纳米脂质体 农产品 基因编辑 基因沉默

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

Highly efficient gene delivery systems are essential for genetic engineering in plants. Traditional delivery methods have been widely used, such as Agrobacterium-mediated transformation, polyethylene glycol (PEG)-mediated delivery, biolistic particle bombardment, and viral transfection. However, genotype dependence and other drawbacks of these techniques limit the application of genetic engineering, particularly genome editing in many crop plants. There is a great need to develop newer gene delivery vectors or methods. Recently, nanomaterials such as mesoporous silica particles (MSNs), AuNPs, carbon nanotubes (CNTs), and layer double hydroxides (LDHs), have emerged as promising vectors for the delivery of genome engineering tools (DNA, RNA, proteins, and RNPs) to plants in a species-independent manner with high efficiency. Some exciting results have been reported, such as the successful delivery of cargo genes into plants and the generation of genome stable transgenic cotton and maize plants, which have provided some new routines for genome engineering in plants. Thus, in this review, we summarized recent progress in the utilization of nanomaterials for plant genetic transformation and discussed the advantages and limitations of different methods. Furthermore, we emphasized the advantages and potential broad applications of nanomaterials in plant genome editing, which provides guidance for future applications of nanomaterials in plant genetic engineering and crop breeding.

The genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants. Here, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1. Our results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.

… The development of a highly efficient Cas9/gRNA RNP delivery will pave the way for more robust and high-throughput genome editing in plant cells. Liposomes can be easily produced …

Plant genetic engineering is essential for improving crop yield, quality, and resistance to abiotic/biotic stresses for sustainable agriculture. Agrobacterium‐, biolistic bombardment‐, electroporation‐, and poly(ethylene glycol) (PEG)‐mediated genetic‐transformation systems are extensively used in plant genetic engineering. However, these systems have limitations, including species dependency, destruction of plant tissues, low transformation efficiency, and high cost. Recently, nanotechnology‐based gene‐delivery methods have been developed for plant genetic transformation. This nanostrategy shows excellent transformation efficiency, good biocompatibility, adequate protection of exogenous nucleic acids, and the potential for plant regeneration. However, the nanomaterial‐mediated gene‐delivery system in plants is still in its infancy, and there are many challenges for its broad applications. Herein, the conventional genetic transformation techniques used in plants are briefly discussed. After that, the progress in the development of nanomaterial‐based gene‐delivery systems is considered. CRISPR‐Cas‐mediated genome editing and its combined applications with plant nanotechnology are also discussed. The conceptual innovations, methods, and practical applications of nanomaterial‐mediated genetic transformation summarized herein will be beneficial for promoting plant genetic engineering in modern agriculture.

Plant genetic engineering is crucial for enhancing crop yield, quality, and resilience to both abiotic and biotic stresses, thereby promoting sustainable agriculture. Agrobacterium-mediated, biolistic bombardment, electroporation, and poly (ethylene glycol) (PEG)-mediated genetic transformation systems are widely applied in plant genetic engineering. However, these systems have limitations, including species dependency, destruction of plant tissues, low transformation efficiency, and high cost. Recently, gene-delivery methods based on nanotechnology have been developed for plant genetic transformation. This nanostrategy demonstrates remarkable transformation efficiency, excellent biocompatibility, effective protection of exogenous nucleic acids, and the potential for plant regeneration. However, the application of nanomaterial-mediated gene-delivery systems in plants is still in its early stages and faces numerous challenges for widespread adoption. Herein, the conventional genetic transformation techniques utilized in plants are succinctly examined. Subsequently, the advancements in nanomaterial-based gene-delivery systems are reviewed. The applications of CRISPR-Cas-mediated genome editing and its integration with plant nanotechnology are also examined. The innovations, methods, and practical applications of nanomaterial-mediated genetic transformation summarized herein are expected to facilitate the progress of plant genetic engineering in modern agriculture.

Abstract The conversion of bacterial CRISPR/Cas defense system into a simple and efficient tool for genome manipulations brought experimental biology into new dimensions. Suddenly, genome editing reached many groups most of which were interested in it but not able to employ the available time- and labor-consuming approaches of the pre-CRISPR era. In plant biology and biotechnology, CRISPR/Cas gene editing became the second most important technology after plant transformation. Actually, it relies on the available array of methods of gene delivery. While sufficient for most purposes, the classic gene transfer methods might become a problem for some experimental settings. The main obstacle is that they include DNA delivery and, frequently, its subsequent integration into cellular genome. For this reason novel methods to achieve gene editing without the need of stable transformation and even without DNA delivery were developed. These new approaches include in vitro ribonucleoprotein complexes formulations (delivered by microinjection, particle bombardment, electroporation, liposomes etc.), use of virus-like particles and employment of bacterial secretory systems for Cas/gRNA delivery. The first attempts to achieve DNA-free editing were made less than ten years ago. Later, different types of animal and plant cells were addressed. In this mini review we try to summarize the current developments and emerging trends in the field of DNA-free editing in plants.

… of efficient liposomes, for appropriate fusion conditions and for an understanding of the nature of liposome-eell interactions. Various characteristics and techniques of the liposome-cell …

Abstract Using modern genome editing tools, scientists are increasingly able to engineer animals and plants for better traits and improved downstream outcomes that benefit humans. As part of the CRISPR-Cas system, guide RNA (gRNA) is used to identify the target sequence, while Cas is an endonuclease that performs the nucleotide cleavage. It is imperative that these two components are delivered to the nucleus of the cell in order to ensure an optimal editing process. As a consequence of differences in the cellular structure and biomolecular composition of the outer membrane, plants are not capable of being cloned genetically in the same manner as animal cells. A more optimized method and pipeline must be developed to improve the efficiency of transformations and genome editing for plants. In this book chapter, we highlight traditional and novel delivery methods used for optimal delivery of plant genome editing components. We discuss the potential and limitations of these methods in the light of recent literature and available experimental validations.

… complex as RNP and perform DNA-free editing, they demonstrated the efficiency of CRISPR RNP in plant genome editing. But this elaborate protoplast isolation and regeneration …

The 21st century witnessed a boom in plant genomics and gene characterization studies through RNA interference and site-directed mutagenesis. Specifically, the last 15 years marked a rapid increase in discovering and implementing different genome editing techniques. Methods to deliver gene editing reagents have also attempted to keep pace with the discovery and implementation of gene editing tools in plants. As a result, various transient/stable, quick/lengthy, expensive (requiring specialized equipment)/inexpensive, and versatile/specific (species, developmental stage, or tissue) methods were developed. A brief account of these methods with emphasis on recent developments is provided in this review article. Additionally, the strengths and limitations of each method are listed to allow the reader to select the most appropriate method for their specific studies. Finally, a perspective for future developments and needs in this research area is presented.

