The process of genetic transformation is generally categorized into two primary types: stable transformation and transient transformation (Miki and Shimamoto, 2004). Stable transformation is a labor-intensive process with relatively low efficiency (Guidarelli and Baraldi, 2015; Vyacheslavova et al., 2012). In contrast, transient transformation offers a simpler and more efficient approach to gene delivery, addressing several limitations of stable transformation, such as low transformation efficiency, difficulties in selecting transformants, and challenges associated with plant regeneration (Toldi et al., 2009; Canto, 2016).

Syringe infiltration is recognized as an efficient technique for transient transformation, appreciated for its simplicity, speed, and versatility (Schöb et al., 1997; Wroblewski et al., 2005; Orzaez et al., 2006). This method involves using a needleless syringe to incorporate molecular cargo into matured intact leaves, making it broadly applicable for various plant biology studies (Santi et al., 2008; Sparkes et al., 2006; Xu et al., 2014; Bhaskar et al., 2009). The syringe infiltration process offers several key advantages, including the ability to introduce a single target gene across an entire leaf or insert multiple genes into different regions of a single leaf, thus enabling the execution of multiple experiments on the single leaf (Vaghchhipawala et al., 2011). Consequently, syringe infiltration has broad applications in plant biotechnology, enabling the evaluation of transient gene expression, gene knockdown, and gene knockout using siRNA, dsRNA, or CRISPR technologies, regardless of the carriers utilized (Přibylová et al., 2022; Kaur et al., 2021).

The delivery of nanoparticle-cargo complexes into leaf tissues begins with the preparation of nanoparticles. These nanoparticles are then mixed with the desired cargos, which may include plasmid DNA, dsRNA, siRNA, or CRISPR constructs, at the appropriate concentration. This mixture is incubated at for 30 min to 1 h to facilitate the conjugation of the nanoparticles with the cargos (Figure 1) (Demirer et al., 2019). After conjugation, the complexes are loaded into a needleless syringe for delivery. Injection into the leaf tissue occurs from the abaxial surface of intact leaves. To improve infiltration efficiency, a small puncture is created on the abaxial surface using a tip or small needle, and the conjugate enters through this puncture (Figure 2). Wipe any leftover solutions on the surfaces of leaves post-infiltration using a Kimwipe and delicately rub the leaf surface. If the leaves are infiltrated with plasmids containing fluorescent reporter genes such as GFP, RFP, and dsRed, it is preferable to transfer these plants in a growth chamber having low intensity light and room temperature instead of placing in a chamber with high-intensity light, as this may lead to photobleaching of the reporter genes. This approach allows for the efficient delivery of the nanoparticle-cargo complex into the leaf tissue (Demirer et al., 2019). Although fewer studies explore syringe infiltration for delivering nanoparticle and cargo complexes into leaf tissues compared to agroinfiltration, this technique holds significant potential for effectively delivering these complexes into plant leaf tissues. For example, the functionalization of quantum dots (QD) with β-cyclodextrin molecular baskets allowed the grafting and transportation of a variety of substances into plant chloroplasts. Nanoparticle coating with a guiding peptide allowed targeted delivery into Arabidopsis chloroplasts through syringe infiltration. Peptide biorecognition provided efficient and specific transport of QDs with chemical payloads into chloroplasts in living plant cells and resulted in a high delivery efficiency of 74.6% ± 10.8% (Santana et al., 2020; Kwak et al., 2019) successfully showed syringe infiltration of chitosan functionalized SWCNTs to deliver genes to chloroplasts in matured intact leaves arugula, watercress, spinach, and tobacco plants. This nanoparticle mediated approach allowed for targeted transgene expression and offered advantages over traditional methods such as Agrobacterium mediated transformation, offering a simpler, more convenient and cheaper alternative. This demonstrates syringe infiltration as a promising method for chloroplast transformation in mature plants and opens the door for plant bioengineering and research. Yong et al. (2022) demonstrated that 40-nm layered double hydroxide nanoparticles, when applied via needleless syringe