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>

<ol>
	<li>Jiang, F., Zhu, J., Liu, H. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protoplasts:+A+useful+research+system+for+plant+cell+biology,+especially+dedifferentiation.">Protoplasts: A useful research system for plant cell biology, especially dedifferentiation.</a> Protoplasma. 250 (6), 1231-1238 (2013).</li><li>Wang, S., Tiwari, S. B., Hagen, G., Guilfoyle, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AUXIN+RESPONSE+FACTOR7+restores+the+expression+of+auxin-responsive+genes+in+mutant+Arabidopsis+leaf+mesophyll+protoplasts.">AUXIN RESPONSE FACTOR7 restores the expression of auxin-responsive genes in mutant Arabidopsis leaf mesophyll protoplasts.</a> The Plant Cell. 17 (7), 1979-1993 (2005).</li><li>Hirner, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arabidopsis+LHT1+is+a+high-affinity+transporter+for+cellular+amino+acid+uptake+in+both+root+epidermis+and+leaf+mesophyll.">Arabidopsis LHT1 is a high-affinity transporter for cellular amino acid uptake in both root epidermis and leaf mesophyll.</a> The Plant Cell. 18 (8), 1931-1946 (2006).</li><li>Hwang, I., Sheen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-component+circuitry+in+Arabidopsis+cytokinin+signal+transduction.">Two-component circuitry in Arabidopsis cytokinin signal transduction.</a> Nature. 413 (6854), 383-389 (2001).</li><li>Tan, M. -. L. M. C., Rietveld, E. M., van Marrewijk, G. A. M., Kool, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+leaf+mesophyll+protoplasts+of+tomato+cultivars+(L.+esculentum):+Factors+important+for+efficient+protoplast+culture+and+plant+regeneration.">Regeneration of leaf mesophyll protoplasts of tomato cultivars (L. esculentum): Factors important for efficient protoplast culture and plant regeneration.</a> Plant Cell Reports. 6 (3), 172-175 (1987).</li><li>Brandt, K. M., Gunn, H., Moretti, N., Zemetra, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+streamlined+protocol+for+wheat+(Triticum+aestivum)+protoplast+isolation+and+transformation+with+CRISPR-Cas+ribonucleoprotein+complexes.">A streamlined protocol for wheat (Triticum aestivum) protoplast isolation and transformation with CRISPR-Cas ribonucleoprotein complexes.</a> Frontiers in Plant Science. 11, 769 (2020).</li><li>Nadakuduti, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methods+to+assess+in+vivo+activity+of+engineered+genome-editing+nucleases+in+protoplasts.">Evaluation of methods to assess in vivo activity of engineered genome-editing nucleases in protoplasts.</a> Frontiers in Plant Science. 10, 110 (2019).</li><li>OECD, Food and Agriculture Organization of the United Nations. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OECD-FAO+Agricultural+Outlook+2021-2030.">OECD-FAO Agricultural Outlook 2021-2030.</a> OECD, Food and Agriculture Organization of the United Nations. , (2021).</li><li>Gallois, P., Marinho, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leaf+disk+transformation+using+Agrobacterium+tumefaciens-expression+of+heterologous+genes+in+tobacco.">Leaf disk transformation using Agrobacterium tumefaciens-expression of heterologous genes in tobacco.</a> Plant Gene Transfer and Expression Protocols. 49, 39-48 (1995).</li><li>Sch&#228;ffner, A. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 &#176;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 &#38; 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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Biology

2.5K 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

Gene-gun Transfection of Hippocampal Neurons

Hi, I'm Kma. I work in Case Lab at Brown University Clean Slicer stage with 70%ethanol. Place plate insert into six.Well plate with medium and incubate at 37 degree whole brain with tweezers between midbrain and four brainin. Isolate hippocampus on one side, isolate hippocampus from the second side. Place hippocampi onto A PBS drop.Aspirate out the PBS make sagittal sections using the tissue slicer. Repeat transfer slicers onto a perish by squatting with PBS wash sections by adding and aspirating PBS and medium gently. Separate sections with tweezers.Wash sections with PBS and media. Again, transfer sections onto the plate and insert. Aspirate out the media in sections using tweezers.Attach gene gun hose to the helium cylinder. Attach barrel to gene gun. Fill the magazine with bullets.Insert magazine into the Jing gun. Adjust the pressure on the helium tank shoot. EGFP coated beads onto the sections.And now.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Protein Transfection of Mouse Lung