… Cationic lipids are positively charged liposomes that can bind to negatively charged DNA molecules, forming liposome-DNA complexes that can enter plant cells. This method is efficient…

Abstract The development of nano delivery vectors for plant genetic transformation needs more research for determining the delivery challenges and increasing plant genetic efficiency. Efficient in vivo delivery of the CRISPR/Cas9 system to the target cell—avoiding side effects and delivery barriers that conflict with other transport approaches—remains a challenge. Recently, nonviral vectors have emerged as new classes of gene carriers in terms of safety, simplicity, and adaptability. RNAi and CRISPR systems can be delivered by nonviral vectors that are more easily degraded in vivo, and the expression of this transition facilitates use in medicine, biology, genetics, and agriculture. Polymer and lipid nanoparticles can be produced more easily and the size, composition, and structure of inorganic nanoparticles can be precisely controlled. In this chapter, we present the latest research on RNAi and CRISPR delivery technology based on lipids and polymers that are produced through synthetic techniques.

: Current trends in plant genetic transformation technologies, i.e., designing and applying molecules like miRNA, RNAi, and CRISPR-Cas, largely enable researchers to target speci fi c sites in the plant genome to avert the growing biotic and abiotic threats to plants. However, the delivery of these molecules through conventional techniques brings an array of drawbacks such as low e ffi ciency due to the cell wall barrier, tissue damage that leads to browning or necrosis, degradation of these biomolecules by physiological conditions (high temperature, harsh pH, and light), and plant-speci fi c protocols. The advancements in nanotechnology o ff er an excellent alternative for the safe and highly e ffi cient delivery of biomolecules such as miRNA, CRISPR-Cas, and RNAi without damaging the plant tissues. Nanoparticle (polymeric, metallic, magnetic, silica, carbon, etc.)-based delivery of biomolecules can be e ffi ciently utilized especially for plant protection applications. Herein, we present a comprehensive overview of current trends (with a focus on the previous fi ve years) in nanoparticle-based delivery of miRNA, RNAi, CRISPR-Cas and simillar biomolecules for plant protection applications. In addition, a future perspective focuses on the research gaps and unexplored potentials of nanoparticles for the delivery of biomolecules.

The fall armyworm (FAW, Spodoptera frugiperda) is an invasive lepidopteran pest of staple crops. Its broad host range, ability to spread rapidly, and increasing resistance to pesticides pose a major threat to global food security. RNA interference (RNAi) offers a sustainable and targeted alternative to broad-spectrum chemical pesticides, but its efficacy is limited in lepidopterans primarily by the rapid degradation of double-stranded RNA (dsRNA) in the midgut and poor epithelial uptake. Here, we investigated lipid nanoparticles (LNPs) as a delivery strategy to enhance dsRNA stability and uptake in FAW larvae. LNP–dsRNA complexes (40–50 nm, +39 to +56 mV) were generated by the microfluidic mixing of a ternary lipid blend. Encapsulation protected dsRNA from degradation by gut enzyme extracts for up to 1 h, even under highly alkaline conditions (pH 11.5). The analysis of larvae exposed to Cy3-labeled dsRNA by fluorescence microscopy demonstrated that LNPs improved internal distribution beyond the gut lumen, whereas unformulated dsRNA mainly accumulated at the peritrophic membrane. These results indicate that LNPs resist the gut environment and overcome limited systemic uptake, the two major physiological barriers to RNAi in lepidopterans, enabling the more efficient delivery of dsRNA. This study establishes a lipid nanoparticle-based dsRNA delivery platform that overcomes key physiological barriers in FAW, providing a prerequisite for future in vivo gene knockdown and efficacy studies.

Nucleic acid-based therapeutics have the ability to tackle a wide range of diseases and stress tolerance that present significant obstacles for conventional approaches in agriculture. RNA-based medicines have become a promising approach, using nanoformulation treatments to specifically target certain diseases. Nanoformulations offer numerous benefits in comparison to alternative treatment methods, such as precise administration, minimal toxicity, and medication loading compatibility due to their bioactivity. There are a variety of nanoformulations available today, such as liposomes, polymeric nanoparticles (NPs), magnetic NPs, nanogels, and solid lipid nanoparticles (SLNs). RNA-based therapy employs intracellular gene nanoparticles containing messenger RNA (mRNA), which play an important role in stress management and pest as well as disease control. The adoption of mRNA-based technology paves the way for future technological progress. This review focuses on elucidating the process underlying the development of RNA interference (RNAi) and the diverse array of nanocarriers employed for the transportation of RNAi. Currently, this technique is being employed in the field of crop protection to combat diseases, pests, and environmental stress. The article highlights the benefits of RNAi mediated nanoformulations and discusses the significant obstacles that must be overcome to improve the viability of this technology for future applications.

… Lipid nanocarriers, which include liposomes, lipid nanoparticles (LNPs), and nanostructured lipid … Lipid nanoparticles (LNPs) have attracted interest for their biocompatibility, …

Most crop viruses are carried and spread by seeds. Virus-infected seeds are seed-borne viral disease infections, and thus, reducing the rate of seed infection is an urgent problem in the seed-production industry. The objective of this study was to use nanoparticles (NPs) to directly deliver dsRNA into plants or pollen to initiate RNA interference (RNAi) to reduce viral carryover in seeds. Chitosan quaternary ammonium salt (HACC), complexed with dsRNAs, was selected for targeting the genes for the tobacco mosaic virus (TMV) coat protein (CP) and TMV RNA-dependent RNA polymerase (RdRP) to form HACC-dsRNA NPs. These NP-based dsRNAs were delivered to the plants using four different methods, including infiltration, spraying, root soaking, and pollen internalization. All four methods were able to reduce the seed-carrying rate of offspring seeds of the TMV-infected plants, with pollen internalization being the most effective in reducing the TMV-carrying rate from 95.1 to 61.1% in the control group. By measuring the plant uptake of fluorescence-labeled NPs and dsRNAs, the transportation of the HACC-dsRNA NPs into the plants was observed, and the uptake of dsRNA in combination with small RNA sequencing was further confirmed, resulting in the silencing of homologous RNA molecules during the topical application. The results demonstrated that the incidence of TMV infection was reduced by various degrees via RNAi induction without the need to develop transgenic plants. These results demonstrate the advantages of NP-based RNAi technology in breeding for disease resistance and developing a new strategy for virus-resistant breeding in plants.

