Name: Scalable, Flexible, and Cost-Effective Seedling Grafting (Video) | JoVE Journal
Uploaded: 2023-01-06T13:20:00.000+00:00
Duration: 9 min 33 s
Description: Learn a scalable seedling grafting method for plant biology research using Arabidopsis and silicone elastomer devices. This low-cost protocol requires no prior experience and enables long-distance signaling studies.

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5 minutes

Thanks to Javier Brumos for initial training and guidance in grafting Arabidopsis seedlings.

1. Device preparation
<ol>
	<li>Make the silicone grafting device by casting silicone elastomer solution in a square Petri dish (100 mm x 100 mm). Prepare 15 mL of the elastomer solution, following the manufacturer&#39;s guidelines. 
	NOTE: Silicone elastomer kits typically include a silicone-based liquid and a curing agent, that when mixed together allow the silicone to solidify.</li>
	<li>Prepare the square Petri dish by laying four straight pieces of 29 G wire in the square Petri dish, equidistant from one another (Figure 1A). Ensure that the wire lies flush with the bottom of the mold. To fully straighten the wire, roll it on a hard uniform surface with a heavy and flat object (e.g., a metal tube rack). 
	NOTE:&#160;Twist ties often contain 29 G wire and can be used after removing the outer paper coating with acetone.</li>
	<li>Pour the mixed silicone elastomer solution on top of the wires and cover with the top of the Petri dish. Allow the silicone to cure for 24-48 h at room temperature.</li>
	<li>Remove the silicone sheet from the Petri dish using clean forceps and move to a clean flat surface.</li>
	<li>Remove the wires from the silicone sheet. Remove the thin layer of silicone remaining on the outside of the channel with fine point forceps, to allow the channel to be open on one side (Figure 1A).</li>
	<li>Cut the silicone sheet perpendicularly to the channels into 3 mm strips using clean scissors. Move each strip to an aluminum foil envelope and seal with autoclave tape.</li>
	<li>Autoclave the strips at 121 &#176;C for at least 30 min and store until ready for use.</li>
</ol>
2. Seedling preparation
<ol>
	<li>Sterilize and vernalize seeds.
	<ol>
		<li>Suspend up to 100 Arabidopsis seeds in 1 mL of 50% bleach solution containing 0.1% Tween 20 in a 1.5 mL microcentrifuge tube, and incubate for 5-10 min. Remove the bleach solution through pipetting or aspiration under sterile conditions. Rinse the seeds with 1 mL of sterilized dH2O. Be sure to invert the tubes to adequately rinse the seeds and remove any bleach solution left at the top of the tube. Repeat the rinsing 4x.</li>
		<li>Leave approximately 0.25 mL of water in the tubes with the seeds and store at 4 &#176;C for 3 days in the dark.</li>
	</ol>
	</li>
	<li>Plate the seeds in preparation for grafting.
	<ol>
		<li>Prepare a 1% agar MS plate as follows: for 1 L of MS (0.5% sucrose) solid medium, mix 4.4 g of MS salt, 5 g of sucrose, and 10 g of agar in 800 mL of water, adjust the pH to 5.7 with KOH, and then bring the total volume to 1 L with additional water. Autoclave for at least 20, min before pouring ~25 mL into the square Petri dishes.</li>
		<li>Under sterile conditions, move the appropriate number of prepared seeds to the plate, using a 20 &#181;L pipette tip to aspirate and transfer the seeds.</li>
		<li>Place a sterile strip on the plate surface to guide seed positioning, so the seeds are aligned with the channels on the strip. Remove the strip once the seeds are plated. 
		NOTE: One 100 mm x 100 mm square plate can accommodate two rows of seedlings (Figure 1B).</li>
		<li>Once the plates are standing up, allow the liquid to evaporate out of the solid medium and pool at the bottom of the plate. After the seeds are placed onto the plate, put on the plate cover and seal one side of the plate that is parallel to the two rows of seeds (indicated by the blue highlighted region in Figure 1B) with parafilm. Wrap breathable tape on top of the parafilm and around all the other edges of the plate.</li>
	</ol>
	</li>
	<li>Carefully stand up two plates with the parafilm-sealed side facing down. Separate the two plates at the bottom by placing a horizontal 15 mL centrifuge tube between them and secure with a rubber band. Ensure that the plate surfaces form a 100&#176;-110&#176; angle with the benchtop surface (Figure 1C).</li>
	<li>Store the plates in this orientation for 72 h in total darkness at 21 &#176;C, to allow the seedling hypocotyls to grow ~5 mm in length. After 72 h, remove the plates from the dark and grow under 16 h light (intensity of 100 &#181;E m-2 sec-1) and 8 h dark cycles for 2-4 more days at the same temperature before grafting.</li>
	<li>Graft the seedlings between 5 and 7 days after being plated. Place a grafting strip over the seedlings, fitting their hypocotyls into the channels. Gently position the seedling so the root-hypocotyl junction is positioned at the bottom of the silicone strip to prepare the seedling for cutting (Figure 1D).</li>
</ol>
3. Grafting procedure
<ol>
	<li>Prepare a sterile working environment by sanitizing a dissection scope with 70% ethanol and autoclaving two pairs of fine tipped forceps and a scalpel handle. Perform all grafting procedures in a sterile hood and with the aid of a dissection scope as needed. Perform most of the grafting using a magnification of 10.5x.</li>
	<li>Prepare the scions. Use a fresh scalpel blade to cut the hypocotyl perpendicularly to create a straight clean cut. Push the blade forward rather than pressing down into the plant to prevent the seedling from getting pushed into the agar (Video 1).</li>
	<li>Remove the shoot. Take care to keep the cut portion of the shoot hydrated by ensuring contact with the media surface. Alternatively, move the shoot to a designated holding area, such as the top of a Petri dish filled with sterile dH2O, until ready for use.</li>
	<li>Prepare the rootstocks. Gently pull the root by catching the root in the space left between the closed forceps and turning them, leaving the cut section of the rootstocks in the middle of the strip (Video 2). 
	NOTE: The fragile root will be damaged if crushed between the closed forceps directly, necessitating wedging the root in the sharp angle of the tweezer ends to manipulate the tissue.</li>
	<li>Gently pick up the desired shoot using the fine tipped forceps and insert into the top of the channel. 
	NOTE: It is critical to visually confirm contact between the scions and rootstocks to obtain a successful graft (Video 3).</li>
	<li>After all the grafts have been made, wrap the plates with parafilm and breathable tape and set up the plates in the same way as before, without disturbing the seedlings or silicone strips. Carefully move the plates to a growth chamber set at 26 &#176;C with 16 h light/8 h dark cycles.</li>
	<li>Evaluate the grafted seedlings under sterile conditions after 7-10 days. Carefully remove the silicone strip using forceps by peeling up one side, allowing the channels to free the seedlings. Remove any adventitious roots growing from the scion by cutting them from the scion with a fresh scalpel blade or crushing them using fine tipped forceps. Visually evaluate whether the rootstock has become firmly attached to the scion to form a successful graft (Figure 2).</li>
	<li>Move successful grafts to seedling propagation soil to grow for as long as required. Cover the soil with transparent plastic for a few days as the seedlings get established. After transferring the plants to the soil, grow under the previously mentioned light and dark cycles at 21 &#176;C.</li>
</ol>

