This work was supported by the National Human Genome Research Institute Interdisciplinary Training in Genomic Sciences (T32 training grant no. HG000035 to J.T.) and the National Institute of Health (NIGMS 1R35GM139532 to C.Q.) and by the National Science Foundation (RESEARCH-PGR IOS-1748843 and PlantSynBio 2240888 to CQ). We thank Andrea Gallovatti at Rutgers University for the gift of the maize seeds.

1. Growth of the plant material
<ol>
	<li>Rinse maize kernels twice with tap water. Soak the kernels in tap water overnight at room temperature.</li>
	<li>The next day, fill a 72 cell seed starter tray with wetted 1:1 vermiculite:peat moss. Rinse the kernels once and plant them tip cap down, one kernel per cell.</li>
	<li>Grow under long-day conditions (16 h light, 8 h dark) at 25 &#176;C for 3 days.</li>
	<li>Transfer to constant darkness at 25 &#176;C, and grow for 9-11 days.</li>
</ol>
2. Plasmid isolation
<ol>
	<li>Transform commercial chemically competent E. coli with the plasmid(s) of interest according to the manufacturer&#39;s instructions.</li>
	<li>Grow the transformed E. coli in 50 mL of liquid LB medium with appropriate antibiotics overnight at 37 &#176;C with shaking.</li>
	<li>Pellet the&#160;E. coli by centrifuging for 10 min at 3,000 x g at room temperature. Extract the plasmids using a commercially available midiprep plasmid DNA isolation kit.</li>
	<li>Estimate the plasmid DNA concentration using a microvolume spectrophotometer. 
	NOTE: Material suitable for transfection should have a concentration of &#8805;800 ng/&#181;L and an A260/A280 of &#8805;1.8.</li>
</ol>
3. Protoplast isolation
<ol>
	<li>Prepare 20 mL of enzyme solution.
	<ol>
		<li>Add 2.18 g of mannitol, 0.30 g of cellulase R-10, and 0.06 g of macerozyme R-10 to a 50 mL centrifuge tube. Invert to mix the powders. Add deionized H2O to 15 mL. Add 200 &#181;L of 1 M MES, pH 5.7. Vortex to mix.</li>
		<li>Heat the solution at 55 &#176;C for 10 min. Cool at room temperature for 10 min.</li>
		<li>Add 20 &#181;L of 1 M CaCl2, 0.02 g of bovine serum albumin, and 100 &#181;L of 1 M &#946;-mercaptoethanol.</li>
		<li>Fill to 20 mL with deionized H2O. Pour into a foil-wrapped 250 mL beaker.</li>
	</ol>
	</li>
	<li>Take the maize seedlings out of the dark. Cut them a few centimeters above the soil using a new razor blade. Place the cut ends in water. Keep them in the dark at room temperature while not in use.</li>
	<li>Select the second and third leaves of each seedling, taking care to exclude leaves with brown or damaged areas. Select 12-16 leaves. Cut ~1 cm off the tip of each leaf and discard. Then, cut a 10 cm section from each leaf. 
	NOTE: If more tissue is needed, use an additional 20 mL volume of enzyme solution in an additional 250 mL beaker. Too much leaf material in a given amount of enzyme solution can decrease the viability.</li>
	<li>Stack the leaf sections on top of each other with the basal ends together. Clamp the bundle together at the basal end using a binder clip. Place the bundle of leaves on a piece of white paper.</li>
	<li>Grip the leaves ~1 cm from the distal end. Using a new razor blade, cut the leaves from the distal end perpendicular to the veins into thin (0.5-1 mm) slices. Avoid chopping straight down, but rather make short, forward slices through the tissue.
	<ol>
		<li>Swap the razor blades whenever visible residue begins to be deposited on the paper. Visible residue indicates mashing of the tissue due to a dull blade or poor technique.</li>
	</ol>
	</li>
	<li>After cutting ~2 cm of the length of the leaf bundle, promptly transfer the slices to the beaker of enzyme solution. Do not allow the material to dry out, as this will reduce the number of protoplasts obtained. Gently swirl the enzyme solution to wet the material.
	<ol>
		<li>Place a foil lid over the beaker to block light. Repeat the slicing and transferring process until the entire bundle is cut close to the binder clip.</li>
	</ol>
	</li>
	<li>Transfer the beaker of enzyme solution to a bell jar. Vacuum-infiltrate between &#8722;50.8 kPa and &#8722;67.7 kPa relative to ambient pressure for 3 min at room temperature.</li>
	<li>Transfer the beakerto a tabletop shaker. Shake at 40 RPM for 2.5 h at room temperature. Gently swirl the beaker by hand. When fully digested, the enzyme solution will appear slightly milky. If the solution is still clear, continue shaking at 40 RPM for up to one additional hour. Do not exceed 3.5 h.</li>
	<li>During digestion, make a mannitol + MES + MgCl2 (MMG) solution, a polyethylene glycol (PEG) solution, and an incubation solution.
	<ol>
		<li>MMG: Add 5.47 g of mannitol, 200 &#181;L of 1 M MES, pH 5.7, and 750 &#181;L of 1 M MgCl2 to a 50 mL centrifuge tube. Fill to 50 mL with distilled H2O. Vortex to dissolve. Place on ice.</li>
		<li>Incubation solution: Add 5.47 g of mannitol, 200 &#181;L of 1 M MES at pH 5.7, and 200 &#181;L of 1 M KCl to a 50 mL centrifuge tube. Fill to 50 mL with distilled H2O. Vortex to dissolve. Place on ice.</li>
