Measuring Plasmodesmata‐mediated Intercellular Trafficking Using Microparticle Bombardment in Arabidopsis and Crops

Abstract

Plasmodesmata (PD) are highly specialized, nanoscopic pores that traverse the cell wall to connect the cytoplasm of adjacent plant cells, enabling direct cell‐to‐cell communication. PD provides the continuity of three key cellular components: the plasma membrane, the endoplasmic reticulum (ER), and the cytosol. The compressed ER within PD is known as the desmotubule. PD mediates the intercellular trafficking of ions, metabolites, hormones, proteins, and RNA molecules between adjacent cells. Although several methods have been developed to quantify PD‐mediated molecular trafficking, it remains a technical challenge. Among these, PD‐mediated movement of fluorescent proteins is one of the most commonly used approaches. Here we present a microparticle bombardment method using a biolistic particle delivery system to investigate the PD‐mediated movement of fluorescent proteins. We equipped the delivery system with a flow guiding barrel to improve bombardment efficiency and consistency. We demonstrated the effects of gold particle aggregation and plant age on transformation efficiency and protein movement in Arabidopsis. We also showed the feasibility of the method in determining PD‐mediated movement in tomato, pepper, and soybean. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC.

Basic Protocol: Microparticle bombardment assay for measuring plasmodesmata‐mediated trafficking

INTRODUCTION

Plasmodesmata (PD) are plasma membrane‐lined pores that traverse cell walls, containing a central endoplasmic reticulum (ER)‐derived structure known as the desmotubule. By mediating the intercellular trafficking of ions, metabolites, proteins, and RNA molecules, PD play critical roles in plant growth, development, and stress responses (Bayer & Benitez‐Alfonso, 2024; Tee & Faulkner, 2024). PD permeability is tightly regulated by developmental cues and environmental signals, ensuring local cell coordination and maintaining overall plant fitness (Alazem & Burch‐Smith, 2024). Due to the nanoscale dimensions of PD and the dynamic regulation of molecular trafficking, determining PD‐mediated intercellular movement remains a significant experimental challenge.

Monitoring the movement of symplasmic fluorescent probes between cells is a proxy for measuring PD‐mediated intercellular trafficking. These probes fall into two main categories: small fluorescent tracer molecules and fluorescent proteins. A major technical hurdle lies in the delivery of small fluorescent tracer molecules or the expression of fluorescent proteins in plant cells. Early efforts involved the microinjection of fluorescent dyes, such as fluorescence‐labeled dextrans of various sizes, into single mesophyll cells to determine the size exclusion limit and assess PD permeability (Kragler, 2015). Successful microinjection requires experience and skill, and has a high failure rate due to the limited cytosolic space. Localized loading of fluorescent tracers, including dextran and 8‐hydroxypyrene‐1,3,6‐trisulfonic acid (HPTS), was used to study the PD transport capacity of cells during different stages of embryonic development (Kim et al., 2002; Xu et al., 2012). Another fluorescent dye, carboxyfluorescein diacetate (CFDA), is widely used to determine PD‐mediated intercellular molecular flow. CFDA, a nonfluorescent, cell‐permeant compound, can be cleaved by intercellular esterases, yielding the fluorescent but membrane‐impermeable probe carboxyfluorescein (CF). The movement of CF between cells can be detected to measure PD‐mediated trafficking. This dye has been used in Drop‐ANd‐See (DANS), CFDA feeding, and fluorescence recovery after photobleaching (FRAP)‐based CF mobility assays. DANS was designed to test overall PD permeability in leaves by dropping CFDA on the adaxial side and detecting CF signals on the abaxial side (Lee et al., 2011; Wang et al., 2020). CFDA feeding measures CF unloading and diffusion in the root tip after CFDA loading on the cotyledons of seedlings. This method can be used to test long‐distance symplasmic transport and local intercellular trafficking (Huang et al., 2019; Li et al., 2024). The CF mobility assay, measured by FRAP, is used to determine PD permeability at the cellular level (Perez‐Sancho et al., 2025). Additionally, photoactivable caged fluorescein enables the in situ activation of fluorescence in a single cell, and the subsequent diffusion of the signal to neighboring cells is monitored (Ayyoub et al., 2024).