<ol><li>Mudge, K., Janick, J., Scofield, S., Goldschmidt, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+history+of+grafting%5BTitle%5D)+AND+%22Horticultural+Reviews%22%5BJournal%5D)">A history of grafting.</a> Horticultural Reviews. 35, 437-493 (2009).</li>
<li>Holbrook, N. M., Shashidhar, V. R., James, R. A., Munns, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Stomatal+control+in+tomato+with+ABA-deficient+roots%3A+Response+of+grafted+plants+to+soil+drying%5BTitle%5D)+AND+%22Journal+of+Experimental+Botany%22%5BJournal%5D)">Stomatal control in tomato with ABA-deficient roots: Response of grafted plants to soil drying.</a> Journal of Experimental Botany. 53 (373), 1503-1514 (2002).</li>
<li>Notaguchi, M., Okamoto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dynamics+of+long-distance+signaling+via+plant+vascular+tissues%5BTitle%5D)+AND+%22Frontiers+in+Plant+Science%22%5BJournal%5D)">Dynamics of long-distance signaling via plant vascular tissues.</a> Frontiers in Plant Science. 6, 161(2015).</li>
<li>Ko, D., Helariutta, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Shoot-root+communication+in+flowering+plants%5BTitle%5D)+AND+%22Current+Biology%22%5BJournal%5D)">Shoot-root communication in flowering plants.</a> Current Biology. 27 (17), 973-978 (2017).</li>
<li>Thomas, H. R., Frank, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Connecting+the+pieces%3A+uncovering+the+molecular+basis+for+long-distance+communication+through+plant+grafting%5BTitle%5D)+AND+%22New+Phytologist%22%5BJournal%5D)">Connecting the pieces: uncovering the molecular basis for long-distance communication through plant grafting.</a> New Phytologist. 223 (2), 582-589 (2019).</li>
<li>Takahashi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+small+peptide+modulates+stomatal+control+via+abscisic+acid+in+long-distance+signalling%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A small peptide modulates stomatal control via abscisic acid in long-distance signalling.</a> Nature. 556 (7700), 235-238 (2018).</li>
<li>Brumos, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Local+auxin+biosynthesis+is+a+key+regulator+of+plant+development%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Local auxin biosynthesis is a key regulator of plant development.</a> Developmental Cell. 47 (3), 306-318 (2018).</li>
<li>Corbesier, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FT+protein+movement+contributes+to+long-distance+signaling+in+floral+induction+of+Arabidopsis%5BTitle%5D)+AND+%22Science%22%5BJournal%5D)">FT protein movement contributes to long-distance signaling in floral induction of Arabidopsis.</a> Science. 316 (5827), 1030-1033 (2007).</li>
<li>Yin, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Graft-union+development%3A+A+delicate+process+that+involves+cell-cell+communication+between+scion+and+stock+for+local+auxin+accumulation%5BTitle%5D)+AND+%22Journal+of+Experimental+Botany%22%5BJournal%5D)">Graft-union development: A delicate process that involves cell-cell communication between scion and stock for local auxin accumulation.</a> Journal of Experimental Botany. 63 (11), 4219-4232 (2012).</li>
<li>Turnbull, C. G. N., Booker, J. P., Leyser, H. M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micrografting+techniques+for+testing+long-distance+signalling%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">Micrografting techniques for testing long-distance signalling.</a> The Plant Journal. 32 (2), 255-262 (2002).</li>
