupled selection assay and a directed evolution approach to rapidly identify origin of replication mutations that influence copy number and screen for mutants that improve Agrobacterium-mediated transformation (AMT) efficiency. By introducing these mutations into binary vectors within the plasmid backbone used for AMT, we observe improved transient transformation of Nicotiana benthamiana in four diverse tested origins (pVS1, RK2, pSa and BBR1). For the best-performing origin, pVS1, we isolate higher-copy-number variants that increase stable transformation efficiencies by 60-100% in Arabidopsis thaliana and 390% in the oleaginous yeast Rhodosporidium toruloides. Our work provides an easily deployable framework to generate plasmid copy number variants that will enable greater precision in prokaryotic genetic engineering, in addition to improving AMT efficiency.

Exploration of the advantages of targeted isolation of deep-sea microorganisms and genetically engineered strains.

Oil, mineral processing and environmental restoration can be dangerous processes. Attempts are often made to apply microorganisms to reduce the risks, but the adaptability of terrestrial organisms is often weak. Although genetically engineered strains can improve their environmental adaptability through targeted modification, there are problems such as metabolite accumulation, poor plasmid stability and potential pathogenicity. Screening of extremophiles from the natural environment has become an inevitable choice. The special environment in the deep sea (high pressure, low temperature, low nutrition, high salinity) is a natural place for extremophiles to grow and survive, thus screening of extremophiles from the deep sea is conducive to the green and sustainable development of industry. In this paper, the application status and problems of genetically engineered strains are reviewed based on the microorganisms needed for extreme industry. This paper focuses on the application status and advantages of deep-sea microorganisms. It is found that their advantages are strong adaptability, stable gene, friendly environment, simple and convenient technology (compared with genetic engineering), which has a broad industry processes application prospect. This review broadens the scope of microbial applications.

Microbial Ecology of Denitrification Process and Its Application in Wastewater: Treatment, Challenges and Opportunities

Denitrification is a crucial microbial process in the nitrogen cycle, transforming nitrate (NO₃⁻) into nitrogen gas (N₂), thereby mitigating nitrogen pollution in aquatic ecosystems. This microbial activity plays a vital role in wastewater treatment by removing excess nitrogen, which contributes to eutrophication and water contamination. The denitrification process involves various microbial communities, including bacteria such as Pseudomonas, Paracoccus, and Bacillus, which operate under anoxic conditions to achieve nitrogen reduction, an optimizing denitrification in wastewater treatment presents several challenges, such as maintaining ideal environmental conditions (e.g., carbon availability, oxygen levels, pH) and overcoming issues related to incomplete denitrification, which can lead to the production of harmful intermediates like nitrous oxide (N₂O). Despite these hurdles, recent advancements in microbial ecology, such as the use of biofilms, bioreactors, and genetic engineering, offer promising opportunities to enhance denitrification efficiency. This review explores the microbial ecology of the denitrification process, its application in wastewater treatment, and the challenges and opportunities associated with its practical implementation in reducing nitrogen pollution.

Critical analysis of enhanced microbial bioremediation strategies of PAHs contaminated sites: Toxicity and techno-economic analysis

Polycyclic aromatic hydrocarbons (PAHs) pose significant threats to environmental integrity and public health due to their high toxicity, persistence, and potential for bioaccumulation. In contaminated soils, PAH concentrations typically range from 1 to 100 mg/kg, with severely polluted areas reaching up to 1000 mg/kg. Conventional bioremediation techniques, limited to 30–50% efficiency, underscore the need for more effective solutions. This review highlights recent advancements in microbial bioremediation strategies, demonstrating removal efficiencies of 80–90% through the utilization of functional microorganisms, which metabolize PAHs into non-toxic compounds. Innovative techniques such as genetic engineering, microbial immobilization, and nanotechnology are shown to achieve over 90% pollutant removal. The review discusses key metabolic pathways and enzymatic processes driving PAH degradation, such as ring-hydroxylation and oxygenation. Techno-economic assessments indicate up to 40% cost savings and improved energy efficiency compared to conventional methods, facilitating scalability for large-scale environmental restoration projects. Microbial solutions for groundwater pollution, where PAH levels often exceed the maximum contaminant level (MCL) of 0.2 μg/L, are found to be highly effective in mitigating ecological risks and protecting public health. This comprehensive analysis highlights the promising role of advanced microbial bioremediation techniques in addressing PAH contamination across diverse ecosystems, including soils, sediments, and aquatic environments.