Fluorescent proteins are utilized to measure macromolecule movement through PD. Green fluorescent protein (GFP) can be transiently expressed in single cells via microparticle bombardment or Agrobacterium tumefaciens‐mediated transformation. In microparticle bombardment, GFP‐expressing plasmid DNA is coated onto gold particles and delivered into plant cell nuclei (Aung et al., 2017; Cheval et al., 2020; Fernandez & Burch‐Smith, 2022; Fernandez et al., 2025; Tee et al., 2022). Agrobacterium‐based methods involve infiltrating plasmid‐bearing Agrobacterium at a low inoculum into the leaves of highly susceptible plants, such as Nicotiana benthamiana (Brunkard et al., 2020; Horner & Brunkard, 2022; Li et al., 2021). Both methods generate isolated transformed single cells, and the diffusion of GFP from the transformed cells to their neighboring cells is detected to determine PD‐dependent permeability. GFP can also be expressed under cell‐type or tissue‐specific promoters in transgenic plants, which can be used to visualize the PD‐mediated movement of molecules at defined cell interfaces and reveal the directionality of intercellular trafficking (Yan et al., 2019; Li et al., 2024; Jacquier et al., 2024). Engineered variants of fluorescent proteins, including photoactivable GFP (PaGFP) (Nicolas et al., 2017), photoswitchable GFP (DRONPA‐s) (Gerlitz et al., 2018; Perez‐Sancho et al., 2025), and photoconvertible GFP (Dendra 2) (Kitagawa & Fujita, 2013), are also powerful tools to study the dynamics of PD transport at the cellular level. Fluorescent signals can be activated in single cells, and the loss of signal in the activated cells and/or the gain in the neighboring cells can be detected through time‐lapse imaging to monitor real‐time intercellular trafficking. Each of the approaches described above has distinct advantages and limitations. The selection of the most appropriate method should be guided by the desired resolution of the analysis and the specific cell type, tissue, or species being studied.

Here, we present a detailed protocol for a microparticle bombardment assay using a biolistic particle delivery system equipped with a flow guiding barrel. Biolistic plant transformation and genome editing efficiencies are significantly enhanced by using the flow guiding barrel (Thorpe et al., 2025). Although the microparticle bombardment assay is confined to epidermal tissues, it enables direct DNA delivery, making it broadly applicable across both model and non‐model plant species. As the technique is somewhat specialized, attention to certain technical details is essential for successful implementation. We highlight how gold particle quality and leaf developmental stage influence transformation efficiency and GFP movement in Arabidopsis. Additionally, we demonstrate the applicability of the method in assessing PD‐mediated GFP diffusion in other plant species, such as tomato, pepper, and soybean. These insights provide valuable guidance for troubleshooting and optimizing the assay in diverse plant systems.

MICROPARTICLE BOMBARDMENT ASSAY FOR MEASURING PLASMODESMATA‐MEDIATED TRAFFICKING

This protocol details a microparticle bombardment assay for assessing PD permeability. We employ a biolistic system fitted with a flow guiding barrel (Fig. 1), which significantly enhances transformation efficiency and consistency (Thorpe et al., 2025). The steps include preparing reagents for particle coating, loading microcarriers, performing bombardments, acquiring confocal images, and quantifying GFP movement. Critical considerations, such as gold particle quality and leaf developmental stage, are addressed in the Critical Parameters and Troubleshooting sections to optimize assay performance.

Figure 1.

Overview of PDS‐1000/He biolistic particle delivery system equipped with a flow guiding barrel. (A) A schematic diagram demonstrating the mechanics of the biolistic procedure. (B) Photographs of the physical setup show the components of the bombardment system.