<li>Marsch-Martínez, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+efficient+flat-surface+collar-free+grafting+method+for+Arabidopsis+thaliana+seedlings%5BTitle%5D)+AND+%22Plant+Methods%22%5BJournal%5D)">An efficient flat-surface collar-free grafting method for Arabidopsis thaliana seedlings.</a> Plant Methods. 9 (1), 14(2013).</li>
<li>Tsutsui, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Micrografting+device+for+testing+systemic+signaling+in+Arabidopsis%5BTitle%5D)+AND+%22The+Plant+Journal%22%5BJournal%5D)">Micrografting device for testing systemic signaling in Arabidopsis.</a> The Plant Journal. 103 (2), 918-929 (2020).</li>
<li>Xia, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elucidation+of+the+mechanisms+of+long-distance+mRNA+movement+in+a+Nicotiana+benthamiana%2Ftomato+heterograft+system%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">Elucidation of the mechanisms of long-distance mRNA movement in a Nicotiana benthamiana/tomato heterograft system.</a> Plant Physiology. 177 (2), 745-758 (2018).</li>
<li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Unidirectional+movement+of+small+RNAs+from+shoots+to+roots+in+interspecific+heterografts%5BTitle%5D)+AND+%22Nature+Plants%22%5BJournal%5D)">Unidirectional movement of small RNAs from shoots to roots in interspecific heterografts.</a> Nature Plants. 7 (1), 50-59 (2021).</li>
<li>Ragni, L., Hardtke, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+but+thick+enough-the+Arabidopsis+hypocotyl+as+a+model+to+study+secondary+growth%5BTitle%5D)+AND+%22Physiologia+Plantarum%22%5BJournal%5D)">Small but thick enough-the Arabidopsis hypocotyl as a model to study secondary growth.</a> Physiologia Plantarum. 151 (2), 164-171 (2014).</li>
<li>Chen, I. -J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+chemical+genetics+approach+reveals+a+role+of+brassinolide+and+cellulose+synthase+in+hypocotyl+elongation+of+etiolated+Arabidopsis+seedlings%5BTitle%5D)+AND+%22Plant+Science%22%5BJournal%5D)">A chemical genetics approach reveals a role of brassinolide and cellulose synthase in hypocotyl elongation of etiolated Arabidopsis seedlings.</a> Plant Science. 209, 46-57 (2013).</li>
<li>An, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Coordinated+regulation+of+apical+hook+development+by+gibberellins+and+ethylene+in+etiolated+Arabidopsis+seedlings%5BTitle%5D)+AND+%22Cell+Research%22%5BJournal%5D)">Coordinated regulation of apical hook development by gibberellins and ethylene in etiolated Arabidopsis seedlings.</a> Cell Research. 22 (5), 915-927 (2012).</li>
<li>Vandenbussche, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ethylene-induced+Arabidopsis+hypocotyl+elongation+is+dependent+on+but+not+mediated+by+gibberellins%5BTitle%5D)+AND+%22Journal+of+Experimental+Botany%22%5BJournal%5D)">Ethylene-induced Arabidopsis hypocotyl elongation is dependent on but not mediated by gibberellins.</a> Journal of Experimental Botany. 58 (15-16), 4269-4281 (2007).</li>
<li>Vandenbussche, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Arabidopsis+mutant+alh1+illustrates+a+cross+talk+between+ethylene+and+auxin%5BTitle%5D)+AND+%22Plant+Physiology%22%5BJournal%5D)">The Arabidopsis mutant alh1 illustrates a cross talk between ethylene and auxin.</a> Plant Physiology. 131 (3), 1228-1238 (2003).</li>
<li>Deslauriers, S. D., Larsen, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((FERONIA+is+a+key+modulator+of+brassinosteroid+and+ethylene+responsiveness+in+arabidopsis+hypocotyls%5BTitle%5D)+AND+%22Molecular+Plant%22%5BJournal%5D)">FERONIA is a key modulator of brassinosteroid and ethylene responsiveness in arabidopsis hypocotyls.</a> Molecular Plant. 3 (3), 626-640 (2010).</li>
</ol>