infiltration, are rapidly absorbed by mature intact tobacco leaf cells and chloroplasts. Their study also elucidated the movement of these nanoparticles through the apoplast and vascular system. Additionally, they found that 40-nm LDH nanoparticles significantly increased the internalization of molecular cargo into tobacco leaf cells, achieving over 70% downregulation of the targeted transgenic mRNA within a single day after infiltration. Lei et al. (2020) synthesized gold nanoparticles (AuNPs) conjugated with branched polyethylenimine and combined them with small interfering RNAs (siRNAs) targeting the NPR1 gene, resulting in AuNPs-siRNANPR1 complexes. These were delivered into fully developed Arabidopsis thaliana leaves using a needleless syringe. The study demonstrated that AuNPs-siRNANPR1 effectively penetrated Arabidopsis cells, leading to an 80% reduction in NPR1 gene expression. Bacteriostatic and ion leakage assays further validated the successful gene silencing of NPR1 in Arabidopsis leaves following treatment with AuNPs-siRNANPR1. Similarly, several studies have employed syringe infiltration technology to deliver nanocarrier complexes into intact leaves (Table 2). Overall, syringe infiltration is a versatile and efficient technique for transient genetic modification and the delivery of nanoparticle-cargo complexes into plant tissues. Its simplicity, effectiveness, and broad applicability make it an essential tool in plant biotechnology, particularly for experiments involving rapid gene expression, gene knockdown, or precise nanoparticle-cargo delivery. Syringe infiltration holds great potential for advancing research in genetic engineering and nanotechnology. However, its use for delivering nanocarrier complexes has been limited to a narrow range of plant species. Expanding its application to a broader spectrum of species, with nanoparticles facilitating the delivery of diverse cargos, should be a priority for future research. Enhancing its applicability for large-scale studies will significantly increase its value in plant biotechnology, driving further advancements in genetic engineering and nanoparticle-mediated delivery systems.

Vacuum infiltration is a versatile method used for transferring recombinant DNA using different carriers into plant tissues through an infiltration-mediated permeation pathway (Yan et al., 2012; Bian et al., 2024). Basically, there are two types of vacuum infiltration techniques available for the delivery of nanocarrier complex into various tissues. In one case the cargoes will be delivered into small tissues like small plants, leaves, pollens by placing the tissues in the infiltration solution containing the nanoparticle/cargo complex which is formed by mixing and incubating the desired concentration of nanoparticles and cargo for specific period of time, together in the large syringe. By drawing the plunger of the syringe, while obstructing its entrance, a negative pressure is created in both the syringe chamber and the infiltrated tissues. Upon unblocking the syringe aperture and releasing the plunger, the pressure in the tissues is effectively balanced by drawing the surrounding fluid into the intercellular gaps (Zheng et al., 2021; O’Leary et al., 2014; Chen et al., 2015; Matsuo et al., 2016). The main benefit of this technique is the cargo can be delivered into varies plant tissues without any specialized instruments, whereas in case of syringe infiltration the cargoes can be delivered only to matures leave. However, this method of vacuum infiltration can be applied only to small plant tissues. In another method of vacuum infiltration cargoes will be delivered into plant tissues using the vacuum pump (Simmons et al., 2009; Ranjbaran and Datta, 2019; Simmons et al., 2012). Plant parts, including flower buds, leaves, and seeds, are submerged in infiltration media containing nanoparticle/cargo complex. The submerged explants are then exposed to negative atmospheric pressure in a closed vacuum chamber. Negative pressure draws air from the submerged interstitial space of the explant. Releasing the vacuum permits nanoparticle/cargo complex in association with the interstitial spaces of the explant (Figure 2). This method allows the delivery of nanocomplex carriers into large plant tissues in contrast to syringe mediated vacuum infiltration. The infiltrated plant tissues are ideally transferred to a controlled environment with a temperature of 29°C and a light cycle of 14 h of light and 10 h of darkness. As similar to the syringe infiltration, few investigations have been reported, showing the successful implication of vacuum infiltration technique in nanoparticle mediated gene delivery compared to agrobacterium mediated gene delivery using vacuum infiltration. For instance, a nanomaterial synthesized by functionalizing polyethyleneimine with carbon dot can able to deliver the plasmid DNA containing GUS gene into mature rice embryo induced callus through vacuum infiltration method (Wang Z.