Materials

• Plant materials: Arabidopsis thaliana (Col‐0), Solanum lycopersicum (Sun Gold cherry tomato), Capsicum annuum (Shishito pepper), and Glycine max (Edamame) were grown at 22°C with 50% relative humidity under 110 µmol m⁻² s⁻¹ white fluorescent light on a 16‐hr light/8‐hr dark cycle. Arabidopsis leaves (3 or 4 weeks old) and tomato, pepper, and soybean leaves (6 weeks old) were used for microparticle bombardment.

• Plasmids: pL2M‐eGFP‐NLStdTomato (Reyes Caldas et al., 2022)

• 0.6‐µm gold particles (Bio‐Rad, cat. no. 1652262)

• 2.5 M calcium chloride (see recipe)

• 0.1 M spermidine (see recipe)

• 0.6% Agar plates (see recipe)

• 100% ethanol (200 proof)

• 70% ethanol

• ddH₂O

• PDS‐1000/He System (Bio‐Rad, cat. no. 1652257) and a vacuum pump

• 450 psi rupture disks (Bio‐Rad, cat. no. 1652326)

• Macrocarrier (Bio‐Rad, cat. no. 1652335)

• Macrocarrier Holder (Bio‐Rad, cat. no. 1652322)

• Stopping screens (Hermes Biomaterials Inc.)

• Flow guiding barrel (Hermes Biomaterials Inc.)

• Metal or plastic mesh

• 1.5‐ml centrifuge tubes (Eppendorf, cat. no. EP0030108051)

• 250‐ml glass bottle (Fisherbrand, cat. no. FB800250)

• 1‐L glass bottle (Fisherbrand, cat. no. FB8001000)

• Pipette tips (RAININ)

• Petri dishes (Fisher, cat. no. FB0875713A)

• Centrifuge (VWR, cat. no. 76019‐132)

• Sonication bath (Fisher Scientific, cat. no. 22‐066282)

• Vortex mixer (Millipore Sigma, cat. no. CLS6775)

• FIJI/ImageJ (version: 2.0.0‐rc‐68/1.52e)

• 1Pour 70% ethanol into Petri dishes for surface sterilization of the stopping screens and rupture disks. Soak the stopping screens for 5 min and rupture disks for 2 min in ethanol. Remove disks and stopping screens from the ethanol with tweezers and place them into empty Petri dishes for drying (lean them against the wall of the Petri dish).
• 2Autoclave the flow guiding barrel or sterilize with 70% ethanol.
• 3Switch on the vacuum pump of the gene gun for half an hour to reach a pressure of 27–28 in. Hg before starting biolistic transformation to ensure a thorough vacuum during experiments.

• 4Cut leaves from plants and place abaxial side up on 0.6% agar plates. Cover the plates with lids until bombardment.

  To ensure consistency across genotypes and treatment conditions, use leaves at the same developmental stage from plants grown under the same conditions.

  For Arabidopsis, arrange 4 to 10 leaves in a circular pattern on the plate, avoiding the center if possible, as shown in Figure 1.

• 5Weigh 10 mg gold particles into a 1.5‐ml centrifuge tube.
• 6Add 1 ml ddH₂O to the stock centrifuge tube to make 10 mg/ml gold particles.
• 7Ultrasonicate and vortex the gold stock tube rigorously for 1 min each until the gold particles are well suspended.

  Avoid using aggregated gold particles that cannot be well‐dispersed.

• 8Add 100 µl of 10 mg/ml gold stock solution to a new 1.5‐ml centrifuge tube.
• 9Add 40 µl of 250 ng/µl plasmids to the tube (total 10 µg).

  The plasmids were isolated and purified from bacterial cultures using the Midiprep kit.

• 10Add 50 µl of 2.5 M calcium chloride.
• 11Quickly add 20 µl of 0.1 M Spermidine and vortex for 30 s.

  Freshly prepared spermidine solution is recommended.

