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>

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A., Munns, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stomatal+control+in+tomato+with+ABA-deficient+roots:+Response+of+grafted+plants+to+soil+drying.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+long-distance+signaling+via+plant+vascular+tissues.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shoot-root+communication+in+flowering+plants.">Shoot-root communication in flowering plants.</a> Current Biology. 27 (17), 973-978 (2017).</li><li>Thomas, H. 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G. N., Booker, J. P., Leyser, H. M. 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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 junction10. While this method is highly effective in protecting the two graft parts from separation, it is not an easy task to insert the fragile scion and rootstock into a narrow-bore tube. Moreover, the thickening at the junction from growth often makes it difficult to remove the narrow-bore silicone tubes without damaging the grafted seedlings. Other methods used alternative techniques to avoid the use of a grafting collar while preventing seedlings from moving during growth, including removing both cotyledons from the scions and growing grafted seedlings on an oblique surface9,11. While these methods require few resources to complete, when trialed, these methods resulted in high levels of scion migration away from the rootstock, resulting in lower grafting success than reported (80% on average for the recommended conditions in both studies) using these techniques. Additionally, they both require the user to align the interface between the graft seedling parts without any aids, a task requiring a high level of skill. A micrografting chip designed specifically for Arabidopsis seedling grafting has recently been reported12. Each chip contains four micro-chambers to allow four seedlings to grow, with the special design of the chamber space enabling the hypocotyl to grow inside a micro-channel. The rootstocks are generated in-place on the chip, and the scions are inserted into the micro-channels. This eliminates the need to handle both rootstocks and scions for grafting, and the micro-channel confines the grafting in place without sacrificing the ease of removing seedlings once the graft union is formed. While this new method greatly reduces the technical barrier of seedling grafting, the fabrication of the micrografting chip requires Micro-Electro Mechanical Systems (MEMS), a technology not readily accessible to most biology laboratories. The dimensions of the micro-chamber on the micrografting chip were optimized for Arabidopsis Col-012. This prevents use of the chips for other species or different Arabidopsis accessions/mutants that have hypocotyls that vary in height or thickness15,16,17.
The low-cost, flexible, and easy-to-follow method described here was inspired by previous micrografting chip work to facilitate young seedling grafting. In this method, the manipulation of delicate seedlings is minimal, easing the use for new users. The channels in the strips aid new users in aligning the grafted seedlings and keep the root and shoot in good contact during graft site formation, while also ensuring easy removal after the graft has been formed. Due to the high-throughput production of the silicone strips and relatively low cost of the starting materials, grafting large numbers of seedlings can be achieved at a fraction of the cost of the micrografting chips. This homemade device is estimated to cost $0.12 per grafting site, making this an economic option for labs working with a variety of plant species or seedling phenotypes (Table 2). While the handmade aspect of the grafting strips decreases the costs associated with this method, it also introduces the chance for variability between different strips. As previously emphasized, the quality of the grafting strips is key for the success of this method. The rise in use of early-stage seedling grafting in molecular genetics has given the field of plant genetics a powerful tool for studying long-distance signaling in plants. This simple, low-cost, and easily accessible method will provide another tool to help facilitate successful adoption of the seedling grafting technique by labs with minimal prior training and experience.
Critical considerations 
Processes such as initiating a wound repair response, establishing cell-to-cell communication between the scion and rootstock, and ultimately vasculature formation, are necessary for the formation of a successful graft9. During the grafting process, it is important to keep the seedlings hydrated and undamaged beyond necessity. Using this method, the cut ends of the roots and scions must be clean and perpendicular to ensure a flush connection between the scion and rootstock. If the grafting site is damaged during cutting, or the grafting pieces are not cut at the same angle, successful grafting is unlikely due to a lack of close contact between cell layers across the grafting junction. If any of the grafting parts of the plant are allowed to dry or are damaged, the previously mentioned processes will be inhibited, and grafting will not be successful. Young seedlings are fragile and easily crushed by forceps. If users find it necessary, the forceps can be used to hold onto one of the two cotyledons to maneuver the scion. Compared to the previously reported grafting chip method, the chance to damage the rootstock while pulling the hypocotyl down into the silicone strip is increased. Handling techniques to minimize this risk are described in protocol step 3.4.
A total of four grafting sites per strip is the recommended number of positions to ensure that the seedlings do not touch one another during the graft-junction formation stage. To scale up the total number of grafts performed at one time, it is recommended that users increase the number of plates used, rather than decrease the amount of space used per graft.
Modification of the protocol is possible to accommodate divergent hypocotyl growth or non-Arabidopsis species 
Some mutants etiolate at different rates to wild-type plants. Phytohormones such as gibberellins, brassinosteroids, ethylene, and auxin play an extensive role in seedling growth regulation18,19,20. Mutant lines defective in these hormone pathways may experience atypical etiolation rates16,17. The user-controlled design of the grafting strip allows for accommodation of these phenotypic differences, but sufficient testing in these cases is required. If a genetic line is used that experiences etiolation at significantly different rates from the wild type, users should determine if the 3 day etiolation period described here is appropriate for their lines before beginning. Inadequately etiolated seedlings will result in difficult grafting due to a shortened or excessively extended (and thinner) hypocotyl. Users interested in grafting seedlings of species other than Arabidopsis can adapt this protocol to suit their needs through changing the grafting channel diameter. Tomato and tobacco seedlings grown to appropriate grafting age appear to require 0.8 and 0.4 mm diameter grafting channels, respectively9.