-P. et al., 2020). CNTs carrying plasmid DNA can effectively penetrate plant cell walls, facilitating the expression of reporter genes. This was observed in excised embryos that were vacuum-infiltrated with a CNT-reporter gene complex at a pressure of 500 mm Hg for 5 min (Dunbar et al., 2022). Izuegbunam et al. (2021) demonstrated that the vacuum infiltration method significantly contributed to the successful uptake of a nano-biomimetic carrier containing plasmid DNA and the subsequent transient activation of a reporter gene in the leaves of Arabidopsis, common ice plant, and tobacco, as well as in the developing seed tissues of Arabidopsis, field mustard, barley, and wheat. This technique enabled efficient delivery of plasmid DNA into plant tissues. The study suggests that this nano-biomimetic transformation strategy, utilizing vacuum infiltration, may facilitate stable transformation in crop species without requiring multiple rounds of selection and breeding to eliminate the plasmid nanocarrier, a step often necessary with carbon-based nanocarriers. The transient activation of a reporter gene and effective uptake of the plasmid DNA carrier were also observed in the leaves of Arabidopsis, common ice plant, and tobacco, as well as in the seed tissues of Arabidopsis, field mustard, barley, and wheat. The study utilized vacuum infiltration at a pressure of −0.01 MPa for 1 minute to introduce the pDNA-R-nHA complex into the detached healthy leaves. The infiltration process for the seeds was repeated three times at 15-minute intervals, ensuring consistent delivery of the plasmid DNA (Izuegbunam et al., 2021). Similarly, few studies have employed vacuum infiltration technology to deliver nanocarrier complexes into plant tissues (Table 2). Overall, vacuum infiltration has proven to be a versatile and effective method for delivering nanoparticle/cargo complexes into various plant tissues, in contrast to syringe infiltration, which is primarily effective for gene delivery into only leaf tissues. While vacuum infiltration has been successfully employed for delivering plasmid DNA into plant tissues, there are no reported instances of its use for delivering siRNA or dsRNA, as seen with syringe infiltration. Additionally, syringe-mediated vacuum infiltration has not yet been explored for the delivery of nanocarrier complexes. Nonetheless, this technique holds significant potential, particularly for applications involving small tissues. Future research should investigate the use of siRNA, dsRNA, and CRISPR constructs as cargos, in combination with nanoparticles as carriers, delivered into plant tissues via both vacuum pump-mediated and syringe-mediated vacuum infiltration approaches. Such advancements could significantly expand the application of vacuum infiltration in plant genetic engineering, offering new possibilities for precision gene editing and crop improvement.

This physical method accelerates micro-sized, DNA-coated projectiles into plant cells or tissues under vacuum conditions (Maliga and Tungsuchat-Huang, 2014; Liu Q. et al., 2020). Utilizing gold or tungsten particles, the technique employs spermidine and CaCl₂ for the incorporation of gene of interest (Macklin et al., 1999). The optimal size of the microprojectiles for plant transformation typically ranges from 0.6 to 1.0 μm in diameter (Tian and Seguin, 2004). The biolistic method applies a selective mechanical force to deliver DNA-coated microprojectiles into plant cells, effectively minimizing damage to the surrounding plant tissue (Liu et al., 2019). As the biolistic approach majorly relies on physical force, this technique do not have species constraint and can deliver gene of interest into wide range of crop species (Cho et al., 2004; Zhang et al., 2015). Further, this method is effective across different plant tissues, including leaf tissues, callus, and even seeds. The biolistic method proved to be a rapid and efficient method of generating transgenic plants via transient and stable transformation (Abdallah et al., 2010; Lacroix and Citovsky, 2020). This approach facilitates the