• 12Centrifuge at 2490 × g and room temperature for 1 min.
• 13Remove 210 µl supernatant.
• 14Resuspend in 100 µl of 100% ethanol.
• 15Ultrasonicate for 15–30 s and vortex for 15–30 s to ensure the gold particles are resuspended well.
• 16Centrifuge down at 2490 × g and room temperature for 1 min.
• 17Remove 100 µl supernatant.
• 18Resuspend in 100 µl of 100% ethanol.
• 19Ultrasonicate the resulting gold/DNA pellet quickly until it is resuspended well.
• 20Immediately pipette 10 µl gold‐DNA solution onto the center of each macrocarrier.
• 21After every three macrocarriers, re‐vortex the gold‐DNA tube to ensure a consistent mixture and pipette onto the remaining macrocarriers.
• 22Let air dry or vacuum dry for 5–10 min until completely dry.

• 23Set helium pressure on the tank regulator to 900–1100 psi.
• 24Place one 450 psi rupture disk into the recess of the rupture disk retaining cap, and screw the retaining cap onto the end of the gas acceleration tube.

  Both low‐ and high‐pressure bombardment have been employed to assess PD‐mediated trafficking of fluorescent proteins. Low‐pressure delivery (e.g., 60 psi; Fernandez & Burch‐Smith, 2022) is thought to minimize mechanical stress on leaves, thereby reducing PD closure due to the bombardment. In contrast, high‐pressure bombardment (1100–1350 psi) has also been effectively used to investigate PD function, both with and without elicitor treatments that induce PD closure (Aung et al., 2020; Faulkner et al., 2013; Tee et al., 2023). In this study, we used an intermediate pressure of 450 psi to deliver plasmid DNA into plant cells.

• 25Prepare microcarrier launch assembly: insert the flow guiding barrel into the launch shelf; place a stopping screen on the top of the flow guiding barrel; install the macrocarrier holder containing the loaded macrocarrier on the top rim of the fixed nest; then place the macrocarrier cover lid on the assembly and tighten.

  The flow guiding barrel can significantly enhance delivery efficiency and consistency (Thorpe et al., 2025).

  The dry macrocarriers should be facing down towards the stopping screen.

• 26Place the microcarrier launch assembly in the top slot in the bombardment chamber.
• 27Use metal or plastic mesh to cover the leaves. Place the target shelf at the desired level and place the leaf sample plate on it.

  In our setup, the distance between the microcarrier launch assembly and the target shelf was approximately 9 cm.

• 28Close and latch the chamber door.
• 29Set the vacuum control switch on the main unit control panel to the VAC position.
• 30Evacuate the chamber to the desired level (27–28 in. Hg). The FIRE switch on the right side of the panel will be illuminated once the chamber is under vacuum. Hold the vacuum by quickly pressing the vacuum control switch through the middle VENT position to the bottom HOLD position.
• 31Press and hold the FIRE button continuously until the rupture disk bursts and the helium pressure gauge drops to zero. Release the FIRE button immediately to avoid wasting helium.

  A small pop will be heard when the rupture disk bursts. Note that the helium pressure gauge only acts as a guide, while the actual release pressure is determined by the rating of the rupture disk used.

• 32Release the vacuum from the chamber by setting the vacuum control switch to the middle VENT position.
• 33Open the chamber door and remove the leaf sample plate from the chamber. Put the lid back on the plate.
• 34Unscrew the lid and remove the macrocarrier holder. Unload and discard the macrocarrier and the stopping screen.
• 35Unscrew the retaining cap and remove the remains of the rupture disk.
• 36Repeat steps 23–35 for the next sample.
• 37After completing all bombardments, release the residual pressure from the system: close the main valve on the helium cylinder; close and latch the chamber door; draw 5 in. of mercury; activate the FIRE button; then set the vacuum control switch to VENT.
• 38Cover the sample plates with aluminum foil and leave them on the lab bench until imaging.