		<li>PEG solution: Add 0.44 g of mannitol, 1.0 g of PEG, and 400 &#181;L of 1 M CaCl2 to a 15 mL centrifuge tube. Fill to 4 mL with distilled H2O. Vortex to mix. Incubate at 37 &#176;C with shaking for 30 min until fully dissolved. Keep at room temperature.</li>
	</ol>
	</li>
	<li>Shake the enzyme solution at 80 RPM for 10 min at room temperature.</li>
	<li>Place the beaker with the enzyme solution on ice. Do not remove the foil covering. Add 20 mL of ice-cold MMG to the enzyme solution. Filter through a 40 &#181;m cell strainer into a 50 mL centrifuge tube. 
	NOTE: Keep all the solutions on ice for the following steps until the addition of the PEG. Keep the protoplasts covered with foil to minimize light exposure.</li>
	<li>Split the filtrate into equal volumes of 10 mL each. Pour each volume into a round-bottom glass centrifuge tube, and spin down for 4 min at 100 x g at room temperature.</li>
	<li>Discard the supernatant and add 1 mL of ice-cold MMG to each tube. Resuspend the pellets by gently swirling and tapping the tubes. Combine the samples into a single round-bottom glass centrifuge tube. Add MMG to a total volume of 5 mL. Spin down for 3 min at 100 x g at room temperature.</li>
	<li>Wash with 5 mL of MMG, and spin down for 3 min at 100 x g at room temperature.</li>
	<li>Repeat the wash with 5 mL of MMG, and spin down for 3 min at 100 x g at room temperature. After spinning down the second wash, observe whether the supernatant is clear. If it remains cloudy, perform a third wash.</li>
	<li>Resuspend the protoplasts in 1 mL of MMG. Cover loosely with foil and keep on ice.</li>
	<li>Determine the protoplast yield and check the viability.
	<ol>
		<li>In a 1.5 mL microcentrifuge tube, add together 4 &#181;L of protoplasts and 72 &#181;L of MMG.</li>
		<li>In a 1.5 mL microcentrifuge tube, add 1 &#181;L of 0.5% fluorescein diacetate (FDA) stock solution to 49 &#181;L of MMG to make a 20x working solution. Add 4 &#181;L of FDA working solution to the tube with the diluted protoplasts. Incubate for 5 min in the dark at room temperature.</li>
		<li>Load 11 &#181;L of the protoplast mixture onto a hemocytometer. Place under a brightfield light microscope. Visually confirm that the cells lack cell walls. Then, count the number of protoplasts. Use this count to estimate the total number of protoplasts obtained. Then, count the number of protoplasts that fluoresce green under UV light when stained with FDA. Successful protoplast isolations yield a viability of 70%-90%.</li>
	</ol>
	</li>
</ol>
4. PEG-mediated transfection
<ol>
	<li>Starting with the estimated protoplast concentration from step 3.17, adjust to ~10,000 protoplasts/&#181;L. If the concentration is low, centrifuge for 3 min at 100 x g, and resuspend in MMG to a concentration of ~10,000 protoplasts/&#181;L.</li>
	<li>Mix ~1,000,000 protoplasts with 15 &#181;g of DNA in a 1.5 mL microcentrifuge tube. Top up the mixture with MMG to 114.4 &#181;L, and incubate on ice for 30 min.</li>
	<li>Resuspend the protoplasts by tapping the side of the tube. Add 105.6 &#181;L of 25% PEG solution to the protoplasts to reach a final weight/volume of 12% PEG. Gently mix by inverting 5-10 times. Protoplasts in PEG solution are fragile; do not shake the tube. Incubate for 10 min in the dark at room temperature. 
	NOTE: The transfection can be scaled up in multiples of the volumes given in steps 4.2 and step 4.3. For example, 10 million protoplasts and 150 &#181;g of DNA can be suspended in 1,444 &#181;L of MMG and treated with 1,056 &#181;L PEG solution in a 50 mL centrifuge tube.</li>
	<li>Dilute with 5 volumes of incubation solution. Gently mix by inversion. Spin down for 4 min at 100 x g at room temperature. 
	NOTE: If scaling up, split the protoplasts into multiple round-bottom glass tubes containing no more than 10 mL each to ensure complete pelleting. Proportionately scale up the incubation solution volume used for washing.</li>
	<li>Remove the supernatant by pipetting. Wash with 1-5 mL of incubation solution, and spin down for 3 min at 100 x g at room temperature.</li>
	<li>Resuspend the protoplasts in the incubation solution to a concentration of 500-1,000 cells/&#181;L.</li>
	<li>Incubate the protoplasts in the dark overnight (~16 h) at room temperature.</li>
</ol>
5.&#160; Protoplast recovery and expression verification
<ol>
	<li>Spin down the protoplasts for 4 min at 100 x g at room temperature. NOTE: If scaling up, split the protoplasts into multiple round-bottom glass tubes containing no more than 10 mL each.</li>
	<li>Wash with 1-5 mL of incubation solution, and spin down for 3 min at 100 x g at room temperature.</li>
	<li>Resuspend, and combine in 1-5 mL of incubation solution.</li>
	<li>If the protoplasts are transfected with a fluorescent reporter gene (e.g., GFP), take an aliquot to check the transfection efficiency. Using a hemocytometer, first count the total number of protoplasts using brightfield light microscopy. Then, count the fraction of fluorescent protoplasts under UV light.</li>
	<li>Centrifuge the protoplasts for 3 min at 100 x g at room temperature. 
	NOTE: The protoplasts are now ready for downstream applications such as RNA extraction with phenol and guanidine isothiocyanate.</li>
</ol>