Increasing protein expression enables researchers to better understand the functional role of that protein in regulating key biological processes1. In the lung, this has been achieved typically through genetic approaches that utilize transgenic mice2,3 or viral or non-viral vectors that elevate protein levels via increased gene expression4. Transgenic mice are costly and time-consuming to generate and the random insertion of a transgene or chronic gene expression can alter normal lung development and thus limit the utility of the model5. While conditional transgenics avert problems associated with chronic gene expression6, the reverse tetracycline-controlled transactivator (rtTA) mice, which are used to generate conditional expression, develop spontaneous air space enlargement7. As with transgenics, the use of viral and non-viral vectors is expensive8 and can provoke dose-dependent inflammatory responses that confound results9 and hinder expression10. Moreover, the efficacy of repeated doses are limited by enhanced immune responses to the vector11,12. Researchers are developing adeno-associated viral (AAV) vectors that provoke less inflammation and have longer expression within the lung13.
Using &beta;-galactosidase, we present a method for rapidly and effectively increasing protein expression within the lung using a direct protein transfection technique. This protocol mixes a fixed amount of purified protein with 20 &mu;l of a lipid-based transfection reagent (Pro-Ject, Pierce Bio) to allow penetration into the lung tissue itself. The liposomal protein mixture is then injected into the lungs of the mice via the trachea using a microsprayer (Penn Century, Philadelphia, PA). The microsprayer generates a fine plume of liquid aerosol throughout the lungs. Using the technique we have demonstrated uniform deposition of the injected protein throughout the airways and the alveoli of mice14. The lipid transfection technique allows the use of a small amount of protein to achieve effect. This limits the inflammatory response that otherwise would be provoked by high protein administration. Indeed, using this technique we published that we were able to significantly increase PP2A activity in the lung without affecting lung lavage cellularity15. Lung lavage cellularity taken 24 hr after challenge was comparable to controls (27&plusmn;4 control vs. 31&plusmn;5 albumin transfected; N=6 per group). Moreover, it increases protein levels without inducing lung developmental changes or architectural changes that can occur in transgenic models. However, the need for repeated administrations may make this technique less favorable for studies examining the effects of long-term increases in protein expression. This would be particularly true for proteins with short half-lives.

Transgenic mice or viral vectors have been used to increase protein expression within the lung. However, these techniques are time-consuming, technically challenging and have off-target effects that can confound results. Our protein transfection protocol uses a lipid based transfection reagent and an ultrafine microsprayer to uniformly deliver active protein to lung cells.

Increasing protein expression enables researchers to  better understand the functional role of that protein in regulating key  biological processes1. In ...

Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated with specialization, development and increase in cellular size. In plants, endoreduplication seems to facilitate the growth and expansion of certain tissues and organs. Among them is the tuber of potato (Solanum tuberosum), which undergoes considerable cellular expansion in fulfilling its function of carbohydrate storage. Thus, endoreduplication may play an important role in how tubers are able to accommodate this abundance of carbon. However, the cellular debris resulting from crude nuclear isolation methods of tubers, methods that can be used effectively with leaves, precludes the estimation of the tuber endoreduplication index (EI). This article presents a technique for assessing tuber endoreduplication through the isolation of protoplasts while demonstrating representative results obtained from different genotypes and compartmentalized tuber tissues. The major limitations of the protocol are the time and reagent costs required for sample preparation as well as relatively short lifespan of samples after lysis of protoplasts. While the protocol is sensitive to technical variation, it represents an improvement over traditional methods of nuclear isolation from these large specialized cells. Possibilities for improvements to the protocol such as recycling enzyme, the use of fixatives, and other alterations are proposed.

The protocol described herein is a method for measuring endoreduplication within tubers of potato (Solanum tuberosum). It includes plasmolysis and protoplast extraction steps to decrease the noise and debris in downstream flow cytometric analysis.

Endoreduplication, the replication of a cell's nuclear genome without subsequent cytokinesis, yields cells with increased DNA content and is associated ...

Production and Visualization of Bacterial Spheroplasts and Protoplasts to Characterize Antimicrobial Peptide Localization

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of conventional light microscopes. As the resolution for a given microscope cannot be easily enhanced, we present protocols to transform the small rod-shaped gram-negative Escherichia coli (E. coli) and gram-positive Bacillus megaterium (B. megaterium) into larger, easily imaged spherical forms called spheroplasts or protoplasts. This transformation allows observers to rapidly and clearly determine whether peptides lodge themselves into the bacterial membrane (i.e., membrane localizing) or cross the membrane to enter the cell (i.e., translocating). With this approach, we also present a systematic method to characterize peptides as membrane localizing or translocating. While this method can be used for a variety of membrane-active peptides and bacterial strains, we demonstrate the utility of this protocol by observing the interaction of Buforin II P11A (BF2 P11A), an antimicrobial peptide (AMP), with E. coli spheroplasts and B. megaterium protoplasts.