  We typically image samples 24 hr post‐bombardment. Chemical treatment can be applied 4 hr post‐bombardment by transferring the leaves to medium supplemented with the desired elicitors (e.g., hydrogen peroxide or 22‐amino‐acid flagellin peptide).

• 39Prepare the samples for imaging: cut the leaf into two halves and remove the midrib; mount each half leaf in water on a glass slide; cover the samples with a coverslip; then press the coverslip gently using the tip of a tweezer or scissors to flatten the tissue.

  A flat tissue allows for better imaging, which can capture all cells containing the GFP signal at each bombardment site.

• 40Imaging: place the sample under a confocal microscope; use 488 and 555 nm to excite GFP and tdTomato, respectively; use SP 555 and SP 640 emission filters for GFP and tdTomato, respectively; use the ocular lens with 10× magnification to scan the entire tissue to locate the bombardment sites; then capture images showing both GFP and tdTomato signals for each site.

  Nuclear red fluorescent signals from NLStdTomato expression can be used to locate the bombardment sites, which are also characterized by a single strongly GFP‐expressed cell surrounded by neighboring cells with relatively weak signals (Figs. 2, 3, and 4). Ensure that you include every cell showing GFP signal at each bombardment site. We found that collecting Z‐section images is not necessary for reliably quantifying PD‐mediated trafficking.

Figure 2.

The effect of gold particle aggregation on transformation efficiency and protein movement in Arabidopsis leaves. (A) Aggregation of gold particle 2 (Gold2) was observed in microcentrifuge tubes, on the macrocarrier surface, and under microscopy. Scale bars = 50 µm. (B) Confocal images show cell‐to‐cell movement of eGFP at bombardment sites using gold particle 1 (Gold1) and Gold2. Scale bars = 50 µm. (C) Quantitative data show the total number of transformation events in five leaves. (D) Quantitative data show the number of neighboring cells containing eGFP at bombardment sites. Mann–Whitney U Test was used to analyze the data (*, P‐value < 0.05). The number of bombarded sites analyzed is indicated (n).

Figure 3.

The effect of leaf developmental stage on transformation efficiency and protein movement. (A) The 4th leaves from 3‐week‐old and 4‐week‐old plants were collected and mounted on 0.6% agar for bombardment. Scale bars = 1 cm. (B) Confocal images show cell‐to‐cell movement of eGFP at bombardment sites using leaves from 3‐week‐old and 4‐week‐old plants. Scale bars = 50 µm. (C) Quantitative data show the total number of transformation events in five leaves. (D) Quantitative data show the number of neighboring cells containing eGFP at bombardment sites. Mann‐Whitney U Test was used to analyze the data (*, P‐value < 0.05). The number of bombarded sites analyzed is indicated (n).

Figure 4.

Cell‐to‐cell movement of eGFP at bombardment sites in various plant species. (A) Images of leaves from different species were taken following microparticle bombardment. Leaves were mounted on 0.6% agar with the abaxial side upward for the procedure. Scale bars = 1 cm. (B) PD‐mediated trafficking of eGFP from bombarded sites to neighboring cells. Bombarded sites were identified by nuclear‐localized tdTomato expression. Scale bars = 100 µm.

• 41Open the images using FIJI/ImageJ. Count the number of neighboring cells displaying GFP signal at each bombardment site.

  It is recommended to quantify GFP movement from 50 or more bombardment sites per sample for reliable statistical analysis. Box plots or column plots can be used to present the data. The Mann‐Whitney U Test can be performed to test the statistical significance of differences.

REAGENTS AND SOLUTIONS

Use milli‐Q (MQ) water for all preparations.

Agar plates (0.6%)

Dissolve 3 g Difco agar (BD, cat. no. DF0479‐17‐3) in 0.5 L MQ water in a 1‐L glass bottle (Fisherbrand, cat. no. FB8001000), autoclave for 20 min, aliquot 20 ml into each Petri dish (Fisher, cat. no. FB0875713A), and store at 4°C up to 3 months. Warm the plates to room temperature before use.