Developing safe and effective double-stranded RNA (dsRNA) delivery systems remains a major challenge for gene silencing, especially in lepidopteran insects. This study evaluated the protamine sulfate (PS)/lipid/dsRNA nanoparticle (NP) delivery system for RNA interference (RNAi) in cells and larvae of the fall armyworm (FAW), Spodoptera frugiperda, a major worldwide pest. A highly efficient gene delivery formulation was prepared using a cationic biopolymer, PS, and a cationic lipid, Cellfectin (CF), complexed with dsRNA. The NPs were prepared by a two-step self-assembly method. The formation of NPs was revealed by dynamic light scattering and transmission electron microscopy. The formation of CF/dsRNA/PS NPs was spherical in shape and size, ranging from 20 to 100 nm with a positive charge (+23.3 mV). Interestingly, prepared CF/dsRNA/PS NPs could protect dsRNA (95%) from nuclease degradation and thus significantly improve the stability of dsRNA. Formulations prepared by combining EGFP DNA with CF/PS increased transfection efficiency in Sf9 cells compared to PS/EGFP and CF/EGFP NPs. Also, the PS/CF/dsRNA NPs enhanced the endosomal escape for the intracellular delivery of dsRNA. The gene knockdown efficiency was assessed in Sf9 Luciferase (Luc) stable cells after a 72 h incubation with CF/dsRNA/PS, PS/dsRNA, CF/dsRNA, or naked dsRNA. Knockdown of the Luc gene was detected in CF/dsRNA/PS (76%) and PS/dsRNA (42.4%) not CF/dsRNA (19.5%) and naked dsRNA (10.3%) in Sf9 Luc cells. Moreover, CF/dsIAP/PS (25 μg of dsRNA targeting the inhibitor of apoptosis, IAP, gene of FAW) NPs showed knockdown of the IAP gene (39.5%) and mortality (55%) in FAW larvae. These results highlight the potential application of PS/lipid/dsRNA NPs for RNA-mediated control of insect pests.

To fulfil the growing needs of the global population, sustainability in food production must be ensured. Insect pests and pathogens are primarily responsible for one-third of food losses and harmful synthetic pesticides have been applied to protect crops from these pests and other pathogens such as viruses and fungi. An alternative pathogen control mechanism that is more “friendly” to the environment can be developed by externally applying double-stranded RNAs (dsRNAs) to suppress gene expression. However, the use of dsRNA sprays in open fields is complicated with respect to variable efficiencies in the dsRNA delivery, and the stability of the dsRNA on and in the plants, and because the mechanisms of gene silencing may differ between plants and between different pathogen targets. Thus, nanocarrier delivery systems have been especially used with the goal of improving the efficacy of dsRNAs. Here, we highlight recent developments in nanoparticle-mediated nanocarriers to deliver dsRNA, including layered double hydroxide, carbon dots, carbon nanotubes, gold nanoparticles, chitosan nanoparticles, silica nanoparticles, liposomes, and cell-penetrating peptides, by review of the literature and patent landscape. The effects of nanoparticle size and surface modification on the dsRNA uptake efficiency in plants are also discussed. Finally, we emphasize the overall limitation of dsRNA sprays, the risks associated, and the potential safety concerns for spraying dsRNAs on crops.

… that employ RNAi in crop protection. Further, we discuss the factors that affect the efficacy of RNAi-… Furthermore, the review emphasizes the importance of nanoparticle-mediated delivery …

In planta RNAi or host-induced gene silencing (HIGS) has undergone significant advancements that have rendered it efficient and stable at the transgenerational level in plants for regulating host genes and targeting genes of insect pests and plant pathogens. Similarly, topical RNAi or spray-induced gene silencing (SIGS) has garnered considerable attention as an environmentally sustainable, selective, and alternative approach to chemical control of insect pests and plant pathogens. Several biotechnology companies and startups have focused their efforts on RNAi-based solutions for topical application in agriculture. Nevertheless, further technological advancements are required to enhance the efficacy of topical RNAi in agriculture, including improved dsRNA delivery systems, better target gene selection, and addressing biosafety regulatory issues. Herein, this review discusses key advances and bottlenecks in RNAi, and summarizes successful applications of these RNAi-based technologies in agriculture focusing on in planta and topical RNAi to control insect pests and plant pathogens. Furthermore, this review delves into the patenting landscape, biosafety considerations, risk evaluations, and the current regulatory status of RNAi in Latin America. Finally, it explores the contributions of RNAi to plant science, food production, and fostering a more sustainable form of agriculture.

Summary Spray‐induced gene silencing (SIGS) is an innovative and eco‐friendly technology where topical application of pathogen gene‐targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross‐kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNA encapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi‐mediated protection against B. cinerea both on pre‐ and post‐harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection.

The control of agricultural pest insects currently relies on broad-spectrum insecticides, which select for resistance in pest populations while also harming non-target species. In contrast, RNA interference (RNAi) has a species-dependent mode of action based on the delivery of double-stranded RNA (dsRNA) that precisely matches essential genes in pests, minimizing off-target effects. The successful application of RNAi requires the development of sprayable formulations that temporarily protect the dsRNA from environmental degradation (allowing uptake by pest insects) but also ensure the efficient release of the dsRNA within insect cells. Lipid nanoparticles (LNPs) based on pharmaceutical-grade lipids are currently too expensive for agricultural use, making the development of affordable and scalable dsRNA-LNP formulations essential for spray-induced gene silencing. Here we used technical-grade lipid components (available at the ton scale) and demonstrated the cost-effective production of structurally controlled dsRNA-LNP formulations by optimizing formulation recipes and scaling up the microfluidic mixing process. The dispersions contained spherical nanoparticles less than 100 nm in diameter, with a zeta potential exceeding + 20 mV, and an entropy-driven Gibbs free energy change for dsRNA-LNP decomplexation in the moderate range of approximately – 20 kJ/mol. The formulations protected dsRNA from RNase III degradation and hydrolysis at pH 4–11 for at least 24 h while allowing SDS-mediated dsRNA release. Our work provides insight into the structure–property correlations of inexpensive dsRNA-LNP formulations for sustainable RNAi-based pest management systems. Supplementary Information The online version contains supplementary material available at 10.1038/s41598-026-44095-2.