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The authors declare no competing interests.

As reported previously, the quality of plant material used for protoplasting is essential for obtaining high-quality protoplasts16,17,18. It is crucial to select healthy unblemished leaves. This work used the popular research maize cultivar B73. We have not tested other cultivars. The maize seedlings used for this protocol were grown in darkness because etiolated leaves produce protoplasts with greater viability 16 h after transfection compared to protoplasts from green leaves. For applications that do not require overnight incubation, using green or greening leaf material would be possible.
A critical step is properly cutting the leaf material into thin slices before enzymatic digestion. This process increases the surface area for the mesophyll cells to be exposed to the enzyme solution, which increases the number of recovered protoplasts. The distal leaf tips are discarded, and thin (&#8804;1 mm) slices are cut with new razor blades, which are switched out immediately when they begin to dull. For a given amount of tissue, thinner slices yield more protoplasts provided the correct technique is employed to minimize the crushing of the leaves.
Washing the protoplasts correctly is also important, as they are quite delicate after undergoing cell wall removal. After digestion, all the solutions are kept on ice, and the dark-grown protoplasts are protected from light. The osmolarity and pH are buffered by performing the washes in MMG solution. To isolate the protoplasts from the less dense cellular debris, centrifugation takes place at 100 x g. Protoplasts centrifuged at 200 x g show similar viability after isolation but lower viability the following day, and they transform approximately half as well. We recommend checking the viability of the protoplasts by staining with FDA (fluorescein diacetate) immediately after isolation; the normal viability ranges between 70%-90%. Protoplasts with viability lower than 40% after isolation yield few transformants.

