Scalable, Flexible, and Cost-Effective Seedling Grafting

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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.

Wyniki

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 th...

Dyskusje

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...

Materiały

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