Piercing-sucking pests are the most notorious group of pests for global agriculture. RNAi-mediated crop protection by foliar application is a promising approach in field trials. However, the effect of this approach on piercing-sucking pests is far from satisfactory due to the limited uptake and transport of double strand RNA (dsRNA) in plants. Therefore, there is an urgent need for more feasible and biocompatible dsRNA delivery approaches to better control piercing-sucking pests. Here, we report that foliar application of layered double hydroxide (LDH)-loaded dsRNA can effectively disrupt Panonychus citri at multiple developmental stages. MgAl-LDH-dsRNA targeting Chitinase (Chit) gene significantly promoted the RNAi efficiency and then increased the mortality of P. citri nymphs by enhancing dsRNA stability in gut, promoting the adhesion of dsRNA onto leaf surface, facilitating dsRNA internalization into leaf cells, and delivering dsRNA from the stem to the leaf via the vascular system of pomelo plants. Finally, this delivery pathway based on other metal elements such as iron (MgFe-LDH) was also found to significantly improve the protection against P. citri and the nymphs or larvae of Diaphorina citri and Aphis gossypii, two other important piercing-sucking hemipeteran pests, indicating the universality of nanoparticles LDH in promoting the RNAi efficiency and mortality of piercing-sucking pests. Collectively, this study provides insights into the synergistic mechanism for nano-dsRNA systemic translocation in plants, and proposes a potential eco-friendly control strategy for piercing-sucking pests.

RNA interference (RNAi) targeting lethal genes in insects has great potential for sustainable crop protection. Compared with traditional double‐stranded (ds)RNA delivery systems, nanoparticles such as chitosan, liposomes, and cationic dendrimers offer advantages in delivering dsRNA/small interfering (si)RNA to improve RNAi efficiency, thus promoting the development and practice of RNAi‐based pest management strategies. Here, we illustrate the limitations of traditional dsRNA delivery systems, reveal the mechanism of nanoparticle‐mediated RNAi, summarize the recent progress and successful applications of nanoparticle‐mediated RNAi in pest management, and finally address the prospects of nanoparticle‐based RNA pesticides.

Existing, emerging, and reemerging strains of phytopathogenic fungi pose a significant threat to agricultural productivity globally. This risk is further exacerbated by the lack of resistance source(s) in plants or a breakdown of resistance by pathogens through co-evolution. In recent years, attenuation of essential pathogen gene(s) via double-stranded (ds) RNA-mediated RNA interference (RNAi) in host plants, a phenomenon known as host-induced gene silencing, has gained significant attention as a way to combat pathogen attack. Yet, due to biosafety concerns regarding transgenics, country-specific GMO legislation has limited the practical application of desirable attributes in plants. The topical application of dsRNA/siRNA targeting essential fungal gene(s) through spray-induced gene silencing (SIGS) on host plants has opened up a transgene-free avenue for crop protection. However, several factors influence the outcome of RNAi, including but not limited to RNAi mechanism in plant/fungi, dsRNA/siRNA uptake efficiency, dsRNA/siRNA design parameters, dsRNA stability and delivery strategy, off-target effects, etc. This review emphasizes the significance of these factors and suggests appropriate measures to consider while designing in silico and in vitro experiments for successful RNAi in open-field conditions. We also highlight prospective nanoparticles as smart delivery vehicles for deploying RNAi molecules in plant systems for long-term crop protection and ecosystem compatibility. Lastly, we provide specific directions for future investigations that focus on blending nanotechnology and RNAi-based fungal control for practical applications.

… as well as the difficulty in regenerating transformed protoplasts into plants. We hypothesized that cationic lipid nanoparticles could encapsulate and protect the RNP complex from …

Recent research reports have shown that plant pests and pathogens have depleted the crop yield widely, which has led to an increased dependence on commercial pesticides and fungicides. Increased usage of these pesticides has also shown adverse effects on the environment, therefore many techniques have been implemented for solving the issue, some of which include using nanobioconjugates, RNA(i), which put into use double-stranded RNAs to inhibit gene expression. A more innovative and eco-friendly strategy includes spray induced gene silencing, which is being increasingly implemented. This review delves into the eco-friendly approach of spray induced gene silencing (SIGS) in combination with nanobioconjugates, which have been used concerning various plant hosts and their pathogens to provide improved protection. Furthermore, nanotechnological advancements have been understood by addressing the scientific gaps to provide a rationale for the development of updated techniques in crop protection.

Spray-induced gene silencing (SIGS) presents a promising RNA interference (RNAi)-based crop protection strategy against eukaryotic phytopathogens. However, the application of SIGS faces challenges, such as the limited uptake of dsRNA by certain pathogens and the instability of dsRNA in the environment. This study introduces innovative biomimetic nanovesicles, called extracellular vesicle (EV) mimetic chimeric nanovesicles (ECNs), assembled from tomato leaf cell membranes and cationic sterosomes via the freeze-thaw method. Similar to the function of EVs in nucleic acid transport between cells, ECNs serve as a hybrid nanosystem to overcome the challenge of delivering exogenous dsRNA in Phytophthora infestans. When applied to SIGS, the superiority of ECNs in crop protection becomes more apparent, including high loading and protection of dsRNA, improved biosafety, and efficient internalization into pathogen and plant cells, all of which significantly enhance the efficacy of RNAi in preventing early infection of P. infestans to susceptible tomato plants. This study demonstrates that ECNs are promising RNA delivery vehicles and will promote the use of SIGS-based RNA pesticides in sustainable agricultural production.

RNA interference (RNAi) is increasingly used for plant protection against pathogens and pests. However, the traditional delivery method causes plant tissue damage, is affected by environmental factors, and faces difficulties in penetrating the barriers of cell walls and the limitations of plant species, ultimately leading to low delivery efficiency. With advances in nanotechnology, nanomaterials (NMs) have been identified as effective carriers for nucleic acid delivery because of their ability to operate independently of external mechanical forces, prevent degradation by bioenzymes, exhibit good biocompatibility, and offer high loading capacity. This review summarizes the application of NM-mediated RNAi against plant pathogens and pests, focusing on how different NMs break through the cell barriers of plants, pathogens, and pests according to their size, morphology, and charge characteristics. Furthermore, we discuss the advantages and improvement strategies of NMs as nucleic acid delivery carriers, alongside assessing their potential application for the management of plant pathogens and pests.