Pelleting and washing are easier when working with many protoplasts because a clearly visible pellet is obtained after transfection. For this reason, transfection is performed with 1 million or more protoplasts. When scaling up the protocol, an appropriately sized vessel should be selected for the incubation steps (1.5 mL, 15 mL, or 50 mL centrifuge tube), and the volume of solution used for the wash steps should be scaled accordingly. In any case, it is beneficial to centrifuge in &#8804;10 mL aliquots to ensure that the protoplasts do not remain in the supernatant. One innovation of this protocol is the removal of the 1 h incubation and wash step in W5 solution that is listed in other protocols14,19, which proved unnecessary in our hands.
As seen in other studies, the amount and quality of DNA are both critical for successful transfection19. For plasmids in the 4-6 kb range, 15 &#181;g of DNA is used per 1 million protoplasts. Poor-quality DNA preparations can result in reduced viability after transfection, so we recommend using a commercial DNA extraction kit when isolating plasmids from bacteria. Incubating the protoplasts overnight is done at room temperature in the dark to allow for the transcription of the plasmid or plasmid library while minimizing stress. After overnight incubation, approximately half the number of originally transfected protoplasts is recovered. Protoplasts are diluted to a concentration of 500-1,000 cells/&#181;L for overnight incubation. Protoplasts left to incubate overnight at concentrations higher than 1,000 cells/&#181;L have lower recovery rates and lower viability. At higher concentrations, debris from damaged and dead protoplasts may contribute to the death of neighboring protoplasts. Washing the protoplasts after overnight incubation serves both to remove this debris and remove the excess plasmid.
Protocols such as these have the limitation that not all plant species or tissue types are amenable to protoplasting. Furthermore, gene expression differences may be induced by the protoplasting process.
In summary, we have detailed a simple and scalable protocol to isolate and transfect millions of viable protoplasts from the staple agricultural crop Z. mays. This method can transfect many more protoplasts using PEG, with a transformation efficiency nearly as high as electroporation-based methods15. The greater net number of transformants enables the testing of hundreds or thousands of constructs in large-scale experiments such as massively-parallel reporter assays12,13. Future genome-scale experiments in maize will allow scientists to better understand maize gene expression and gene regulation.

Transient gene expression is a powerful tool in plant biology. However, plant cells are difficult to transfect because they are surrounded by a cell wall. This barrier to DNA entry is not present in other commonly studied cell types such as mammalian or insect cells. Past research addressed this challenge by employing protoplasts: plant cells whose cell walls have been digested and removed. In contrast to unprocessed leaf tissue, plant protoplasts are easy to work with in suspension and amenable to transfection techniques mediated by electroporation or polyethylene glycol (PEG)1. Plant cells retain much of their functionality even after cell wall removal. Thus, protoplasts are used in a variety of plants to study gene and protein function2,3, to understand signal transduction4, and to identify possible targets for plant breeding5. Protoplasts are also a convenient vehicle for screening genome editing constructs in diverse crop species including wheat and potato6,7. In short, transient assays in protoplasts are indispensable for agricultural biotechnology.
Maize (Zea mays L.) is the world-leading cereal crop by tonnage; as such, it is a major focus of basic and applied plant science research8. However, unlike in model plants such as Nicotiana tabacum9,&#160;transient transgene expression in intact maize leaves is not routinely performed. Protoplasting has been used to obtain and transfect maize leaf mesophyll cells10,11. Previous methods have achieved transfection efficiencies up to 75% using electroporation and produced transfected protoplasts at the scale of tens of thousands10,15.
This protocol details how to protoplast millions of maize mesophyll cells and transfect them with an efficiency comparable to previous methods. The main steps are growing etiolated maize seedlings, harvesting the leaf tissue, digesting the plant cell wall, and delivering plasmid DNA into the cells using PEG. The key contribution of this protocol is the ability to scale up protoplast transfection while maintaining the cell viability required for functional assays. This enables the use of genome-scale experiments, such as massively parallel reporter assays, that can test the function of hundreds of thousands of regions throughout the maize genome. This method has recently been used to investigate large libraries of cis-regulatory DNA elements including enhancers and promoters12,13,14.