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

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&#39;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 silicone strip, enclosed channels were made by leaving the thin layer of silicone that remains on the back of the strip channels after the wires are removed from the silicone sheets. Instead of germinating the seedlings directly on the plate and later placing the grafting strip over the seedling hypocotyls, the seeds were germinated directly on the strip after inserting the seeds into the channel filled with a small amount of MS medium. Using this technique, half of the seedlings were getting stuck in the channels during germination and failed to elongate. Including the non-germinated and the non-elongated seedlings, successful grafting rates of 25% (n = 16) were achieved (Table 1). To address the observed elongation failure, seeds were oriented so the embryo&#39;s cotyledons and radical were pointing downward during germination. Using this method, 75% of the seeds germinated and elongated successfully, which resulted in a slight increase in grafting efficiency (33% success, n = 12) (Table 1). To increase successful seedling development, the thin layer of silicone closing the channel was removed from the medium side of the grafting strip to allow contact between the seed and the growth medium (Figure 1A). Using this method, 85% of the seedlings germinated and elongated successfully, and 31% (n = 16) were successfully grafted (Table 1). Despite significantly raising the rate of proper seed development on the strip, losing even a small number of seedlings can significantly impact the experimental population size when performing reciprocal grafts between different experimental groups. For this reason, germinating seedlings directly on the medium surface was determined to be ideal.
Maintaining seedling survival rate and minimizing processing time and efforts 
Seedlings were first cultivated for grafting on MS plates (1% sucrose), transferred to a solid surface (such as the lid of a Petri dish filled with water) for cutting, then assembled on a plate with the strip for grafting. Using this technique, a 50% success rate (n = 8) was observed (Table 1). While this method obtained the highest rate of success, it was time-consuming and effort-intensive, requiring 2.5 min for each graft, limiting the number of grafts that could be assembled at once. To lower the time and effort required, seedlings were cultivated vertically on the grafting plate (0.5% sucrose) and then inserted into the strip directly before grafting, as described in this protocol. To further test this method, three trials were conducted of varying sizes. The first two trials both had a success rate of 48% (n = 25 and 64) and the third trial had a success rate of 25% (n = 16) (Table 1). Together, these trials indicate a success rate of 45% (n = 105). This success rate is comparable to the more time-consuming method and requires approximately 1 min per graft to place the grafting strip over the seedlings and to complete the grafting (protocol steps 3.2-3.5).
The rate of grafting success is heavily reliant on the quality of the grafting strip construction. While the first two trials have a success rate of 48%, the third trial has a lower success rate. The handmade quality of the grafting strip inherently results in slight variability between grafting sites. If the wires used for strip construction are not completely straight, then the channels in the strips will not be flush with the bottom of the mold and will result in channels of slightly variable depths. In total, 25 strips are made in each strip casting, resulting in 100 unique grafting sites (Table 2). While the effect of slight variation in the grafting sites is balanced out in the two larger trials, in the smaller trial with lower rates of success, it seems that grafting sites with more variation due to slightly curving wires may negatively impact graft formation. To minimize this type of variation, a wire-straightening technique is described in protocol step 1.2.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64519/64519fig01.jpg" /> 