incorporation of multiple genes into plan cells at the same time, which could be challenging to other conventional transformation methods. This method is widely accepted in delivering various molecular cargoes into plant tissues such as plasmid DNA, siRNA, dsRNA, protein, and Cas9-RNP complex (Ueki et al., 2009; Klein et al., 1987; Kikkert et al., 2004; Wu et al., 2019). The utilization of the nanoparticle in the biolistic DNA delivery by functionalizing the microprojectiles with nanoparticles and using nanoparticle as substitute of gold or tungsten microcarriers has been attempted in the last decade, for instance, Torney et al. (2007) showed the first successful co-delivery of nanoparticles and DNA in Nicotiana benthamiana cotyledons using a biolistic approach. In this investigation gold-coated MSNs with a pore size of 3 nm and a diameter of 100–200 nm were employed. These MSNs were loaded with plasmid DNA encoding the reporter gene GFP and promoter, and the nanoparticle ends were capped with gold nanoparticles. Following biolistic delivery, the gold nanoparticle caps were removed, releasing a chemical inducer, β-estradiol, in the presence of dithiothreitol in the regeneration media. This release activated transient GFP expression in the cotyledons. Martin-Ortigosa et al. (2014) reported the development and evaluation of a novel platform composed of gold nanoparticles attached to mesoporous silica nanoparticles (Au-MSN) using a biolistic particle delivery mechanism. Specifically, the microprojectile, which consisted of gold nanoparticles functionalized with mesoporous silica nanoparticles (Au-MSN), was employed for this purpose. To demonstrate proof-of-concept, the study showed that Au-MSN with relatively large pore sizes (approximately 10 nm) could effectively carry and release both proteins and plasmid DNA within the same cell. This was achieved after the particles penetrated the plant cell wall through bombardment. This advancement represents the first instance of the biolistic-mediated co-delivery of proteins and plasmid DNA to plant cells using gold-functionalized mesoporous silica nanoparticles. In order to avoid the functionalization of microparticle with nanoparticle and to use nanoparticle alone as microcarrier in the biolistic DNA delivery in plants, Rajkumari et al. (2021) investigated the use of silver nanoparticles as substitute for gold microcarriers for biolistic gene transfer in tobacco. The study compared the efficacy of biolistic transformation using silver nanoparticles (100 nm) and gold microcarriers (0.6 microns) across various concentrations. The silver nanoparticles were delivered using helium pressure of 650 psi and a target distance of 9 cm, while the gold microcarriers were delivered at a pressure of 900 psi and a distance of 6 cm. The highest GUS expression was observed with both the gold microcarriers and silver nanoparticles. Notably, the use of silver nanoparticles resulted in significantly higher transformation efficiency compared to gold microcarriers. This improvement in efficiency also led to a substantial reduction in the cost of consumables, with expenses decreasing by up to 37.5 times. Additionally, in the biolistic DNA delivery method, relatively little attention has been given to the critical steps of DNA binding to and release from gold particles, both of which are essential for the success of the delivery process. A crucial element in these processes is the use of cationic molecules, as their positively charged amine groups enable electrostatic binding between negatively charged gold particles and DNA. Although various molecules contain amine groups, spermidine is almost universally used in biolistic protocols (Fadeev et al., 2006; Wang and Frame, 2009). Despite its widespread use, the formation of the DNA-gold complex has not been thoroughly investigated. Moreover, spermidine is expensive and cannot be synthesized under typical laboratory conditions, which adds to the cost and complexity of biolistic DNA delivery. Therefore, future research should focus on evaluating the efficiency of amine-functionalized nanoparticles as a potential alternative to spermidine for the effective binding of DNA to microcarriers. Additionally, studies should explore the use of amine-coated nanoparticles and silver nanoparticles, as suggested by Rajkumari et al. (2021) as cost-effective replacements for spermidine and gold microparticles, respectively (Figure 3). These advancements could significantly reduce the expenses associated with biolistic DNA delivery technology.