Versatile Xylose and Arabinose Genetic Switches development for Yeasts

Inducible transcription systems are essential tools in genetic engineering, where tight control, strong inducibility and fast response with cost-effective inducers are highly desired. However, existing systems in yeasts are rarely used in large-scale fermentations due to either cost-prohibitive inducers or incompatible performance. Here, we developed powerful xylose and arabinose induction systems in Saccharomyces cerevisiae, utilizing eukaryotic activators XlnR and AraRA from Aspergillus species and bacterial repressors XylR and AraRR. By integrating these signals into a highly-structured synthetic promoter, we created dual-mode systems with strong outputs and minimal leakiness. These systems demonstrated over 4000- and 300-fold regulation with strong activation and rapid response. The dual-mode xylose system was fully activated by xylose-rich agricultural residues like corncob hydrolysate, outperforming existing systems in terms of leakiness, inducibility, dynamic range, induction rate, and growth impact on host. We validated their utility in metabolic engineering with high-titer linalool production and demonstrated the transferability of the XlnR-based xylose induction system to Pichia pastoris, Candida glabrata and Candida albicans. This work provides robust genetic switches for yeasts and a general strategy for integrating activation-repression signals into synthetic promoters to achieve optimal performance.

Saga of Soggy Sauerkraut

The creation of undesirable (soggy) sauerkraut resulted in the loss of $1,000,000 worth of organic sauerkraut in 2022, which prompted a multistep investigation of the cause and potential solution. The cause of this condition has been previously reported as unique fermentation conditions and the lack of key trace nutrients essential for cabbage (Brassica oleracea var. capitata) cell wall integrity. Because the condition was limited to organic sauerkraut in 2022, this investigation initially focused on differences in fermentation conditions between organic and conventional sauerkraut. No differences in fermentation conditions accounted for the condition; therefore, attention was focused on analyzing the mineral content of cabbage grown for sauerkraut production that pinpointed a deficiency in critical micronutrients such as iron, copper, manganese, boron, and zinc. This deficiency was traced to the use of poultry manure that was contaminated with glyphosate residue from conventionally fed turkeys and chickens that consumed genetically engineered (GE) feed and used as the fertilizer for organic cabbage production. The presence of glyphosate, a potent mineral chelator and antibiotic, was identified as a significant factor that impairs the absorption and physiological function of essential minerals in the shikimate metabolic pathway whereby cell walls and lignin are produced, thus compromising the structural quality of the sauerkraut. After this discovery, the study progressed to evaluate various remediation strategies aimed at eliminating glyphosate from the soil and restoring nutrient uptake. Corn grain and silage were selected as the test crops for this phase. Among the tested remediation solutions were raw sauerkraut juice containing Lactobacillus plantarum, which is reported to degrade glyphosate in the rumen of dairy cows and two patented proprietary microbial mixtures, PB027 and PB027SK, that degrade glyphosate by all three of the known metabolic pathways. These treatments were specifically formulated to degrade residual glyphosate in the soil. The results showed that these interventions could reduce soil glyphosate levels by 80% to 90% within 6 to 7 months to significantly enhance both the yield and quality of corn grain and silage. The increase in corn grain yield from glyphosate degradation on the Shiocton silt loam soil was 907.89 kg·ha−1 (13.5 bushels/acre). The increase in yield on the irrigated Kidder sandy loam soil was quantified at 726.31 kg·ha−1 (10.8 bushels/acre) for corn grain and 6.62 t·ha−1 (2.68 t/acre) for silage, with an additional improvement in silage feed quality beneficial for milk production. The findings underscore the importance of addressing both micronutrient sufficiency and glyphosate residue in soil to ensure the optimal growth of cabbage and the quality of sauerkraut produced. By successfully identifying manure as a subtle source of nutrient immobilization and implementing effective soil remediation techniques, this research highlights a clear path forward for improving crop yield and quality to ultimately enhance the structural integrity and consumer acceptance of sauerkraut. This study has broader applications for the nutritional content and crop yields of many organic crops that use conventional poultry and animal manures that may contain glyphosate in desiccated plant tissues or GE feeding operations.