Figure 1: Illustration of the grafting preparation steps. (A) Model of the silicone strip mold design. This panel demonstrates the square plate mold used for casting the silicone strips. Four sections of 29 G wires of equal length are laid at the bottom of the plate, and silicone elastomer mix is poured on top of them (above). After the silicone has fully cured, the silicone square is removed from the plate and the wires are removed to create the grafting channels. A thin layer of silicone remains on the bottom of the channels after the wires are removed (indicated by *). This layer should be removed to leave the channels open on the bottom (below). The square is then cut perpendicularly to the wire channels into 3 mm strips, as shown by the dotted lines (above). (B) Seed positioning on the grafting plate. Using the aid of a sterile strip, two rows of seeds are positioned on the plate in line with the strip channels. After placing the seeds, this strip should be removed to prevent any germination hindrance. To prepare MS plates for vertical growth, parafilm should be wrapped along the bottom (root-side) edge of the plate, marked here with blue highlighting. (C) Vertical plate position for seed germination. After plating, the seeds are germinated vertically on angled plates. A 15 mL conical tube is placed at the base of two plates to create a slightly obtuse angle between the plate surface and the benchtop. The parafilmed edge (marked with blue) of the plate is oriented toward the benchtop surface. A rubber band is wrapped around the top of the two plates to keep them in place. (D) Seedling positioning in the strip directly before grafting. Sterile grafting strips are placed on top of the seedlings with hypocotyls inside the strip channels. <a href="https://www.jove.com/files/ftp_upload/64519/64519fig01large.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/64519/64519fig02.jpg" /> 
Figure 2: Image captured on a dissection scope showing a healed grafting site. The black arrow indicates the graft junction site, and the red arrows indicate adventitious roots that were removed during the graft evaluation process. Scale bar = 0.5 mm. <a href="https://www.jove.com/files/ftp_upload/64519/64519fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Video 1: Demonstration of active grafting using live plant imaging. The placement and motion of the scalpel blade to create a clean cut. The seedling hypocotyl is cut directly above the grafting strip by pushing the scalpel blade forward. <a href="https://www.jove.com/files/ftp_upload/64519/Video 1 - cutting.mp4" target="_blank">Please click here to download this Video.</a>
Video 2: Positioning the cut rootstock hypocotyl for grafting within the strip. The rootstock is gently pulled usingforceps to position the graft junction site in the middle of the strip. <a href="https://www.jove.com/files/ftp_upload/64519/Video 2 - root positioning.mp4" target="_blank">Please click here to download this Video.</a>
Video 3: Placing the scion and completing the graft. The scion is replaced and pushed into the strip until it connects with the rootstock. Proper alignment and contact is evaluated by seeing the hypocotyl respond when the scion is lightly pushed. <a href="https://www.jove.com/files/ftp_upload/64519/Video 3 - scion positioning.mp4" target="_blank">Please click here to download this Video.</a>
Table 1:&#160;Grafting trial variables and success rates. This table summarizes the results of the grafting trials used to determine the optimal grafting protocol for the highest rates of success. The variables examined in these trials include seedling germination conditions, silicone strip construction, and when seedlings were inserted into the silicone strip channels during grafting. <a href="https://www.jove.com/files/ftp_upload/64519/64519_Table 1.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: The number of grafting sites that can be generated from a 250 mL bottle of silicone elastomer. The price for each unit of production is also shown here (at the time of publication), and can be easily adjusted upon fluctuations in the market value of the silicone elastomer reagents. <a href="https://www.jove.com/files/ftp_upload/64519/64519_Table 2.xlsx" target="_blank">Please click here to download this Table.</a>

Watch this Scientific Journal Video about Scalable, Flexible, and Cost-Effective Seedling Grafting at JoVE.com

Scalable, Flexible, and Cost-Effective Seedling Grafting

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

Nanyang Technological University

Research

JoVE Journal

Biology

1.7K Views.  North Carolina State University. 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.