<table><tbody><tr><td>1.5 mL graduated microcentrifuge tubes</td><td>VWR</td><td>490004-444</td><td></td></tr><tr><td>15 mL Falcon conical centrifuge tube</td><td>Corning</td><td>05-527-90</td><td></td></tr><tr><td>250 mL Pyrex glass beaker</td><td>Fisher</td><td>50-121-5005</td><td></td></tr><tr><td>40 um nylon mesh cell strainer</td><td>Fisher</td><td>22363547</td><td></td></tr><tr><td>50 mL Falcon tube</td><td>Corning</td><td>14-432-22</td><td></td></tr><tr><td>72 cell propagation tray</td><td>T.O. Plastics</td><td>725607C</td><td></td></tr><tr><td>Aluminum foil</td><td>VWR</td><td>89107-732</td><td></td></tr><tr><td>Bell jar</td><td>Bel-Art</td><td>F42022-0000</td><td></td></tr><tr><td>Benchtop centrifuge</td><td>Eppendorf</td><td>5424-R</td><td></td></tr><tr><td>Beta-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250-250ML</td><td>Dissolve in ultrapure H2O to make a 1M stock solution. Store at room temperature.</td></tr><tr><td>Bovine serum albumin</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Calcium chloride dihydrate (CaCl2*2H2O)</td><td>PhytoTech Labs</td><td>C135</td><td>Dissolve in ultrapure H2O to make a 1M stock solution. Store at room temperature.</td></tr><tr><td>Cellulase Onozuka R-10</td><td>Duchefa Biochemie</td><td>C8001.0010</td><td></td></tr><tr><td>D-Mannitol</td><td>PhytoTech Labs</td><td>M562</td><td></td></tr><tr><td>Dissecting scope</td><td>Reichert</td><td>Microstar IV</td><td></td></tr><tr><td>Fluorescein Diacetate (FDA)</td><td>Thermofischer Scientific</td><td>F1303</td><td>Dissolve in acetone at 0.5% weight/volume to make a 1000X stock solution. Protect from light and store at -20 &amp;deg;C.</td></tr><tr><td>Fluorescence Illumination Systems</td><td>Prior</td><td>Lumen200</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Leica</td><td>MDG36</td><td></td></tr><tr><td>High-capacity centrifuge</td><td>Eppendorf</td><td>5810 R</td><td></td></tr><tr><td>Macerozyme</td><td>Duchefa Biochemie</td><td>M8002.0005</td><td></td></tr><tr><td>Magnesium chloride (MgCl2)</td><td>Sigma-Aldrich</td><td>M292-1KG</td><td>Dissolve in ultrapure H2O to make a 1M stock solution. Store at room temperature.</td></tr><tr><td>Maize seeds</td><td>B73</td><td>USDA-ARS</td><td>https://npgsweb.ars-grin.gov/gringlobal/accessiondetail?accid=PI+550473</td></tr><tr><td>Medium binder clip</td><td>Uline</td><td>S-24649</td><td></td></tr><tr><td>MES</td><td>Sigma-Aldrich</td><td>M5287-250G</td><td>Dissolve in ultrapure H2O to make a 1M stock solution. Adjust pH to 5.7 using KOH. Protect from light and store at room temperature.</td></tr><tr><td>Micropipette 20-200 uL</td><td>Gilson</td><td>F123601G</td><td></td></tr><tr><td>Nanodrop microvolume spectrophotometer</td><td>Thermofischer Scientific</td><td>ND-1000</td><td></td></tr><tr><td>NEB 5-alpha competent E. coli</td><td>New England BioLabs</td><td>C2987H</td><td></td></tr><tr><td>Neubauer hemocytometer</td><td>Daigger Scientific</td><td>EF16034F</td><td></td></tr><tr><td>No. 9 Single-edge razor blade</td><td>Excel</td><td>20009</td><td></td></tr><tr><td>Orbital shaker</td><td>VWR</td><td>89032-104</td><td></td></tr><tr><td>Poly(ethylene) glycol 4,000</td><td>Sigma-Aldrich</td><td>81240-1KG</td><td></td></tr><tr><td>Potassium chloride (KCl)</td><td>Sigma-Aldrich</td><td>P9541-1KG</td><td>Dissolve in ultrapure H2O to make a 1M stock solution. Store at room temperature.</td></tr><tr><td>Professional Grade Vermiculite</td><td>NK Lawn &amp;amp; Garden</td><td>G208</td><td></td></tr><tr><td>Round-bottom glass centrifuge tube 30 mL</td><td>Kimble Chase</td><td>45500-30</td><td></td></tr><tr><td>Rubber adapter for Corex centrifuge tubes</td><td>Corning</td><td>CLS8445AO</td><td></td></tr><tr><td>Sphagnum peat moss</td><td>Premier Horticulture</td><td>0128P</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Thermofischer Scientific</td><td>15596018</td><td></td></tr><tr><td>Tygon vacuum tubing</td><td>VWR</td><td>76336-844</td><td></td></tr><tr><td>Vacuum gauge</td><td>Wika</td><td>4269978</td><td></td></tr></tbody></table>

scalable transfection of maize mesophyll protoplasts

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation or polyethylene glycol (PEG). Previous methods were developed to produce tens of thousands of transfected protoplasts at once. Here,&#160;we describe a straightforward method to isolate and transfect millions of leaf mesophyll protoplasts in maize (Zea mays L.). This streamlined process removes certain common protoplasting steps, such as washing in W5. Additionally, steps such as centrifugation, PEG-mediated transfection, and incubation have been modified to work with a greater number of protoplasts. The ability to express large libraries of plasmid constructs enables genome-scale experiments, such as massively parallel reporter assays in maize.