Two-step biocatalytic conversion of post-consumer polyethylene terephthalate into value-added products facilitated by genetic and bioprocess engineering

Two-step biocatalytic conversion of post-consumer polyethylene terephthalate into value-added products facilitated by genetic and bioprocess engineering

Metabolome and transcriptome association study reveals biosynthesis of specialized benzylisoquinoline alkaloids in Phellodendron amurense

ObjectiveBenzylisoquinoline alkaloids (BIAs) have pharmacological functions and clinical use. BIAs are mainly distributed in plant species across the order Ranunculales and the genus Phellodendron from Sapindales. The BIA biosynthesis has been intensively investigated in Ranunculales species. However, the accumulation mechanism of BIAs in Phellodendron is largely unknown. The aim of this study is to unravel the biosynthetic pathways of BIAs in Phellodendron amurens. MethodsThe transcriptome and metabolome data from 18 different tissues of P. amurense were meticulously sequenced and subsequently subjected to a thorough analysis. Weighted gene co-expression network analysis (WGCNA), a powerful systems biology approach that facilitates the construction and subsequent analysis of co-expression networks, was utilized to identify candidate genes involved in BIAs biosynthesis. Following this, recombinant plasmids containing candidate genes were expressed in Escherichia coli, a widely used prokaryotic expression system. The purpose of this genetic engineering endeavor was to express the candidate genes within the bacteria, thereby enabling the assessment of the resultant enzyme activity. ResultsThe synonymous substitutions per synonymous site for paralogs indicated that at least one whole genome duplication event has occurred. The potential BIA biosynthetic pathway of P. amurense was proposed, and two PR10/Bet v1 members, 14 CYP450s, and 33 methyltransferases were selected as related to BIA biosynthesis. One PR10/Bet v1 was identified as norcoclaurine synthase, which could catalyze dopamine and 4-hydroxyphenylacetaldehyde into (S)-norcoclaurine. ConclusionOur studies provide important insights into the biosynthesis and evolution of BIAs in non-Ranunculales species.

Markerless deletion of the putative type I and III restriction-modification systems in the cellulolytic bacterium Clostridium cellulovorans using a codBA-based counterselection technique

Cellulose from lignocellulosic biomass (LB) is of increasing interest for the production of commodity chemicals. However, its use as substrate for fermentations is a challenge due to its structural complexity. In this context, the highly cellulolytic Clostridium cellulovorans has been considered an interesting microorganism for the breakdown of LB. C. cellulovorans does not naturally produce solvents in useful concentrations, but this could be achieved by metabolic engineering. Unfortunately, this is hampered by the lack of tools for genetic engineering. We describe a genetic system that allows strain engineering by the allelic-coupled exchange method. First, the Gram-positive origin of pUB110 was identified as a suitable clostridial 'pseudo-suicide' origin of replication for the construction of deletion vectors. Second, an efficient counterselection strategy based on a codBA cassette and the use of 5-fluorocytosine as the counterselective compound was employed. Third, since the prevention of DNA transfer by host restriction-modification (RM) systems is a critical barrier to genome engineering, deletion plasmids containing flanking regions for the putative type I (Clocel_1114) and III (Clocel_2651) RM systems were constructed and transferred into C. cellulovorans. The restriction-less strains C. cellulovorans ΔClocel_1114 and C. cellulovorans ΔClocel_2651 exhibit high conjugation efficiency and can be easily used for further metabolic engineering.

Whole-pathway stimulation of anthocyanin biosynthesis by UDP-glycose: flavonoid glycosyltransferase gene (UFGT) providing new insights into natural product development based on plant cell factory

Anthocyanins are a type of plant natural products (PNPs) that play a crucial role in various fields, including food, medicine, cosmetics, and dyes. However, their primary source is plant extraction, leading to significant ecological damage and resource wastage issues. To address the challenge of achieving ecologically sustainable high-throughput anthocyanin production, this study proposes a plant cell factory (PCF) strategy based on suspension cell culture (SCC). In this study, the key gene VdUFGT involved in anthocyanin biosynthesis was cloned from Vitis davidii, and gene overexpression was conducted using Agrobacterium-mediated methods. Notably, the vgt3 transgenic cell line exhibited 4.4-fold increase in anthocyanin content, reaching 286.9 μg/g (FW) in solid medium, along with enhanced levels of flavonoids and proanthocyanidins. Integrative analysis of transcriptomics and metabolomics demonstrated that VdUFGT significantly promoted the expression of genes involved in anthocyanin biosynthesis, leading to the enhanced accumulation of various flavonoids, including anthocyanins. A small-scale plant cell factory was established, achieving an impressive production of 4.11 mg/g (DW) of anthocyanin within just 12 days, which is 8.5 times higher than that of the WT cell line, with the yield of purified freeze-dried anthocyanin powder reaching 3.17%. The plant cell factory, employing genetic engineering and suspension cell culture, offers a safe, ecological, economical, and efficient approach for the high-throughput production of plant natural products such as anthocyanins.