Video: Scalable, Flexible, and Cost-Effective Seedling Grafting

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression and activation of signal transduction pathways. This technique allows the delivery of signaling molecules at precisely defined developmental stages for specific times. After this embryos can be harvested and gene expression examined, for example by in situ hybridization, or activation of signal transduction pathways observed with immunostaining.
In this video heparin beads soaked in FGF18 or AG 1-X2 beads soaked in U0126, a MEK inhibitor, are grafted into the limb bud in ovo. This shows that FGF18 induces expression of MyoD and ERK phosphorylation and both endogenous and FGF18 induced MyoD expression is inhibited by U0126. Beads soaked in a retinoic acid antagonist can potentiate premature MyoD induction by FGF18.
This approach can be used with a wide range of different growth factors and inhibitors and is easily adapted to other tissues in the developing embryo.

By grafting beads soaked in growth factors or specific inhibitors of signaling pathways into developing embryos it is possible to directly test their effects in vivo. In this protocol beads are grafted into the limb bud to determine the effects of these molecules on gene expression and signal transduction.

Using chicken embryos it is possible to test directly the effects of either growth factors or specific inhibitors of signaling pathways on gene expression ...

Developmental Biology

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Environment

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is an accumulating evidence showing that the expressions of certain miRNAs are altered when grafting, an agricultural practice commonly used by farmers to improve crop tolerance to biotic and abiotic stresses. Bottle gourd is an inherently climate-resilient crop compared to many other major cucurbits, including watermelon, rendering it one of the most widely used rootstocks for the latter. The recent advancement of high-throughput sequencing technologies has provided great opportunities to investigate cold-responsive miRNAs and their contributions to heterograft advantages; yet, adequate experimental procedures are a prerequisite for this purpose. Here, we present a detailed protocol for efficiently generating homo- and heterografts between the cold-susceptible watermelon and the cold-tolerant bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis. The presented methods are also useful for other plant-grafting systems, to interrogate miRNA regulations under various environmental stresses, such as heat, drought, and salinity.

Here we present a detailed protocol for efficiently making homo- and heterografts between watermelon and bottle gourd, in addition to methods of tissue sampling, data generation, and data analysis, for the investigation of cold-responsive microRNAs.

MicroRNAs (miRNAs) are endogenous small non-coding RNAs of about 20 - 24 nt, known to play important roles in plant development and adaptation. There is ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Bioengineering

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, engineered grafts offer great potential for patient treatment. However, engineered vascular grafts are generally not easily scalable, requiring manufacture of custom molds or polymer tubes in order to customize to different sizes, constituting a time-consuming and costly practice. Human arteries range in lumen diameter from about 2.0-38 mm and in wall thickness from about 0.5-2.5 mm. We have created a method, termed the "Ring Stacking Method," in which variable size rings of tissue of the desired cell type, demonstrated here with vascular smooth muscle cells (SMCs), can be created using guides of center posts to control lumen diameter and outer shells to dictate vessel wall thickness. These tissue rings are then stacked to create a tubular construct, mimicking the natural form of a blood vessel. The vessel length can be tailored by simply stacking the number of rings required to constitute the length needed. With our technique, tissues of tubular forms, similar to a blood vessel, can be readily manufactured in a variety of dimensions and lengths to meet the needs of the clinic and patient.

Scalable engineered blood vessels would improve clinical applicability. Using easily sizable 3D-printed guides, rings of vascular smooth muscle were created and stacked into a tubular form, forming a vascular graft. Grafts can be sized to meet the range of human coronary artery dimensions by simply changing the 3D-printed guide size.

Coronary artery disease remains a leading cause of death, affecting millions of Americans. With the lack of autologous vascular grafts available, ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Embryo Rescue Protocol for Interspecific Hybridization in Squash

Interspecific hybridization in Cucurbita crops (squash) is desirable for widening genetic variation and for the introgression of useful alleles. Immature embryos generated from these wide crosses must be regenerated using appropriate embryo rescue techniques. Although this technique is well established for many crops, a detailed description of the appropriate methodology for squash that would allow its routine application is lacking. Here, we describe an embryo rescue protocol useful for interspecific hybridization of C. pepo and C. moschata. To identify viable combinations for embryo rescue, 24 interspecific crosses were performed. Fruit set was obtained from twenty-two crosses, indicating a 92% success rate. However, most of the fruits obtained were parthenocarpic, with seeds devoid of embryos (empty seeds). Only one cross combination contained immature embryos that could be regenerated using basal plant growth media. A total of 10 embryos were rescued from the interspecific F1 fruit, and the success rate of embryo rescue was 80%. The embryo rescue protocol developed here will be useful for interspecific hybridization in squash breeding programs.