Magnetofection is a technique of incorporating exogenous nucleic acid into plant cells using a magnetic field (Gong et al., 2021; Wang B. et al., 2020). This method uses MNPs or MONPS, usually made up of Fe3O4, with sizes ranging from 5 nm to 14 nm in diameter (Alonso et al., 2013; Ludwig et al., 2017). The exogenous nucleic acid-coated magnetic nanoparticles are incubated with plant cell suspension to allow the nanoparticle to associate with the cells. Applying the magnetic field strength ranging from 0.1 to 1 T for a duration of 10 min to 1 h facilitates efficient uptake of exogenous nucleic acid-coated magnetic nanoparticles into plant cells (Figure 4). However, the strength and duration of the magnetic field greatly depend on plant species and type of nanoparticles (Wang et al., 2022b). Magnetofection is an effective method for stable pollen transformation, often called as pollen magnetofection, which involves the direct delivery of exogenous nucleic acid-coated magnetic nanoparticles into pollen grains to attain genomic edits transmitted directly to their progeny without involving the regeneration process (Park et al., 2024b). For instance, Zhao et al. (2017) introduced an innovative transformation platform known as pollen magnetofection, which enables the direct production of transgenic seeds without the need for regeneration. This method involves using a magnetic field to deliver magnetic nanoparticles carrying exogenous DNA into pollen. When plants are pollinated with this magnetofected pollen, transgenic plants are directly produced from the transformed seeds. The exogenous DNA is successfully integrated into the plant genome, expressed, and stably passed on to subsequent generations. Wang et al. (2022) presented an innovative, culture-free, and genotype-independent method for maize transfection. To improve DNA delivery efficiency while maintaining high pollen viability, MNPs coated with plasmid DNA encoding the reporter genes like RFP, GUS, EGFP, or the bialaphos resistance gene were introduced into maize pollen grains using the magnetofection technique. After pollination of female florets from maize inbred lines with the magnetofected pollen, red fluorescence was observed in 22% of the transfected pollen grains, and GUS staining was detected in 55% of embryos 18 days after pollination. This method demonstrates the potential for efficient gene delivery in maize, offering a promising tool for plant genetic engineering. Zhang et al. (2023) introduced an optimized transient transformation method using pollen magnetofection in Lilium regale. The study achieved the highest pollen germination rate (85.73%) and transformation efficiency (88.32%) with a specific ratio of nanomagnetic beads to DNA plasmid and a short transformation time. Transgenic seedlings showed strong GUS activity, indicating successful gene transfer. However, transformation efficiency varied among lily germplasm, suggesting species or cultivar specificity in the effectiveness of this method. George (2018) explored the use of magnetofection as a transformation system for tobacco mesophyll protoplasts, optimizing conditions and assessing the impact on protoplast viability. By applying a strong external magnetic field, magnetofectins were directed towards protoplasts, achieving transient GUS gene expression 48 h post-transfection. The highest transformation efficiency was observed with MNP weight ratios of 1:5 and 1:10. However, increasing the density of MNPs negatively impacted protoplast viability. An optimal MNP density of 1.0 μg/mL was identified, which supported gene expression without compromising protoplast viability. Farooq et al. (2022) reported the synthesis of green iron nanoparticles using Camellia sinensis leaf extract and iron chloride. These nanoparticles were employed to develop a novel method for gene transformation in okra (Abelmoschus esculentus) plants, incorporating various magnetofection factors. The study found that the highest gene transformation efficiency was achieved with a DNA-to-iron nanoparticle ratio of 1:20, by rotating the mixture of plasmid DNA, iron nanoparticles, and seed embryos at 800 rpm for 5 h during the magnetofection process. This method successfully facilitated the transformation of the GFP gene in okra plants, highlighting the effectiveness of this approach for plant genetic engineering. Overall, the delivery of nanoparticle-cargo complexes into plant tissues through magnetofection technology shows great promise for achieving both transient and stable gene expression. However, this gene delivery technique has predominantly been used to introduce genes of interest into pollen, with limited studies exploring its application in other plant tissues, such as protoplasts and seeds. Therefore, future research should focus on expanding the use of magnetofection to deliver cargos into various plant tissues, including protoplasts, seeds, and embryos. Moreover, successful magnetofection requires careful optimization of several parameters, including the size and coating of magnetic nanoparticles, the strength of the magnetic field, and the duration of exposure. Fine-tuning these factors is essential to enhance the efficiency and reliability of this technology for broader applications in plant biotechnology.