The authors declare no conflicts of interests.

Summary and significance 
Formation of a graft union is crucial for successful grafting, which requires direct and undisturbed contact between the rootstock and scion. The miniature size and fragility of seedlings of small plants such as Arabidopsis makes it technically challenging to meet this requirement. One technique developed in early Arabidopsis seedling grafting methods was to insert both the scion and the rootstock into a short silicone tubing collar to support the graft ju...

Grafting is an ancient horticultural technique that became an established agricultural practice by 500 BCE1. Grafting different varieties of crop plants to improve yields was the first use of this technique, and continues to be used for this purpose today. In the past decade, grafting has attracted an increasing amount of attention as a tool for molecular biologists to study long-distance signaling in plants2,3,4,5. While grafting adult plants is relatively easy, grafting plants soon after germination is challenging. Despite this, it is sometimes required to assess the effects of long-distance signaling in processes such as plant development, environmental responses, and flowering6,7,8.
Arabidopsis thaliana has been established as the model organism in plant biology for many reasons, including its relatively small size, rendering it easy to grow inside a lab. However, the small size and fragility of Arabidopsis seedlings makes grafting young seedlings very challenging. In many cases, extensive hands-on training is required to successfully obtain seedling grafts. There have been many methodological improvements over the years that have identified ideal growing conditions and new techniques to increase the success rate of seedling grafting9,10,11. The most recent tool introduced was an Arabidopsis seedling grafting chip, that allows even inexperienced users to achieve acceptable levels of grafting success12. While this advance has significantly lowered the technical barrier of seedling grafting, the chip device is expensive, and the number of grafts that can be conducted in parallel quickly becomes cost-prohibitive.
Additionally, this device can only be used for Arabidopsis seedlings that have hypocotyl dimensions that are similar to wild-type seedlings. While Arabidopsis is the keystone species in the world of plant molecular genetics, recent work has been done in other species using seedling grafting. Examples include the grafting of soybean and the common bean, tobacco to tomato, and canola to Arabidopsis, and subsequently sampling both tissues for small RNAs13,14. Therefore, a grafting method that is accessible to most laboratories and can be easily adapted to a wide range of plant species without any major technique changes is highly desirable.
This protocol details a method that employs in-house production of a simple grafting device that allows for the full customization of grafting channel diameter and length to accommodate any seedling morphology across most plant species. The production of these devices is very affordable and highly scalable, as the only components needed are silicone elastomer, wiring or tubing of the correct size, a high precision blade, and a container to serve as a mold. Following the grafting protocol detailed here, users can achieve successful grafting rates of 45% (n = 105), comparable with previously reported grafting results10,12.