The&#160;article describes an embryo rescue protocol for the regeneration of immature embryos derived from the interspecific hybridization of Cucurbita pepo and Cucurbita moschata. The protocol can be easily replicated and will be an important resource for squash breeding programs.

Interspecific hybridization in Cucurbita crops (squash) is desirable for widening genetic variation and for the introgression of useful alleles. Immature ...

A Simple Protocol for Mapping the Plant Root System Architecture Traits

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar tolerance to environmental challenges. An experimental protocol is presented for setting up the hydroponic system, plantlet growth, RSA spreading, and imaging. The approach used a magenta box-based hydroponic system containing polypropylene mesh supported by polycarbonate wedges. Experimental settings are exemplified by assessing the RSA of the plantlets under varying nutrient (phosphate [Pi]) supply. The system was established to examine the RSA of Arabidopsis, but it is readily adaptable to study other plants like Medicago sativa (Alfalfa). Arabidopsis thaliana (Col-0) plantlets are used in this investigation as an example to understand the plant RSA. Seeds are surface sterilized by treating ethanol and diluted commercial bleach, and kept at 4 &#176;C for stratification. The seeds are germinated and grown on a liquid half-MS medium on a polypropylene mesh supported by polycarbonate wedges. The plantlets are grown under standard growth conditions for the desired number days, gently picked out from the mesh, and submersed in water-containing agar plates. Each root system of the plantlets is spread gently on the water-filled plate with the help of a round art brush. These Petri plates are photographed or scanned at high resolution to document the RSA traits. The root traits, such as primary root, lateral roots, and branching zone, are measured using the freely available ImageJ software. This study provides techniques for measuring plant root characteristics in controlled environmental settings. We discuss how to (1) grow the plantlets, and collect and spread root samples, (2) obtain pictures of spread RSA samples, (3) capture the images, and (4) use image analysis software to quantify root attributes. The advantage of the present method is the versatile, easy, and efficient measurement of the RSA traits.

We use simple laboratory tools to examine the root system architecture (RSA) of Arabidopsis and Medicago. The plantlets are grown hydroponically over mesh and spread using an art brush to reveal the RSA. Images are taken using scanning or a high-resolution camera, then analyzed with ImageJ to map traits.

Comprehensive knowledge of plant root system architecture (RSA) development is critical for improving nutrient use efficiency and increasing crop cultivar ...

One-Step Hairy Root Transformation Method for Advancing Root-Related Biology

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a powerful tool to study root-related biology. Hairy root transformation is established in a wide range of dicotyledons and in several monocotyledon species and is almost independent of the genotype. The traditional method of hypocotyl injection with A. rhizogenes to obtain composite plants is inefficient, time-consuming, laborious, and frequently causes the death of tender and tiny hypocotyl plants. A highly efficient, one-step hairy root transformation mediated by A. rhizogenes was established previously, which eliminates the need for transplanting after producing hairy roots. In this study, a partial hypocotyl and primary root were removed, the hypocotyl incision site was coated with A. rhizogenes, and&#160;then hypocotyls were planted in sterile vermiculite. After 12 days of cultivation, the hypocotyl incision expanded and new hairy roots were induced. This article provides the detailed protocol of a one-step transformation method mediated by A. rhizogenes, with its effectiveness demonstrated by producing composite plants of wild soybean, Solanum americanum, and pumpkin.

Author Spotlight: Streamlining Composite Plant Production via Agrobacterium rhizogenes-Mediated Hairy Root Transformation 

Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...

Scalable, Flexible, and Cost-Effective Seedling Grafting

Summary

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Chapters in this video

Grafting of Beads into Developing Chicken Embryo Limbs to Identify Signal Transduction Pathways Affecting Gene Expression

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

Generating Homo- and Heterografts Between Watermelon and Bottle Gourd for the Study of Cold-responsive MicroRNAs

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

Scaling of Engineered Vascular Grafts Using 3D Printed Guides and the Ring Stacking Method

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

Embryo Rescue Protocol for Interspecific Hybridization in Squash

A Simple Protocol for Mapping the Plant Root System Architecture Traits

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation