Scalable, Flexible, and Cost-Effective Seedling Grafting

Summary

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.

Abstract

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.

Introduction

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 plants^2,3,4,5. 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 flowering^6,7,8.

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 grafting^9,10,11. 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 RNAs^13,14. 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 results^10,12.

Protocol

1. Device preparation

1. 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'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.
2. 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: Twist ties often contain 29 G wire and can be used after removing the outer paper coating with acetone.
3. 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.
4. Remove the silicone sheet from the Petri dish using clean forceps and move to a clean flat surface.
5. 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).
6. 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.
7. Autoclave the strips at 121 °C for at least 30 min and store until ready for use.

2. Seedling preparation

1. Sterilize and vernalize seeds.
   1. 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.
   2. Leave approximately 0.25 mL of water in the tubes with the seeds and store at 4 °C for 3 days in the dark.
2. Plate the seeds in preparation for grafting.
   1. 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.
   2. Under sterile conditions, move the appropriate number of prepared seeds to the plate, using a 20 µL pipette tip to aspirate and transfer the seeds.
   3. 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).
   4. 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.
3. 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°-110° angle with the benchtop surface (Figure 1C).
4. Store the plates in this orientation for 72 h in total darkness at 21 °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 µE m^-2 sec^-1) and 8 h dark cycles for 2-4 more days at the same temperature before grafting.
5. 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).

3. Grafting procedure

1. 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.
2. 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).
3. 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.
4. 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.
5. 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).
6. 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 °C with 16 h light/8 h dark cycles.
7. 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).
8. 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 °C.

Representative Results

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 medium^11,12.

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'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.

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. Please click here to view a larger version of this figure.

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. Please click here to view a larger version of this figure.

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. Please click here to download this Video.

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. Please click here to download this Video.

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. Please click here to download this Video.

Table 1: 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. Please click here to download this Table.

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. Please click here to download this Table.

Discussion

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 surface^9,11. 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 thickness^15,16,17.

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 regulation^18,19,20. Mutant lines defective in these hormone pathways may experience atypical etiolation rates^16,17. 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.

Materials

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