Engineering and evolution of Yarrowia lipolytica for producing lipids from lignocellulosic hydrolysates

Yarrowia lipolytica, an oleaginous yeast, shows promise for industrial fermentation due to its robust acetyl-CoA flux and well-developed genetic engineering tools. However, its lack of an active xylose metabolism restricts the conversion of cellulosic sugars to valuable products. To address this, metabolic engineering, and adaptive laboratory evolution (ALE) were applied to the Y. lipolytica PO1f strain, resulting in an efficient xylose-assimilating strain (XEV). Whole-genome sequencing (WGS) of the XEV followed by reverse engineering revealed that the amplification of the heterologous oxidoreductase pathway and a mutation in the GTPase-activating protein gene (YALI0B12100g) might be the primary reasons for improved xylose assimilation in the XEV strain. When a sorghum hydrolysate was used, the XEV strain showed superior xylose consumption and lipid production compared to its parental strain (X123). This study advances our understanding of xylose metabolism in Y. lipolytica and proposes effective metabolic engineering strategies for optimizing lignocellulosic hydrolysates.

A duty to enhance? Genetic engineering for the human Mars settlement.

Humans living off-world will face numerous physical, psychological and social challenges and are likely to suffer negative health effects due to their lack of evolutionary adaptation to space environments. While some of the necessary adaptations may develop naturally over many generations, genetic technologies could be used to speed this process along, potentially improving the wellbeing of early space settlers and their offspring. With broad support, such a program could lead to significant genetic modification of off-world communities, for example, to limit radiation damage on body systems or prevent bone and muscle loss in reduced gravity conditions. Given the extreme stressors of living off-world, and the need to have a healthy workforce to support a fledgling human settlement, those in favour of using genetic technologies to enhance settlers might even claim there is a moral imperative to protect their health in the face of the unique threats of space travel, especially for children born in settlements who did not take on these risks voluntarily. For some, this might simply be an extension of procreative beneficence. However, ethical concerns arise regarding the risks of embracing a eugenicist agenda and the potential impacts on the rights of future settlers to refuse such genetic enhancements for themselves or their children.

Phase separation drives the folding of recombinant collagen

Recombinantly produced collagens present a sustainable, ethical, and safe substitute for collagens derived from natural sources. However, controlling the folding of the recombinant collagens, crucial for replicating the mechanical properties of natural materials, remains a formidable task. Collagen-like proteins from willow sawfly are relatively small and contain no hydroxyprolines, presenting an attractive alternative to the large and post-translationally modified mammalian collagens. Utilizing CD spectroscopy and analytical ultracentrifugation, we demonstrate that recombinant willow sawfly collagen assembles into collagen triple helices in a concentration-dependent manner. Interestingly, we observed that the lower concentration threshold for the folding can be overcome by freezing or adding crowding agents. Microscopy data show that both freezing and the addition of crowding agents induce phase separation. We propose that the increase in local protein concentration during phase separation drives the nucleation-step of collagen folding. Finally, we show that freezing also induces the folding of recombinant human collagen fragments and accelerates the folding of natural bovine collagen, indicating the potential to apply phase separation as a universal mechanism to control the folding of recombinant collagens. We anticipate that the results provide a method to induce the nucleation of collagen folding without any requirements for genetic engineering or crosslinking.

Multiomics analysis reveals the involvement of OnDIVARICATA 3 in controlling dynamic flower coloring of Oncidium hybridum

Flower color is one of the main quality and economic traits of ornamental plants, and a large amount of research on flower color mainly focuses on the differences between varieties, while there were few reports on the change of flower color at different developmental stages. In this study, the metabolome and transcriptome of a new strain 'XM-1' with dynamic color changes of Oncidium were analyzed. The results showed that rutin, quercetin and carotenoids metabolism decreased significantly during the change of color from yellow to white. Analyzing the correlation network between metabolites and differential expressed genes, 25 key structural genes were detected and regulated by multiple MYB-related transcription factors. The MYB-related transcription factor Cluster-100966.1_OnDIVARICATA 3 was selected for further analysis. The phylogenetic tree of DIVARICATA in different species of Orchidaceae and Arabidopsis thaliana was constructed and the most closely related members were selected for sequence comparison. The results showed that OnDIVARICATA 3 contained MYB-like conserved domains. Subcellular localization results showed that OnDIVARICATA 3 was located in the nucleus. In overexpressing OnDIVARICATA 3 transgenic hairy roots, the expression of flower color related genes FLS, ZEP, and CHYB were significantly up-regulated. In summary, this study characterized the key metabolic pathways in the formation of the dynamic flower color of Oncidium hybridum, and constructed the regulatory network of the MYB-related. These results laid a theoretical foundation for the subsequent research on flower color and genetic engineering technology breeding of Oncidium hybridum.