RNA interference (RNAi)-based control technologies are gaining popularity as potential alternatives to synthetic fungicides in the ongoing effort to manage plant pathogenic fungi. Among these methods, spray-induced gene silencing (SIGS) emerges as particularly promising due to its convenience and feasibility for development. This approach is a new technology for plant disease management, in which double-stranded RNAs (dsRNAs) targeting essential or virulence genes are applied to plants or plant products and subsequently absorbed by plant pathogens, triggering a gene silencing effect and the inhibition of the infection process. Spray-induced gene silencing has demonstrated efficacy in laboratory settings against various fungal pathogens. However, as research progressed from the laboratory to the greenhouse and field environments, novel challenges arose, such as ensuring the stability of dsRNAs and their effective delivery to fungal targets. Here, we provide a practical guide to SIGS for the control of plant pathogenic fungi. This guide outlines the essential steps and considerations needed for designing and assessing dsRNA molecules. It also addresses key challenges inherent to SIGS, including delivery and stability of dsRNA molecules, and how nanoencapsulation of dsRNAs can aid in overcoming these obstacles. Additionally, the guide underscores existing knowledge gaps that warrant further research and aims to provide assistance to researchers, especially those new to the field, encouraging the advancement of SIGS for the control of a broad range of fungal pathogens.

… frugiperda is known notorious in agriculture that results in huge crop losses. The study explored the use of nanoliposomes to pass the dsRNA for methoprene tolerance and control the …

… gene silencing of important developmental genes, resulting in distinct phenotypes. A study based on the post-transcriptional gene silencing … and knock down targeted genes in BY-2 …

Genetic engineering in plants serves as a crucial method for enhancing crop quality, yield, and climate resilience through the manipulation of genetic circuits. A novel genetic transformation approach utilizing nanocarriers as a sound plant genetic engineering technique enables the delivery of DNAs or RNAs into the plant cells. Significant advances have recently been made on the nanotechnology-based delivery of nucleic acids in plants. In this review, several nanoparticle-mediated DNA and RNA delivery systems are discussed respectively, involving latest progresses and drawbacks of these approaches used in plant genetic engineering. We also underscores the current challenges that must be addressed in the implementation of nanoparticles-based strategies for plant gene delivery. Furthermore and more importantly, plant-derived exosome-like nanoparticles that facilitate nucleic acids transfer between organisms was initially proposed as a novel and promising nanodelivery platform for the CRISPR/Cas9 genome editing toolkit in plants. We believe that this review will be beneficial for an effective exploration of nucleic acid nanodelivery to aid the plant genetic engineering in modern agriculture.

… Through this study, we have found that silencing Ferredoxin 3 (… Based on this finding, we have developed a liposome … infection in cereal crops through RNA interference (RNAi). …

Spray-induced gene silencing represents an eco-friendly approach for crop protection through the use of double-stranded RNA (dsRNA) to activate the RNA interference (RNAi) pathway, thereby silencing crucial genes in pathogens. The major challenges associated with dsRNA are its limited stability and poor cellular uptake, necessitating repeated applications for effective crop protection. In this study, RNA nanoparticles (NPs) were proposed as effectors in plants and pathogens by inducing the RNAi pathway and silencing gene expression. RNA structural motifs, such as hairpin-loop, kissing-loop, and tetra-U motifs, were used to link multiple siRNAs into a long, single-stranded RNA (lssRNA). The lssRNA, synthesized in Escherichia coli, self-assembled into stable RNA nanostructures via local base pairing. Comparative analyses between dsRNA and RNA NPs revealed that the latter displayed superior efficacy in inhibiting spore germination and mycelial growth of Botrytis cinerea. Moreover, RNA NPs had a more robust protective effect on plants against B. cinerea than did dsRNA. In addition, RNA squares are processed into expected siRNA in plants, thereby inhibiting the expression of the target gene. These findings suggest the potential of RNA NPs for use in plant disease control by providing a more efficient and specific alternative to dsRNA without requiring nanocarriers.

The global agricultural sector faces unprecedented challenges in meeting the projected food demand of 9.7 billion people by 2050, exacerbated by the adverse impacts of climate change, such as increased droughts and temperature extremes. Nanobiotechnology, the synergistic integration of nanotechnology and biotechnology, offers transformative solutions in plant genetic engineering to enhance agricultural sustainability and ensure food security. Nanobiotechnology exploits the unique physicochemical properties of nanomaterials, enabling the precise delivery of genetic materials, advanced gene editing, and real-time monitoring of cellular processes. Innovative nanoparticle-mediated methods facilitate the transfer of nucleic acids, proteins, and other biomolecules into plant cells, overcoming the limitations of conventional genetic transformation methods such as Agrobacterium-mediated transformation and gene gun technologies. For example, magnetic nanoparticles and carbon nanotubes have shown promise in genotype-independent genetic material delivery and efficient transgene expression. This review highlights groundbreaking applications of nanobiotechnology, including enhanced delivery of CRISPR/Cas9 components for accurate gene editing, nanoscale sensors for intracellular process monitoring, and the use of mesoporous silica nanoparticles for stable gene silencing. Despite these advancements, barriers such as nanoparticle biocompatibility, potential toxicity, and scalability in agricultural systems must be addressed. Regulatory frameworks ensuring the safe adoption of nanomaterials in agricultural practices are equally critical. Nanobiotechnology holds the potential to revolutionize plant genetic engineering by enabling precise trait manipulation, increased crop resilience, and reduced environmental impact. Leveraging these advancements can foster sustainable agricultural practices and mitigate the challenges posed by global food demands and climate change.

Efficient delivery of exogenous genetic material remains a core challenge in plant biotechnology, holding profound implications for sustainable agricultural and forestry development. Although traditional delivery methods such as Agrobacterium-mediated transformation, gene gun bombardment, and electroporation have been widely applied in plant genetic engineering, these systems exhibit limitations including species-dependent efficacy, propensity to cause plant tissue damage, low transformation efficiency, susceptibility to environmental factors. In recent years, with the advancement of nanotechnology, nanoparticle-based nucleic acid delivery systems are emerging as novel tools for applications such as novel tools for dsRNA or transgene delivery. These systems leverage the unique physicochemical properties of nanomaterials, including size-dependent phenomena, tunable surface charge, and enhanced membrane penetration capabilities, to achieve targeted delivery and stable expression of genetic payloads. Nevertheless, nanomaterial-mediated gene delivery systems for plants are still in their nascent stages, and their widespread application faces numerous challenges. This article briefly introduces traditional delivery methods, systematically reviews the applications and progress of nanoparticle-based nucleic acid delivery systems, and discusses the cross-species applicability of nanoparticles, as well as the associated biosafety concerns. We aim to offer insights for tackling the prevailing technical bottlenecks and to provide guidance for the rational design of nanomaterials that efficiently traverse the plant cell wall–plasma membrane barrier and stably deliver nucleic acids without eliciting phytotoxicity.