The tissue best suited for protoplast isolation is the second and third leaves of 10-11 day old seedlings (Figure 1). In this work, we obtained approximately 10 million protoplasts from 16 leaf sections, each of which was 10 cm long. The number of protoplasts isolated is dependent on the mass of the leaf material and the width of the leaf slices that are digested in the enzyme solution.
Under a brightfield light microscope, undamaged protoplasts appear roughly spherical, with plastids speckling the surface (Figure 2A). Before proceeding to transfection, the viability can be checked by staining a small aliquot of protoplasts with fluorescein diacetate (FDA). FDA-stained protoplasts that fluoresce under UV light are considered viable (Figure 2B,C). Not all protoplasts that appear undamaged are still viable, and some viable protoplasts may be in the process of dying by evacuolation (Figure 2C). In these cells, the vacuole is swelling and will eventually be ejected from the cell membrane.
Figure 3&#160;shows protoplasts transformed with pJT01, a 4.3 kb maize expression plasmid derived from CD3-911 (ABRC)15. Transfection results in the expression of sGFP tagged with a C-terminal nuclear localization signal from c-Myc (Figure 3A). The proportion of sGFP-expressing protoplasts was measured over multiple experiments using a hemocytometer. The median transfection efficiency achieved by this method was 40% after overnight incubation (n = 9, Figure 4, Table 1), which is comparable to previously published methods15. Depending on the experimental application, if the plasmid or plasmid library lacks a fluorescent reporter, it is recommended to include a positive control to confirm transfection.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64991/64991fig01.jpg" /> 
Figure 1: Representative etiolated maize seedling grown in darkness for 10 days after germination. The arrows indicate the second and third leaves, which are best suited for protoplasting. <a href="https://www.jove.com/files/ftp_upload/64991/64991fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64991/64991fig02.jpg" /> 
Figure 2: Fluorescence microscopy of the protoplast viability after isolation. (A) Brightfield image of protoplasts immediately after isolation. (B) The fluorescence signal of the FDA-stained protoplasts. (C) Overlap of brightfield and fluorescence showing the viable protoplasts. The arrow shows a protoplast undergoing evacuolation. <a href="https://www.jove.com/files/ftp_upload/64991/64991fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64991/64991fig03.jpg" /> 
Figure 3: Fluorescence microscopy of protoplasts transfected with sGFP. (A) Brightfield image of protoplasts after transfection. (B) The fluorescence signal of the transfected protoplasts in the GFP channel. (C) Overlap of brightfield and fluorescence showing the transformed protoplasts. <a href="https://www.jove.com/files/ftp_upload/64991/64991fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64991/64991fig04.jpg" /> 
Figure 4: Percentage of protoplasts showing GFP fluorescence after 16 h of incubation. The transfection efficiency varied among independent trials, with the lowest efficiency around 20% and the highest close to 50%. <a href="https://www.jove.com/files/ftp_upload/64991/64991fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Trial</td>
			<td>GFP cells counted</td>
			<td>Total cells counted</td>
			<td>GFP percentage</td>
		</tr>
		<tr>
			<td>1</td>
			<td>104</td>
			<td>261</td>
			<td>39.8</td>
		</tr>
		<tr>
			<td>2</td>
			<td>148</td>
			<td>298</td>
			<td>49.5</td>
		</tr>
		<tr>
			<td>3</td>
			<td>84</td>
			<td>258</td>
			<td>32.6</td>
		</tr>
		<tr>
			<td>4</td>
			<td>137</td>
			<td>271</td>
			<td>50.6</td>
		</tr>
		<tr>
			<td>5</td>
			<td>127</td>
			<td>299</td>
			<td>42.5</td>
		</tr>
		<tr>
			<td>6</td>
			<td>107</td>
			<td>235</td>
			<td>45.5</td>
		</tr>
		<tr>
			<td>7</td>
			<td>83</td>
			<td>360</td>
			<td>23.1</td>
		</tr>
		<tr>
			<td>8</td>
			<td>134</td>
			<td>549</td>
			<td>24.4</td>
		</tr>
		<tr>
			<td>9</td>
			<td>61</td>
			<td>201</td>
			<td>30.3</td>
		</tr>
		<tr>
			<td>Mean</td>
			<td>109</td>
			<td>304</td>
			<td>36</td>
		</tr>
		<tr>
			<td>Standard deviation</td>
			<td>29</td>
			<td>102</td>
			<td>10</td>
		</tr>
	</tbody>
</table>
Table 1: Representative protoplast transfection efficiency. Data from nine recent protoplasting trials in the authors&#39; laboratory.
Supplemental File 1: GenBank-formatted text file with the sequence of pJT01. A 4.3 kb sGFP expression plasmid serves as a positive control in transfected maize protoplasts. <a href="https://www.jove.com/files/ftp_upload/64991/pJT01_gfp_cmyc.gb.zip" target="_blank">Please click here to download this File.</a>

Watch this Scientific Journal Video about Scalable Transfection of Maize Mesophyll Protoplasts at JoVE.com

Scalable Transfection of Maize Mesophyll Protoplasts

Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

The transfection of maize mesophyll cells often involves digesting the plant cell walls to create protoplasts and then inserting DNA via electroporation ...

scalable-transfection-of-maize-mesophyll-protoplasts

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2.6K Views.  University of Washington. Here, we present a high-throughput protocol to transiently transfect millions of maize protoplasts for testing large libraries of plasmids in maize mesophyll cells.