Exploring Xenorhabdus and Photorhabdus Nematode Symbionts in Search of Novel Therapeutics.

Xenorhabdus and Photorhabdus bacteria, which live in mutualistic symbiosis with entomopathogenic nematodes, are currently recognised as an important source of bioactive compounds. During their extraordinary life cycle, these bacteria are capable of fine regulation of mutualism and pathogenesis towards two different hosts, a nematode and a wide range of insect species, respectively. Consequently, survival in a specific ecological niche favours the richness of biosynthetic gene clusters and respective metabolites with a specific structure and function, providing templates for uncovering new agrochemicals and therapeutics. To date, numerous studies have been published on the genetic ability of Xenorhabdus and Photorhabdus bacteria to produce biosynthetic novelty as well as distinctive classes of their metabolites with their activity and mechanism of action. Research shows diverse techniques and approaches that can lead to the discovery of new natural products, such as extract-based analysis, genetic engineering, and genomics linked with metabolomics. Importantly, the exploration of members of the Xenorhabdus and Photorhabdus genera has led to encouraging developments in compounds that exhibit pharmaceutically important properties, including antibiotics that act against Gram- bacteria, which are extremely difficult to find. This article focuses on recent advances in the discovery of natural products derived from these nematophilic bacteria, with special attention paid to new valuable leads for therapeutics.

Transforming Lignocellulosic Biomass into Biofuels: Recent Innovations in Pretreatment and Bioconversion Techniques

Abstract: This scholarly review investigates contemporary advancements in the conversion of lignocellulosic biomass to biofuels, emphasizing innovative pretreatment and bioconversion technologies that aim to surmount the intrinsic challenges associated with the processing of this complex biomass. Lignocellulosic materials, which are predominantly comprised of cellulose, hemicellulose, and lignin, represent a renewable and plentiful source for biofuel production; however, their structural complexity necessitates sophisticated methodologies for the effective degradation of resistant components. The review scrutinizes a variety of pretreatment methodologies, encompassing physical, chemical, and burgeoning techniques such as plasma-assisted processing, which are engineered to augment cellulose accessibility for enzymatic hydrolysis. Additionally, it elucidates the progress made in bioconversion processes, concentrating on enzymatic hydrolysis, microbial fermentation, and consolidated bioprocessing (CBP), wherein recent endeavors in genetic engineering are refining microbial strains to enhance yield and efficiency. By addressing economic, technological, and environmental challenges, this article emphasizes the role of integrated biorefineries and innovative biotechnologies in facilitating scalable and cost-effective production of lignocellulosic biofuels. Prospective research trajectories include the formulation of sustainable pretreatment techniques and the advancement of synthetic biology to fully harness the potential of lignocellulosic biomass as a renewable energy resource. Ultimately, this review accentuates the significance of lignocellulosic biofuels as a feasible alternative to fossil fuels, thereby contributing to energy sustainability and climate change mitigation through diminished carbon emissions

Advances in Soybean Genetic Improvement.

The soybean (Glycine max) is a globally important crop due to its high protein and oil content, which serves as a key resource for human and animal nutrition, as well as bioenergy production. This review assesses recent advancements in soybean genetic improvement by conducting an extensive literature analysis focusing on enhancing resistance to biotic and abiotic stresses, improving nutritional profiles, and optimizing yield. We also describe the progress in breeding techniques, including traditional approaches, marker-assisted selection, and biotechnological innovations such as genetic engineering and genome editing. The development of transgenic soybean cultivars through Agrobacterium-mediated transformation and biolistic methods aims to introduce traits such as herbicide resistance, pest tolerance, and improved oil composition. However, challenges remain, particularly with respect to genotype recalcitrance to transformation, plant regeneration, and regulatory hurdles. In addition, we examined how wild soybean germplasm and polyploidy contribute to expanding genetic diversity as well as the influence of epigenetic processes and microbiome on stress tolerance. These genetic innovations are crucial for addressing the increasing global demand for soybeans, while mitigating the effects of climate change and environmental stressors. The integration of molecular breeding strategies with sustainable agricultural practices offers a pathway for developing more resilient and productive soybean varieties, thereby contributing to global food security and agricultural sustainability.