Nanocarriers are widely used for efficient delivery of different cargo into mammalian cells; however, delivery into plant cells remains a challenging issue due to physical and mechanical barriers such as the cuticle and cell wall. Here, we discuss recent progress on biodegradable and biosafe nanomaterials that were demonstrated to be applicable to the delivery of nucleic acids into plant cells. This review covers studies the object of which is the plant cell and the cargo for the nanocarrier is either DNA or RNA. The following nanoplatforms that could be potentially used for nucleic acid foliar delivery via spraying are discussed: mesoporous silica nanoparticles, layered double hydroxides (nanoclay), carbon-based materials (carbon dots and single-walled nanotubes), chitosan and, finally, cell-penetrating peptides (CPPs). Hybrid nanomaterials, for example, chitosan- or CPP-functionalized carbon nanotubes, are taken into account. The selected nanocarriers are analyzed according to the following aspects: biosafety, adjustability for the particular cargo and task (e.g., organelle targeting), penetration efficiency and ability to protect nucleic acid from environmental and cellular factors (pH, UV, nucleases, etc.) and to mediate the gradual and timely release of cargo. In addition, we discuss the method of application, experimental system and approaches that are used to assess the efficiency of the tested formulation in the overviewed studies. This review presents recent progress in developing the most promising nanoparticle-based materials that are applicable to both laboratory experiments and field applications.

Plant biotechnology plays a crucial role in developing modern agriculture and plant science research. However, the delivery of exogenous genetic material into plants has been a long-standing obstacle. Nanoparticle-based delivery systems are being established to address this limitation and are proving to be a feasible, versatile, and efficient approach to facilitate the internalization of functional RNA and DNA by plants. The nanoparticle-based delivery systems can also be designed for subcellular delivery and controlled release of the biomolecular cargo. In this review, we provide a concise overview of the recent advances in nanocarriers for the delivery of biomolecules into plants, with a specific focus on applications to enhance RNA interference, foreign gene transfer, and genome editing in plants.

… Nanocarriers like lipid NPs and CDs have shown promise for delivering mRNA in both plant … entry but also on the controlled release of nucleic acids from the nanocarrier matrix. Many …

CRISPR–Cas genetic engineering of plants holds tremendous potential for providing food security, battling biotic and abiotic crop stresses caused by climate change, and for environmental remediation and sustainability. Since the discovery of CRISPR–Cas technology, its usefulness has been demonstrated widely, including for genome editing in plants. Despite the revolutionary nature of genome-editing tools and the notable progress that these tools have enabled in plant genetic engineering, there remain many challenges for CRISPR applications in plant biotechnology. Nanomaterials could address some of the most critical challenges of CRISPR genome editing in plants through improvements in cargo delivery, species independence, germline transformation and gene editing efficiency. This Perspective identifies major barriers preventing CRISPR-mediated plant genetic engineering from reaching its full potential, and discusses ways that nanoparticle technologies can lower or eliminate these barriers. We also describe advances that are needed in nanotechnology to facilitate and accelerate plant genome editing. Timely advancement of the application of CRISPR technologies in plant engineering is crucial for our ability to feed and sustain the growing human population under a changing global climate. Despite its high promise, there are still many challenges for CRISPR-mediated plant genetic engineering, yet nanotechnology can play an important role in lowering and possibly eliminating these challenges.

… Dendrimer lipids having ionizable amine cores can be applied for LNP formulations for encapsulation and delivery of nucleic acids. Basically consisting of dendrimer ionizable lipids …

… how CRISPR–Cas biomolecules can be introduced into plants … delivery, biolistic (or particle-bombardment)-based delivery, and … (LNP) have been successfully used to deliver mRNA …

Microalgae as the photosynthetic organisms offer enormous promise in a variety of industries, such as the generation of high-value byproducts, biofuels, pharmaceuticals, environmental remediation, and others. With the rapid advancement of gene editing technology, CRISPR/Cas system has evolved into an effective tool that revolutionised the genetic engineering of microalgae due to its robustness, high target specificity, and programmability. However, due to the lack of robust delivery system, the efficacy of gene editing is significantly impaired, limiting its application in microalgae. Nanomaterials have become a potential delivery platform for CRISPR/Cas systems due to their advantages of precise targeting, high stability, safety, and improved immune system. Notably, algal-mediated nanoparticles (AMNPs), especially the microalgae-derived nanoparticles, are appealing as a sustainable delivery platform because of their biocompatibility and low toxicity in a homologous relationship. In addition, living microalgae demonstrated effective and regulated distribution into specified areas as the biohybrid microrobots. This review extensively summarised the uses of CRISPR/Cas systems in microalgae and the recent developments of nanoparticle-based CRISPR/Cas delivery systems. A systematic description of the properties and uses of AMNPs, microalgae-derived nanoparticles, and microalgae microrobots has also been discussed. Finally, this review highlights the challenges and future research directions for the development of gene-edited microalgae. Graphical Abstract

Abstract Insect pests and fungal pathogens are estimated to cause 20–40% yield reduction to crops annually, causing $290 billion of economic loss every season worldwide. Pest and pathogen impacts are a persistent and ever-increasing problem for global food production, especially due to climate change and growing populations. Frequent use of chemical pesticides has resulted in increased resistance among pests and pathogens due to the strong selection pressure that the pesticides exert, resulting in the rapid accumulation of mutations that confer behavioural, mechanical and/or biochemical resistance within the pest populations. Due to rising resistance and increasing interest in control measures with low environmental impact, there is an immediate need to find alternative pest and pathogen management strategies. RNA interference (RNAi) has been developed as a control strategy by exploiting inherent cellular defence processes, providing a species-specific biological approach to crop management. Delivery of double-stranded RNA (dsRNA) can be accomplished non-transgenically by spray-induced gene silencing (SIGS), virus-mediated host-induced gene silencing (VmHIGS) or transgenically through host-induced gene silencing (HIGS), specifically targeting pest and pathogen messenger RNAs with sequence homology. Accomplishing effective RNAi strategies requires consideration into how SIGS, VmHIGS, and HIGS approaches intersect with the crop species and pest or pathogen being targeted. Additional technical advancements for the delivery and uptake of dsRNAs, messenger RNA target identification and the possibility of insect or fungal dsRNA resistance are currently being explored. These considerations will enhance the utility, ease of use and implementation of both spray-based and transgenic applications of RNAi technology for improved food security.