Video: Scalable Transfection of Maize Mesophyll Protoplasts

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Genetics

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact plants.

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast permitting in vivo phenotypic analysis. This method does not rely on maize transformation but exploits microbial genetics and secretory capabilities of pathogens. Herein, it allows inspection of in vivo delivered secreted proteins with high spatiotemporal resolution at different kinds of infection sites and tissues. The Trojan horse strategy can be utilized to transiently complement maize loss-of-function phenotypes, to functionally characterize protein domains, to analyze off-target protein effects, or to study onside protein overdosage, making it a powerful tool for protein studies in the maize crop system. This work contains a precise protocol on how to generate a Trojan horse strain followed by standardized infection protocols to apply this method to three different maize tissue types.

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

This work describes the cloning of an Ustilago maydis Trojan horse strain for the in situ delivery of secreted maize proteins into three different types of maize tissues.

Inspired by Homer&#180;s Trojan horse myth, we engineered the maize pathogen Ustilago maydis to deliver secreted proteins into the maize apoplast ...

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a fluorescently tagged-protein of interest is transiently expressed in plant tissue following biolistic delivery of its encoding DNA construct. The intra- and intercellular distribution of the tagged protein is then analyzed by confocal microscopy. We describe this technology in detail, providing step-by-step protocols to assay and evaluate the extent of symplastic protein transport in three plant species, Arabidopsis thaliana, Nicotiana benthamiana and N. tabacum (tobacco).

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Macromolecular trafficking between plant cells can be assessed by transiently expressing a fluorescently-tagged protein of interest and analyzing its intra- and intercellular distribution by confocal microscopy.

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1. Due to its capacity to undergo efficient mitotic homologous recombination, Physcomitrella patens has emerged as an important model system in recent years2. This capacity allows high frequencies of gene targeting3-9, which is not seen in other model plants such as Arabidopsis. To take full advantage of this system, we need an effective and easy method to deliver DNA into moss cells. The most common ways to transform this moss are particle bombardment10 and PEG-mediated DNA uptake11. Although particle bombardment can produce a high transformation efficiency12, gene guns are not readily available to many laboratories and the protocol is difficult to standardize. On the other hand, PEG mediated transformation does not require specialized equipments, and can be performed in any laboratory with a sterile hood. Here, we show a simple and highly efficient method for transformation of moss protoplasts. This method can generate more than 120 transient transformants per microgram of DNA, which is an improvement from the most efficient protocol previously reported13. Because of its simplicity, efficiency, and reproducibility, this method can be applied to projects requiring large number of transformants as well as for routine transformation.

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella protonemal protoplast and Arabidopsis mesophyll protoplast transformation1.

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. Therefore, it is important to establish an efficient transient gene expression system in soybean. Here, we report a simple protocol for the preparation of soybean protoplasts and its application for transient functional analyses. We found that young unifoliate leaves from soybean seedlings resulted in large quantities of high quality protoplasts. By optimizing a PEG-calcium-mediated transformation method, we achieved high transformation efficiency using soybean unifoliate protoplasts. This system provides an efficient and versatile model for examination of complex regulatory and signaling mechanisms in live soybean cells and may help to better understand diverse cellular, developmental and physiological processes of legumes.

We developed a simple and efficient protocol for the preparation of large quantities of soybean protoplasts to study complex regulatory and signaling mechanisms in live cells.

Soybean (Glycine max (L.) Merr.) is an important crop species and has become a legume model for the studies of genetic and biochemical pathways. ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