Ultrasound-based delivery is an innovative technique for introducing genetic material into protoplasts or intact plant cells using high-frequency sound waves (Choudhary and Chin, 1995). Sonication induces acoustic cavitation, producing transient pores in cell membranes, which facilitates the entry of DNA or RNA into the cells. In this process, plant parts are mixed with exogenous nucleic acids in an appropriate buffer or carrier solution. The entire suspension is then exposed to ultrasound waves at various frequencies for a certain duration, minimizing excessive damage to plant cells. This technique has been utilized in nanoparticle-mediated gene delivery to increase the efficacy of gene transfer (Lo et al., 2014) (Figure 5). When the nanoparticle and cargo complex solution is subjected to ultrasound waves, tiny pores form on the cell surfaces, leading to the internalization of the nanocarrier complex into the plant tissues. In comparison to direct DNA delivery methods, sonication (ultrasound) provides a more straightforward and cost-efficient approach that is not limited by plant species. However, it may lead to cell damage or rupture. Thus, optimizing ultrasound conditions is essential to improve DNA uptake without harming the cells. Studies have shown that moderate ultrasound irradiation of 20 kHz–50 kHz is an effective method for transfection, both in vitro and in vivo (Liu et al., 2006). Particularly in gene delivery using sonication technology, it is crucial to prevent DNA degradation caused by ultrasound exposure (Sanzari et al., 2019). This can be achieved by encapsulating or loading the genetic material onto a suitable carrier. At present, a range of nanoparticles, including metal oxides, polymers, and carbon or silica-based nanomaterials, are used as nano-carriers (Li et al., 2019). Sonication enhances the efficiency of cargo delivery by promoting the internalization of the nanocarrier complex through cavitation in plant cells. As a result, the process is mutually beneficial. Zolghadrnasab et al. (2021) developed a highly efficient method for gene transfection in plant suspension cells by employing pDNA-loaded PEI-MSNs in combination with ultrasonic treatment. The results demonstrated that PEI-MSNs could adsorb a significant amount of DNA. When assessing the impact of ultrasonic treatment on the viability of tobacco plant cells, it was found that increasing the ultrasonic intensity from 160 W to 320 W, and then to 640 W, led to substantial cell damage, while extending the duration of the treatment caused less severe degradation. The evaluation of gene transfection efficiency under various conditions indicated that the use of PEI-MSNs as a gene carrier was essential for effective gene delivery into plant cells. Additionally, the incorporation of ultrasonic treatment had a synergistic effect, significantly enhancing gene transfection efficiency. Amani et al. (2018) explored the potential of cationic polyamidoamine (PAMAM) dendrimers, specifically hyperbranched PAMAM (hPAMAM)-G2 with a diethylenetriamine core, as nanocarriers for gene delivery. They assessed the capacity of these dendrimers to bind with DNA, shield it from ultrasonic damage, and facilitate its delivery into alfalfa cells. The study found that without the aid of nanocarriers, naked DNA was unable to penetrate the cell walls and membranes of alfalfa cells, likely due to its morphology, size, and charge. However, when sonication was applied in conjunction with hPAMAM dendrimers, it facilitated the successful delivery of DNA into the cells, highlighting the critical role of sonication in enhancing the efficiency of nanocarrier-based gene delivery. Sangwan et al. (2023) developed 60 nm core–shell piezoelectric nanoparticles, specifically barium titanate nanoparticles (BTNPs) coated with a silica shell, for efficient nucleic acid delivery into monocot and dicot suspension cells using sonoporation. The study optimized ultrasonic treatment times 5 min for dicots and 8 min for monocots at a constant intensity of 160 W and frequency of 40 kHz, ensuring cell viability. BTNPs demonstrated a DNA adsorption capacity of 40.1 μg per mg. GUS analysis showed significant enhancements in transfection efficiency, with increases of 56.9% and 72.6% in dicot and monocot cells, respectively, compared to naked pDNA delivery. This approach shows promise for genotype-independent transfection. Overall, the use of nanoparticle-mediated gene delivery into plant cells via sonication is a promising and straightforward technique that enhances the efficiency of nanocarrier complex uptake. Nanocarriers protect DNA from degradation, while sonication facilitates cavitation, promoting the efficient internalization of the nanocarrier complex. However, despite its potential, the application of sonoporation technology in nanoparticle-mediated gene delivery remains limited. Most studies have focused on plasmid DNA, leaving a gap in research on the use of other genetic materials such as siRNA, dsRNA, and CRISPR constructs. Future investigations should explore these materials with nanoparticle-mediated delivery and sonication to broaden the applicability and effectiveness of this gene delivery approach.