Among the fungal diseases, Fusarium head blight (FHB), caused by Fusarium graminearum, is one of the most destructive disease affecting wheat. This pathogen poses significant threats to global wheat production, leading to substantial yield losses and contaminating grains with harmful mycotoxins. The chemical control of FHB has become increasingly challenging due to the rise of pathogen resistance, environmental concerns, and the effects of climate change. This review introduces a novel approach to disease management through spray-induced gene silencing (SIGS), a cutting-edge technology that uses double-stranded RNA (dsRNA) to silence critical genes in both the fungus and the host plant. This silencing reduces pathogen virulence and enhances plant resilience. A key innovation is the integration of nanotechnology to improve the delivery of dsRNA, addressing challenges related to stability, cellular uptake, and targeting efficiency in field conditions. Nanocarriers have revolutionized dsRNA delivery by improving its encapsulation efficiency, precision, and stability, compared to traditional methods. Advances in cost-effective dsRNA production, particularly through microbial expression systems, enable scalable and sustainable implementation of this technology. This review emphasizes the potential of nanocarrier systems in precision agriculture and highlights their role in replacing harmful chemical treatments with RNA interference (RNAi)-based solutions. RNAi-based approaches not only reduce reliance on synthetic chemicals, but also promote environmental sustainability by addressing fungicide resistance. However, challenges remain in large-scale field application, cost-effectiveness, and regulatory approval processes. Overcoming these hurdles will be crucial to unlocking the full potential of this technology. In conclusion, the combination of nanotechnology and SIGS-based dsRNA delivery offers a groundbreaking approach to managing Fusarium infections in wheat. This innovative strategy has the potential to minimize environmental impacts while enhancing global food security, paving the way for a more sustainable and resilient agricultural future. SIGS technology enhance dsRNA application for green agriculture. Nano capsulation cope key challenges of dsRNA. Innovations in Nanotechnology precisely protect the wheat crop. SIGS technology enhance dsRNA application for green agriculture. Nano capsulation cope key challenges of dsRNA. Innovations in Nanotechnology precisely protect the wheat crop.

Nanocarbon materials have emerged as promising platforms for nucleic acid delivery, bioimaging, and biosensing. Although fullerene derivatives have been employed as delivery carriers, their molecular structures have not been precisely optimized for efficient delivery. Herein, we report the development of a rationally designed cationic perylene derivative, SMZ052, for effective nucleic acid delivery. SMZ052 exhibits delivery efficiencies comparable to those of widely used lipid-based carriers for both plasmid DNA and siRNA. Mechanistic studies have revealed that cellular uptake occurs via cholesterol-dependent pathways including macropinocytosis. Furthermore, SMZ052 enabled the successful transfection of plasmid DNA into tobacco BY-2 protoplasts, demonstrating its applicability in plant science. Our findings highlight the potential of structurally tailored nanocarbon materials as versatile and effective carriers for nucleic acid delivery, offering new avenues for application in plant and animal biotechnologies.

… In this review, we summarized the approaches used for the delivery of the CRISPR/Cas system in mammals, plants, and aquacultures. We further discussed the aspects of delivery that …

Purpose: Nanotechnology has evolved as an effective tool in numerous fields including agriculture, medicine and engineering. Recently it’s potential as an alternative genetic transformation method has been identified. However, a comprehensive understanding over nanoparticles and their behavior in living cells is important to realize the full potential of this technology in biotechnological applications. Therefore, we review the application potential of widely employed nanoparticles in plant transformation here. Literature/Background: Development of new crop varieties with desirable traits via biotechnological applications is a solution for challenges associated with climate change and higher population growth. In such aspects, transformation of plant cells which is known as the process of changing one’s genome by integration exogenous DNA, is an absolute necessity and results far better and improved stable characteristics in original. Rigid and multi layered cell wall impedes penetration of exterior biomolecules and hence causes the transformation process complicated. Even though, numerous conventional methods have been established for plant transformation, lower transformation efficiency, tissue damage and random integration of transgenes warrants the need for novel approaches. In this context, novel techniques have been explored and as a result nanoparticles have been found effective in transformation of protoplasts as well as intact plant cells. Nanoparticles internalized either via endocytosis or direct penetration release transgenes from nanoparticle-DNA complexes and result in transient or stable expression. Nanoparticles ensure higher transformation effi ciency, no transgenic silencing and protection of biogenic molecules from degradation by intracellular nucleases.

… in plant genetic modification by facilitating effective, efficient, and species-unrestricted delivery of DNA, RNA, and protein cargo through the plant … -based gene delivery systems, such as …

The ever-increasing food requirement with globally growing population demands advanced agricultural practices to improve grain yield, to gain crop resilience under unpredictable extreme weather, and to reduce production loss caused by insects and pathogens. To fulfill such requests, genome engineering technology has been applied to various plant species. To date, several generations of genome engineering methods have been developed. Among these methods, the new mainstream technology is clustered regularly interspaced short palindromic repeats (CRISPR) with nucleases. One of the most important processes in genome engineering is to deliver gene cassettes into plant cells. Conventionally used systems have several shortcomings, such as being labor- and time-consuming procedures, potential tissue damage, and low transformation efficiency. Taking advantage of nanotechnology, the nanoparticle-mediated gene delivery method presents technical superiority over conventional approaches due to its high efficiency and adaptability in different plant species. In this review, we summarize the evolution of plant biomolecular delivery methods and discussed their characteristics as well as limitations. We focused on the cutting-edge nanotechnology-based delivery system, and reviewed different types of nanoparticles, preparation of nanomaterials, mechanism of nanoparticle transport, and advanced application in plant genome engineering. On the basis of established methods, we concluded that the combination of genome editing, nanoparticle-mediated gene transformation and de novo regeneration technologies can accelerate crop improvement efficiently in the future.