Calcium Chloride, 2.5 M

Dissolve 2.78 g calcium chloride (Millipore Sigma, cat. no. C4901) in 100 ml MQ water in a 250‐ml glass bottle (Fisherbrand, cat. no. FB800250), autoclave for 20 min, and store at −20°C in 1‐ml aliquots up to 12 months.

Spermidine, 0.1 M

Dissolve 14.5 mg spermidine (Millipore Sigma, cat. no. S2626) in 1 ml MQ water in a 1.5‐ml centrifuge tube (Eppendorf, cat. no. EP0030108051) and store at −20°C in 100‐µl aliquots up to 3 months.

COMMENTARY

Background Information

Monitoring the movement of GFP between cells is a reliable method for assessing PD function. For this approach, GFP must be expressed in isolated single cells, allowing the detection of its movement into neighboring cells via PD. Microparticle bombardment has been utilized to express GFP in isolated single cells in Arabidopsis (Aung et al., 2017; Fernandez & Burch‐Smith, 2022; Tee et al., 2022). Here, we present an optimized microparticle bombardment assay using a biolistic particle delivery system equipped with a flow guiding barrel. The assay employs a plasmid containing two expression cassettes: one encoding free enhanced GFP (eGFP) and the other encoding nuclear‐localized tdTomato (Reyes Caldas et al., 2022). The expression of tdTomato is used to identify the cells that have been bombarded. By assessing the effects of gold particle quality and leaf developmental stage on transformation efficiency and eGFP mobility, we refined the protocol to enhance its robustness and reproducibility, particularly in Arabidopsis. We also demonstrated the application of this method in tomato, pepper, and soybean.

Critical Parameters

Some critical parameters should be considered to ensure the success of this method.

Gold particle quality

The quality of gold particles significantly influences transformation efficiency and subsequent GFP movement. High‐quality gold particles remain well‐dispersed in suspension without visible aggregation. Aggregated particles not only reduce plasmid‐coating efficiency, thereby limiting DNA delivery, but can also cause excessive tissue damage during bombardment. Such damage may induce PD closure, reducing GFP mobility and masking biologically relevant changes in PD permeability. Figure 2 shows the effect of gold particle aggregation on transformation efficiency and protein movement in Arabidopsis leaves.

Optimal plant age and leaf developmental stage

Leaves selected for bombardment should be at an optimal stage of development. In general, younger leaves exhibit higher transformation efficiency. Figure 3 shows that leaves from 3‐week‐old Arabidopsis plants yield a higher number of transformation events and enhanced PD‐mediated movement of GFP. To ensure consistency and reproducibility, it is important to maintain consistent growth conditions, use plants of the same age, and select leaves at comparable developmental stages across different genotypes and treatment conditions. Because plant growth and leaf development vary under different growth conditions, researchers are advised to empirically determine the optimal plant age and stage of leaf development. Moreover, the plants should be healthy and free from signs of biotic or abiotic stress.

Mesh covering

Placing a metal or fabric mesh over the leaf surface during bombardment helps prevent leaf displacement and minimizes physical damage from particle delivery (Fig. 1). This practice can improve transformation efficiency, GFP expression, and GFP movement.

Keeping the leaves hydrated

Leaves should be placed in a hydrated condition (e.g., 0.4%–0.6% agar plates) throughout the entire process until imaging. Ensure the leaves maintain close contact with the agar surface to keep them well hydrated. This is critical not only for maintaining leaf viability but also for effective post‐bombardment treatment by applying chemicals via the plate medium (e.g., hydrogen peroxide or 22‐amino‐acid flagellin peptide).

Post‐bombardment imaging timing

The optimal time between bombardment and imaging depends on the expression of GFP, the efficiency of GFP movement, and the timing of chemical treatment. In Arabidopsis, imaging is typically performed within 24 hr post‐bombardment. However, the timing may vary across species. In general, a longer duration after bombardment (e.g., 48 hr post‐bombardment) may allow for greater protein movement. However, excessive delays could introduce variability and secondary effects.