Here we present a protocol to produce gram-negative Escherichia coli (E. coli) spheroplasts and gram-positive Bacillus megaterium (B. megaterium) protoplasts to clearly visualize and rapidly characterize peptide-bacteria interactions. This provides a systematic method to define membrane localizing and translocating peptides.

The use of confocal microscopy as a method to assess peptide localization patterns within bacteria is commonly inhibited by the resolution limits of ...

Micron-scale Phenotyping Techniques of Maize Vascular Bundles Based on X-ray Microcomputed Tomography

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a 'sample preparation protocol' for maize materials (i.e., stem, leaf, and root) suitable for ordinary microcomputed tomography (micro-CT) scanning. Based on high-resolution CT images of maize stem, leaf, and root, we describe two protocols for the phenotypic analysis of vascular bundles: (1) based on the CT image of maize stem and leaf, we developed a specific image analysis pipeline to automatically extract 31 and 33 phenotypic traits of vascular bundles; (2) based on the CT image series of maize root, we set up an image processing scheme for the three-dimensional (3-D) segmentation of metaxylem vessels, and extracted two-dimensional (2-D) and 3-D phenotypic traits, such as volume, surface area of metaxylem vessels, etc. Compared with traditional manual measurement of vascular bundles of maize materials, the proposed protocols significantly improve the efficiency and accuracy of micron-scale phenotypic quantification.

We provide a novel method to improve the X-ray absorption contrast of maize tissue suitable for ordinary microcomputed tomography scanning. Based on CT images, we introduce a set of image-processing workflows for different maize materials to effectively extract microscopic phenotypes of vascular bundles of maize.

It is necessary to accurately quantify the anatomical structures of maize materials based on high-throughput image analysis techniques. Here, we provide a ...

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative sparsity of information about C. elegans in its natural context comes from the challenges involved in the identification of the small nematode in nature. Despite these challenges, an increasing focus on the ecology of C. elegans has caused a wealth of new information regarding its life outside of the laboratory. The intensified search for C. elegans in nature has contributed to the discovery of many new Caenorhabditis species and revealed that congeneric nematodes frequently cohabitate in the wild, where they feed on microbial blooms associated with rotting plant material. The identification of new species has also revealed that the androdioecious mating system of males and self-fertilizing hermaphrodites has evolved three times independently within Caenorhabditis. The other two selfing species, C. briggsae and C. tropicalis, share the experimental advantages of C. elegans and have enabled comparative studies into the mechanistic basis of important traits, including self-fertilization. Despite these advances, much remains to be learned about the ecology and natural diversity of these important species. For example, we still lack functional information for many of their genes, which might only be attained through an understanding of their natural ecology. To facilitate ecological research of selfing Caenorhabditis nematodes, we developed a highly scalable method to collect nematodes from the wild. Our method makes use of mobile data collection platforms, cloud-based databases, and the R software environment to enhance researchers&#39; ability to collect nematodes from the wild, record associated ecological data, and identify wild nematodes using molecular barcodes.

A Highly Scalable Approach to Perform Ecological Surveys of Selfing Caenorhabditis Nematodes

This protocol can be used to perform large-scale ecological surveys of selfing Caenorhabditis nematodes. The primary advantage of this method is the efficient organization and analysis of ecological and molecular data associated with the nematodes collected from nature.

Caenorhabditis elegans is one of the major model organisms in biology, but only recently have researchers focused on its natural ecology. The relative ...

Scalable, Flexible, and Cost-Effective Seedling Grafting

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage seedlings of the small model plant, Arabidopsis thaliana, is technically challenging and time consuming due to the size and fragility of its seedlings. A growing collection of published methods describe this technique with varying success rates, difficulty, and associated costs. This paper describes a simple procedure to make an in-house reusable grafting device using silicone elastomer mix, and how to use this device for seedling grafting. At the time of this publication, each reusable grafting device costs only $0.47 in consumable materials to produce. Using this method, beginners can have their first successfully grafted seedlings in less than 3 weeks from start to finish. This highly accessible procedure will allow plant molecular genetics labs to establish seedling grafting as a normal part of their experimental process. Due to the full control users have in the creation and design of these grafting devices, this technique could be easily adjusted for use in larger plants, such as tomato or tobacco, if desired.

This protocol describes a robust seedling grafting method that requires no prior experience or training and can be executed at a very low cost using materials easily accessible in most molecular biology labs.

Early-stage seedling grafting has become a popular tool in molecular genetics to study root-shoot relationships within plants. Grafting early-stage ...

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 ...