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Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the injection of maize seedlings near meristematic tissue with Agrobacterium carrying a viral vector. Recombinant foxtail mosaic virus (FoMV) clones engineered for gene silencing and gene expression were used to optimize this method, and its use was expanded to include a recombinant sugarcane mosaic virus (SCMV) engineered for gene expression. Gene fragments or coding sequences of interest are inserted into a modified, infectious viral genome that has been cloned into the binary T-DNA plasmid vector pCAMBIA1380. The resulting plasmid constructs are transformed into Agrobacterium tumefaciens strain GV3101. Maize seedlings as young as 4 days old can be injected near the coleoptilar node with bacteria resuspended in MgSO4 solution. During infection with Agrobacterium, the T-DNA carrying the viral genome is transferred to maize cells, allowing for the transcription of the viral RNA genome. As the recombinant virus replicates and systemically spreads throughout the plant, viral symptoms and phenotypic changes resulting from the silencing of the target genes lesion mimic 22 (les22)&#160;or&#160;phytoene desaturase (pds) can be observed on the leaves, or expression of green fluorescent protein (GFP) can be detected upon illumination with UV light or fluorescence microscopy. To detect the virus and assess the integrity of the insert simultaneously, RNA is extracted from the leaves of the injected plant and RT-PCR is conducted using primers flanking the multiple cloning site (MCS) carrying the inserted sequence. This protocol has been used effectively in several maize genotypes and can readily be expanded to other viral vectors, thereby offering an accessible tool for viral vector introduction in maize.

An Agrobacterium-based injection (agroinjection) protocol is presented for the inoculation of foxtail mosaic virus&#160;and sugarcane mosaic virusclones into maize seedlings. Inoculation in this manner leads to viral infection, virus-induced gene silencing of marker genes, and viral overexpression of GFP.

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Visualization of Cellular Interactions Between the Maize Sheaths and Fungi

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one originally applied to rice leaf sheaths and allows direct microscopic observation of fungal growth and development in living plant cells. Leaf sheaths collected from maize seedlings with two fully emerged leaf collars are inoculated with 20 &#181;L drops of 5 x 105 spores/mL fungal spore suspensions and incubated in humidity chambers at 23 &#176;C under continuous fluorescent light. After 24-72 h, excess tissue is removed with a razor blade to leave a single layer of epidermal cells, an optically clear sample that can be imaged directly without the necessity for chemical fixation or clearing. Plant and fungal cells remain alive for the duration of the experiment and interactions can be visualized in real-time. Sheaths can be stained or subjected to plasmolysis to study the developmental cytology and viability of host and pathogen cells during infection and colonization. Fungal strains transformed to express fluorescent proteins can be inoculated or co-inoculated on the sheaths for increased resolution and to facilitate the evaluation of competitive or synergistic interactions. Fungal strains expressing fluorescent fusion proteins can be used to track and quantify the production and targeting of these individual proteins in planta. Inoculated sheath tissues can be extracted to characterize nucleic acids, proteins, or metabolites. The use of these sheath assays has greatly advanced the detailed studies of the mechanisms of fungal pathogenicity in maize and also of fungal protein effectors and secondary metabolites contributing to pathogenicity.

Author Spotlight: Investigating Fungal Pathogenicity Mechanisms in Maize 

This manuscript details an optimized inoculation protocol that uses detached maize leaf sheaths for reproducible cytological, physiological, and molecular studies of maize interactions with fungal plant pathogens. The leaf sheaths facilitate real-time observation of cellular interactions between the living plant and fungus in unfixed tissues.

We have optimized a protocol to inoculate maize leaf sheaths with hemibiotrophic and necrotrophic foliar pathogenic fungi. The method is modified from one ...

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for various purposes. However, as research in this field increases in diversity and becomes increasingly sophisticated, the need for early, efficient, dependable, and high-throughput transient screening solutions to narrow down strategies proceeding to stable transformation is more apparent. One method that has re-emerged in recent years is the utilization of plant protoplast, for which methods of isolation and transfection are available in numerous species, tissues, and developmental stages. This work describes a simple automated protocol for the randomized preparation of plasmid within a 96-well plate, a method for the isolation of etiolated maize leaf protoplast, and an automated transfection procedure. The adoption of automated solutions in plant biotechnology, exemplified by these novel liquid handling protocols for plant protoplast transfection, represents a significant advancement over manual methods. By leveraging automation, researchers can easily overcome the limitations of traditional methods, enhance efficiency, and accelerate scientific progress.

This protocol describes the creation of a randomized transfection layout using an automated liquid handler, a protoplast isolation protocol for etiolated maize leaf, and a 96-well transfection procedure using a liquid handler.

The field of plant biotechnology has witnessed remarkable advancements in recent years, revolutionizing the ability to manipulate and engineer plants for ...

Scalable Transfection of Maize Mesophyll Protoplasts

Summary

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Chapters in this video

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

Using Ustilago maydis as a Trojan Horse for In Situ Delivery of Maize Proteins

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

A Simple Method for Isolation of Soybean Protoplasts and Application to Transient Gene Expression Analyses

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

Enabling High-throughput Transgene Expression Studies Using Automated Liquid Handling for Etiolated Maize Leaf Protoplasts