This is an innovative approach that involves the integration of exogenous DNA into plants through the application of a spray with a transformation solution. In spray method generally genetic material is blended with carrier solution containing suitable surfactants or adjuvants to enhance the uptake of genetic material (Wang and Jin, 2017a; Rank and Koch, 2021). The transformed solution is applied to the plant surfaces using a standard spray bottle or atomizer. The surfactants soften the lipid bilayer and open pores for internalization of genetic material (Thagun et al., 2022; Wang and Jin, 2017b). While in nanoparticle mediated gene delivery using the spray method, initially the nanoparticle solution would be mixed with the desired concentration of cargos and the mixture is mixed well and subject to the incubation to form a nanoparticle and cargo complex. Then the conjugate would spray to the plant surface, enabling their absorption through stomata or cuticular pores (Figure 6). After being absorbed by the plant, nanoparticles go through the walls and membranes of plant cells, releasing cargo that becomes part of the plant’s cellular machinery, after spraying the formulation the plant should ideally transferred to the growth chamber having the temperature of 25°C. This process has the capability to activate expression of gene of interest in the plant (Mitter et al., 2017). As it is a non-invasive technique and easier to use in a wide range of crop species, this method can be applied to whole plants or large areas of plants, which is helpful for field trials and large-scale applications. This method is used to deliver various molecular cargoes into plant tissues such as dsRNA and siRNA to achive gene knock down in combination with the nanoparticle sinstead of exogenous application od DsRNA or siRNA. Although earlier studies suggested that naked RNAs applied externally could effectively silence genes in plants (Dubrovina et al., 2019), more recent research has demonstrated that this approach is generally ineffective. The likely reason for this ineffectiveness is the negative charge of RNA molecules, which is due to their phosphate groups. This negative charge makes it difficult for RNA to cross the plant cell’s plasma membrane, which is also have a negative charge. This barrier highlights the need for alternative delivery strategies, such as nanoparticle carriers, to increase the molecular cargo uptake and achieve effective gene knockdown in plants (Zhang et al., 2021; Zhang et al., 2022). Nanoparticle/cargo2 complexes with neutralized surfaces have been shown to effectively silence genes, likely due to their capability to cross the plasma membrane. These complexes have demonstrated high efficacy in the knockdown of gene of interest within living plant cells, making them a promising tool for gene regulation (Azeem et al., 2021; Demirer et al., 2021). Therefore, the application of dsRNA or siRNA using nanoparticle as carrier has been using as powefull method of gene regulation using different insertion techniques, more importantly using spraying technique. For instance, Functionalized carbon dots were complexed with screened double-stranded RNAs (dsRNA-CDs) through electrostatic interactions, facilitating spray-induced gene silencing to control Phytophthora species. The spray application of dsRNA-CD complexes substantially increased the control efficiency against Phytophthora infestans, Phytophthora sojae, and both wild-type and fungicide-resistant P. capsici (Wang et al., 2023). In another investigation, a spray technique was used to deliver layered double hydroxide (LDH) nanoparticles complexed with dsRNA to Nicotiana benthamiana leaves. The virus-specific dsRNA-LDH complexes, applied through spraying, successfully activated the plant’s RNA interference (RNAi) machinery, reducing the accumulation of the Tomato Yellow Leaf Curl Virus genome and mitigating symptom development (Hernandez, 2021). Polymer-functionalized graphene oxide nanoparticles (GONs) were also utilized in a spray method to deliver siRNA into intact leaf cells. The spray-applied GON-siRNA complexes penetrated the cell wall and internalized within the plant cells, achieving short-term but highly effective gene silencing, with 97.2% efficiency observed 24 h post-application (Li et al., 2022). Furthermore, Cai et al. (2024) developed a novel spray-based approach using MSNs for siRNA delivery into intact plant leaves. This spray technique achieved up to 98% gene knockdown efficiency at the molecular level without the need for mechanical forces. The method proved to be non-toxic to plant leaves, allowing for repeated siRNA applications for sustained gene silencing. Similarly, several studies have employed spray based technique to deliver nanocarrier complexes into intact leaves (Table 2). Nanoparticle-mediated cargo delivery via the spray method has emerged as one of the most efficient techniques for applying nanocarrier complexes, particularly in achieving gene regulation. However, the efficiency of this spray-based genetic transformation varies depending on the plant species, tissue type, and the formulation of the genetic material used (Sembada and Lenggoro, 2024). While this method is predominantly used to deliver dsRNA and siRNA using nanoparticles as carriers for gene regulation, there remains a need for future research to explore the delivery of CRISPR constructs through nanoparticle-based spray applications.