Troubleshooting Table

See Table 1 for a list of common problems with the protocols, their causes, and potential solutions.

Table 1.

Troubleshooting Guide

Problem	Possible cause	Solution
Massive cell death	Aggregated gold particles Short distance between macrocarrier and sample plate Dehydrated leaves	Use high‐quality gold particles. Adjust the position of the sample plate in the bombardment chamber and use a mesh covering. Keep the leaves well hydrated.
Low GFP expression	Leaves too young or too old Poor‐quality plasmids Aged or oxidized spermidine Massive cell death	Use leaves at an optimal developmental stage. Prepare plasmids with higher purity and concentration. Prepare a fresh batch of spermidine. See solutions for massive cell death.
Less GFP movement	Low GFP expression Short duration after bombardment	See solutions for poor GFP expression. Increase the time between bombardment and imaging.

Understanding Results

The bombardment assay described here will produce images from which GFP movement can be quantified by using Fiji/ImageJ to count the number of neighboring cells showing GFP signal at the bombardment sites. Proper controls should be included to enable a comparison across genotypes and treatments. The number of replicates needed could vary depending on the number of samples and treatment conditions. It is recommended to quantify GFP movement from 50 or more bombardment sites per sample for reliable statistical analysis.

Figure 2 shows the effect of gold particle aggregation on transformation efficiency and protein movement. The aggregated gold particles (Fig. 2A) reduced transformation efficiency, as evaluated by quantifying the total bombardment sites across five leaves (Fig. 2B and C). In addition, less GFP movement was observed when using aggregated gold particles (Fig. 2B and D). The reduction in both transformation efficiency and protein movement is likely due to the tissue damage caused by the aggregated gold particles.

Figure 3 shows the effect of leaf developmental stage on transformation efficiency and protein movement. The 4th leaves from 3‐ or 4‐week‐old plants were used for the bombardment assay. Leaves from 3‐week‐old plants outperformed those from 4‐week‐old plants on both transformation efficiency and protein movement.

Figure 4 shows the successful application of the bombardment assays in Arabidopsis, tomato, pepper, and soybean. GFP and tdTomato signals from the bombardment sites are shown.

Time Considerations

The time for growing plants varies depending on the plant species, growth conditions, and the desired leaf developmental stages for bombardment. For Arabidopsis, 3‐week‐old plants are preferred. The preparation of DNA‐coated microcarriers and bombardment typically takes approximately 2 hr. Imaging is typically performed 24 hr post‐bombardment, with acquisition of 30 images taking approximately 1 hr. To ensure robust statistical analysis, alternate among genotypes or treatments during imaging and collect data from three to four biological replicates.

Author Contributions

Zhongpeng Li: Conceptualization; formal analysis; investigation; methodology; validation; visualization; writing ‐ original draft; writing ‐ review and editing. Connor Thorpe: Conceptualization; investigation; methodology; validation; visualization; writing ‐ original draft; writing ‐ review and editing. Shan Jiang: Conceptualization; funding acquisition; methodology; writing ‐ review and editing. Kyaw Aung: Conceptualization; formal analysis; funding acquisition; investigation; methodology; project administration; supervision; writing ‐ review and editing.

Conflict of Interest

C.T. and S.J. are co‐founders of Hermes Biomaterials Inc., which has licensed the Flow Guiding Barrel technology described in this paper from Iowa State University. These interests have been reviewed and managed by Iowa State University in compliance with its conflict of interest policies. The remaining authors declare no competing interests.

Li, Z. , Thorpe, C. , Jiang, S. , & Aung, K. (2025). Measuring plasmodesmata‐mediated intercellular trafficking using microparticle bombardment in arabidopsis and crops. Current Protocols, 5, e70194. doi: 10.1002/cpz1.70194

Data Availability Statement

Raw data and original images for Figures 2, 3, and 4 are available upon request.