<table><tbody><tr><td>15 mL conical tubes</td><td>VWR International Inc</td><td>10026-076</td><td></td></tr><tr><td>ACETONE (HPLC &amp;amp; ACS Certified Solvent) 4 L</td><td>VWR</td><td>BJAH010-4</td><td></td></tr><tr><td>BactoAgar</td><td>Sigma</td><td>A1296-500g</td><td></td></tr><tr><td>Dow SYLGARD 184 Silicone Encapsulant Clear 0.5 kg Kit</td><td>Dow</td><td>2646340</td><td></td></tr><tr><td>D-Sucrose (Molecular Biology), 1 kg</td><td>Fisher Scientific</td><td>BP220-1</td><td></td></tr><tr><td>Eppendorf Snap-Cap Microcentrifuge Flex-Tube Tubes (1.5 mL), pack of 500</td><td>Fisher Scientific</td><td>20901-551 / 05-402</td><td></td></tr><tr><td>Fisherbrand High Precision #4 Style Scalpel Handle</td><td>Fisher Scientific</td><td>12-000-164</td><td></td></tr><tr><td>Fisherbrand Lead-Free Autoclave Tape</td><td>Fisher Scientific</td><td>15-901-111</td><td></td></tr><tr><td>Fisherbrand square petri dishes</td><td>Fisher Scientific</td><td>FB0875711A</td><td></td></tr><tr><td>Leica Zoom 2000 Stereo Microscope</td><td>Microscope Central</td><td>L-Z2000</td><td></td></tr><tr><td>Micropore Tape</td><td>3M</td><td>B0082A9FEM</td><td></td></tr><tr><td>Murashige and Skoog Basal Medium</td><td>Sigma</td><td>M5519-10L</td><td></td></tr><tr><td>Parafilm</td><td>Genesee Scientific</td><td>16-101</td><td></td></tr><tr><td>potassium hydroxide</td><td>VWR International Inc</td><td>AA13451-36</td><td></td></tr><tr><td>Redi-earth Plug and Seedling Mix</td><td>Sun Gro Horticulture</td><td>SUN239274728CFLP</td><td></td></tr><tr><td>Scotts Osmocote Plus</td><td>Hummert International</td><td>7630600</td><td></td></tr><tr><td>Surgical Design No. 22 Carbon Scalpel Blade</td><td>Fisher Scientific</td><td>22-079-697</td><td></td></tr><tr><td>Tween 20, 500 mL</td><td>Fisher Scientific</td><td>BP337500</td><td></td></tr><tr><td>TWEEZER DUMONT STYL55 DUMOXEL POLS 110 MM</td><td>VWR</td><td>102091-580</td><td></td></tr></tbody></table>

scalable, flexible, and cost-effective seedling grafting

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.

Various aspects of the grafting strip's design were tested to identify the optimal grafting conditions that required the least amount of technical skill (Table 1). All grafting trials were completed on 0.5% sucrose MS medium, which has been previously reported to be an ideal grafting medium11,12.
Optimal seedling growth cannot be achieved with on-strip germination 
In the first iteration of the sili...

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 ...

scalable-flexible-and-cost-effective-seedling-grafting

Research

JoVE Journal

Biology

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Learn a scalable seedling grafting method for plant biology research using Arabidopsis and silicone elastomer devices. This low-cost protocol requires no prior experience and enables long-distance signaling studies.

Scalable, Flexible, and Cost-Effective Seedling Grafting

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