Recent Advances in Genetic Engineering Strategies of Sinorhizobium meliloti.

Sinorhizobium meliloti is a free-living soil Gram-negative bacterium that participates in nitrogen-fixation symbiosis with several legumes. S. meliloti has the potential to be utilized for the production of high-value nutritional compounds, such as vitamin B12. Advances in gene editing tools play a vital role in the development of S. meliloti strains with enhanced characteristics for biotechnological applications. Several novel genetic engineering strategies have emerged in recent years to investigate genetic modifications in S. meliloti. This review provides a comprehensive overview of the mechanism and application of the extensively used Tn5-mediated genetic engineering strategies. Strategies based on homologous recombination and site-specific recombination were also discussed. Subsequently, the development and application of the genetic engineering strategies utilizing various CRISPR/Cas systems in S. meliloti are summarized. This review may stimulate research interest among scientists, foster studies in the application areas of S. meliloti, and serve as a reference for the utilization of genome editing tools for other Rhizobium species.

Gene editing technology combined with response surface optimization to improve the synthesis ability of lycopene in Pantoea dispersa MSC14.

The aim of this study is to engineer Pantoea dispersa MSC14 into a strain capable of producing lycopene and to enhance its lycopene content. Our laboratory isolated the strain P. dispersa MSC14 from petroleum-contaminated soil in a mining area. Whole-genome sequencing confirmed the existence of a carotenoid synthesis pathway in this strain. This study employed an optimized CRISPR/Cas9 system to perform a traceless gene knockout of the lycopene cyclase gene crtY and to overexpress the octahydrolycopene dehydrogenase gene crtI in the P. dispersa MSC14. This strategic genetic modification successfully constructed the lycopene-producing strain MSC14-LY, which exhibited a notable lycopene content with a biomass productivity of 553μg of lycopene per gram dry cell weight(DCW). Additionally, the components of the lycopene fermentation medium were optimized using Plackett-Burman (PB) design and Response Surface Methodology (RSM). The average lycopene content was increased to 5.13mg g -1DCW in the optimized LY fermentation medium. Through genetic engineering, P. dispersa MSC14 was transformed into a strain capable of producing lycopene, achieving a yield of 5.13mg g-1 DCW after medium optimization. Genetic engineering successfully transformed P. dispersa MSC14 into a strain capable of producing lycopene, achieving a yield of 5.13mg g-1 DCW after medium optimization.

Optimization of a rapid, sensitive, and high throughput molecular sensor to measure canola protoplast respiratory metabolism as a means of screening nanomaterial cytotoxicity

Nanomaterial-mediated plant genetic engineering holds promise for developing new crop cultivars but can be hindered by nanomaterial toxicity to protoplasts. We present a fast, high-throughput method for assessing protoplast viability using resazurin, a non-toxic dye converted to highly fluorescent resorufin during respiration. Protoplasts isolated from hypocotyl canola (Brassica napus L.) were evaluated at varying temperatures (4, 10, 20, 30 ˚C) and time intervals (1–24 h). Optimal conditions for detecting protoplast viability were identified as 20,000 cells incubated with 40 µM resazurin at room temperature for 3 h. The assay was applied to evaluate the cytotoxicity of silver nanospheres, silica nanospheres, cholesteryl-butyrate nanoemulsion, and lipid nanoparticles. The cholesteryl-butyrate nanoemulsion and lipid nanoparticles exhibited toxicity across all tested concentrations (5-500 ng/ml), except at 5 ng/ml. Silver nanospheres were toxic across all tested concentrations (5-500 ng/ml) and sizes (20–100 nm), except for the larger size (100 nm) at 5 ng/ml. Silica nanospheres showed no toxicity at 5 ng/ml across all tested sizes (12–230 nm). Our results highlight that nanoparticle size and concentration significantly impact protoplast toxicity. Overall, the results showed that the resazurin assay is a precise, rapid, and scalable tool for screening nanomaterial cytotoxicity, enabling more accurate evaluations before using nanomaterials in genetic engineering.