This approach leverages the natural mechanisms to introduce the exogenous nucleic acid into plant tissue without the aid of external forces. In contrast to external force-assisted gene delivery, passive gene delivery typically involves plant system routes such as leaves and roots for the introduction of nucleic acids (Sembada and Lenggoro, 2024). To carry exogenous nucleic acids, nanoparticles must be compatible with the pore sizes of stomata and root hairs (Figure 6). The ideal size of the nanoparticles used in plant passive transformations ranges from 10 to 40 nm in diameter. The passive gene transformation takes the symplastic routes for the uptake and further translocation of nanoparticles in plant cells. Nanoparticle particles such as oxides of Zn, Au, Ce, Ti, and exclusive carbon are widely employed in passive gene transformation. Due to their microscopic size, nanoparticles are readily absorbed by roots or leaves and taken into the plant vascular route to deliver the exogenous DNA into plant cells without damaging the cell. Generally the passive gene transformation works efficiently where plants require delivering exogenous nucleic acid into plant cells non-invasively (Pal et al., 2024). The study found that plant cell walls can block the entry of particles larger than 5–20 nm without needing any external force (Cunningham et al., 2018; Wang Z.-P. et al., 2020), tested whether 5 nm carbon dots could penetrate walled plant cells and carry a DNA molecule without external aids like a gene gun or ultrasound. Rice Nipponbare plant root tips were soaked in a mixture of carbon dots and plasmid to assess cellular uptake. After 12 h, confocal microscopy showed strong mCherry fluorescence in nearly all root tip cells, indicating that the nanoparticle-plasmid DNA complex can easily pass through the plant cell wall without external assistance, validating the passive diffusion mechanism. Currently, protoplasts are utilized to accelerate plant genetic screening and to facilitate the production of recombinant proteins. This necessitates a transformation platform that is user-friendly and an efficient delivery method that 1can be adapted to various species (Schaumberg et al., 2016). For this purpose, (Demirer et al., 2019) utilized intact, healthy protoplasts derived from arugula leaves to deliver plasmid DNA encoding a nuclear localization signal (UBQ10-GFP), which directs the expressed GFP from the cytosol to the nucleus. They employed carbon nanotubes as carriers to facilitate DNA delivery through a passive diffusion mechanism without the use of any external force. The study demonstrated a protoplast transformation efficiency of 76%. They demonstrated that nanoparticles like carbon nanotubes can swiftly enter plant plastids, with their penetration through the lipid layer happening within seconds of exposure. This nanoparticle-based approach facilitates the rapid and passive delivery of plasmid DNA into protoplasts, achieving high transgene expression efficiency without any noticeable adverse effects on the viability of the protoplasts. Moreover, the use of nanoparticles as carriers has been successfully applied in plants for the delivery of RNA molecules aimed at specific gene knockdown. For instance, (Pal et al., 2024) assessed the effectiveness of dsRNA-induced gene silencing in rice, targeting the phytoene desaturase (PDS) gene using a seedling dip-inoculation method. Three-day-old seedlings were exposed to either naked pds-dsRNA or pds-dsRNA combined with a cationic poly-aspartic acid-derived polymer (CPP6), allowing absorption through the roots. Ten days post-treatment, seedlings exposed to pds-dsRNA showed reduced growth and appeared pale or white. The height of seedlings treated with pds-dsRNA-CPP6 was significantly reduced compared to those treated with CPP6 alone and the control group. This highlights the effectiveness of the cellular uptake method in silencing the targeted gene. This method is used effectively to deliver dsRNA to various plant species, such as Arabidopsis and Cucumber (Pal et al., 2024; Delgado-Martín et al., 2022). Similarly, several studies have employed this technique to deliver nanocarrier complexes into plant tissues (Table 2). Overall, the nanoparticle mediated gene delivery into plant tissues through passive diffusion or cellular uptake mechanism is cost effective and straightforward approach (Sembada and Lenggoro, 2024). However, this method also encounters several challenges, including the size variation in the nanoparticle depending upon pores, channels, or structures within the plant tissue.