… deliver various genetic cargo types (DNA, siRNA, miRNA) but this method … delivery can be used for protoplasts only, thus posing a limitation for several crop species in which protoplast …

Nanoparticles (NPs) derived from RNA interference (RNAi) are considered a potentially revolutionary technique in the field of plant protection in the future. However, the application of NPs in RNAi is hindered by the conflict between the high cost of RNA production and the large quantity of materials required for field application. This study aimed to evaluate the antiviral efficacy of commercially available nanomaterials, such as chitosan quaternary ammonium salt (CQAS), amine functionalized silica nano powder (ASNP), and carbon quantum dots (CQD), that carried double-stranded RNA (dsRNA) via various delivery methods, including infiltration, spraying, and root soaking. ASNP-dsRNA NPs are recommended for root soaking, which is considered the most effective method of antiviral compound application. The most effective antiviral compound tested was CQAS-dsRNA NPs delivered by root soaking. Using fluorescence, FITC-CQAS-dsCP-Cy3, and CQD-dsCP-Cy3 NPs demonstrated the uptake and transport pathways of dsRNA NPs in plants when applied to plants in different modes. The duration of protection with NPs applied in various modes was then compared, providing references for evaluating the retention period of various types of NPs. All three types of NPs effectively silenced genes in plants and afforded at least 14 days of protection against viral infection. Particularly, CQD-dsRNA NPs could protect systemic leaves for 21 days following spraying.

… types of nanoparticles for their suitability in dsRNA delivery for … and advantages of different nanoparticle materials, including … dsRNA in the plants compared to the conventional delivery …

The increasing challenges posed by plant viral diseases demand innovative and sustainable management strategies to minimize agricultural losses. Exogenous double-stranded RNA (dsRNA)-mediated RNA interference (RNAi) represents a transformative approach to combat plant viral pathogens without the need for genetic transformation. This review explores the mechanisms underlying dsRNA-induced RNAi, highlighting its ability to silence specific viral genes through small interfering RNAs (siRNAs). Key advancements in dsRNA production, including cost-effective microbial synthesis and in vitro methods, are examined alongside delivery techniques such as spray-induced gene silencing (SIGS) and nanocarrier-based systems. Strategies for enhancing dsRNA stability, including the use of nanomaterials like layered double hydroxide nanosheets and carbon dots, are discussed to address environmental degradation challenges. Practical applications of this technology against various plant viruses and its potential to ensure food security are emphasized. The review also delves into regulatory considerations, risk assessments, and the challenges associated with off-target effects and pathogen resistance. By evaluating both opportunities and limitations, this review underscores the role of exogenous dsRNA as a sustainable solution for achieving viral disease resistance in plants.

… This study focuses on the role of root hairs as key mediators of dsRNA nanoparticle uptake. … -dsRNA NP uptake, and that the integration of RNA into cell walls across the plant allows for …

… Innovative RNA insecticides, as potential green plant protection products, are … delivery system was constructed for the co-delivery of CYP6CY13 double-stranded RNA (dsRNA) and the …

Fungal pathogens significantly impact global crop production, leading to substantial economic losses and threatening food security. Chemical fungicides remain the primary tools for disease management, deployed to control fungal pathogens. The extensive use of conventional fungicides raises concerns over environmental and human health issues and fungicide resistance, creating demand for alternative control methods for fungal diseases in agriculture. Spray-induced gene silencing (SIGS), a non-transgenic RNA interference (RNAi)-based strategy utilizing the topical application of double-stranded RNAs (dsRNAs), has emerged as a promising alternative for sustainable fungal disease management, including in emerging controlled environment agriculture (CEA) systems. SIGS enables gene-specific silencing in pathogens and offers species-specific control with minimal ecological disruption. However, the environmental instability of naked dsRNAs limits field efficacy. Notably, recent advancements in nanocarrier-based delivery systems, such as chitosan nanoparticles, layered double hydroxide clays, and bacterial minicells have enhanced dsRNA stability, uptake, and bioavailability under field conditions, opening new possibilities for the future of crop protection. Despite these innovations, knowledge gaps remain in understanding the environmental fate, degradation kinetics, and off-target risks of more stable dsRNA, including nanocarrier-formulated dsRNAs, especially CEA systems. We highlight the progress in SIGS mechanisms, nanocarrier-enhanced delivery systems, environmental persistence, and biosafety considerations, and outline future research directions for integrating SIGS-based biopesticides into mainstream crop protection strategies.

Nanotechnology-based RNA interference (RNAi) offers a promising approach to pest control. However, current methods for producing RNAi nanopesticides are mainly implemented in a batch-to-batch manner, lacking consistent quality control. Herein, we present a microfluidic-based nanoplatform for RNA nanopesticide preparation using lipid nanoparticles (LNPs) as nanocarriers, taking advantage of the enhanced mass transfer and continuous processing capabilities of microfluidic technology. The dsRNA@LNPs were rapidly formed within seconds, which showed uniform size distribution, improved leaf wettability, and excellent dispersion properties. The delivery efficiency of dsRNA@LNPs was evaluated by targeting the chitin synthetase B (CHSB) gene ofSpodoptera exigua. The dsRNA@LNPs can effectively resist nuclease-rich midgut fluid degradation. Importantly, dsCHSB@LNPs exhibited increased mortality rates, significant reduction of larvae growth, and enhanced gene suppression efficiency. Therefore, a continuous nanoplatform for RNAi nanopesticide preparation is demonstrated by utilizing microfluidic technology, representing a new route to produce RNAi nanopesticides with enhanced quality control and might accelerate their practical applications.

Delivery of exogenous nucleic acid to intact plants is a desirable but challenging technology due to the dominant transport barrier posed by the plant cell wall. Here, we found that three different morphologies of g‐C3N4 nanomaterials synthesized from urea can assist in the delivery of exogenous nucleic acids into mature leaf cells of Nicotiana benthamiana. Among these, g‐C3N4 carbon dots (CDs) showed a higher ability to deliver exogenous nucleic acid compared to nanoporous and g‐C3N4 nanosheets. The delivery ability of exogenous dsRNA and plasmid DNA by g‐C3N4 could also be restrained by stomatal closure and the endocytosis pathways in plant cells. Furthermore, the coupling of g‐C3N4 CDs with dsCP (CDs@dsCP), which is the dsRNA matching a specific fragment of the Coat protein‐encoding gene of TMV, resulted in a superior antiviral effect compared with other morphologies of g‐C3N4@dsRNAs and other loaded dsRNAs, which match Replicase, RNA‐dependent Replicase or Movement protein of TMV. Significantly, a single spray of CDs@dsCP provided virus protection for at least 5 days. In addition, the g‐C3N4 CDs and g‐C3N4 CDs@dsRNA had no adverse effects on plant growth and development. Overall, our study presents a novel biocompatible and convenient tool for gene expression or gene silencing in intact plant leaves by spraying g‐C3N4 nanomaterials encapsulated with DNA or dsRNA, the efficiency of which is affected by the morphology of the g‐C3N4 nanomaterial, stomatal state and plant endocytosis pathway, and a highly promising solution for plant virus disease, which is an unsolved problem in plant disease control.