Early stage seeding grafting is a powerful tool for setting long-distance signaling in plants. We hope that this method will aid in the successful adoption of seeding grafting without hands-on training. Begin by preparing the square Petri dish, measuring 100x100 millimeters for casting the silicone elastomer.
Take 29 gauge wire in twist ties after removing the outer paper coating on the wire with acetone. And fully straighten the wire by rolling it on a hard, uniform surface with a heavy flat object, like a metal tube rack. Then, lay four pieces of straightened wires equidistant from one another in the square Petri dish, ensuring the wires lie flat on the bottom of the dish.
Pour the mixed silicon elastomer solution on top of the wires in the Petri dish and place the cover on the dish. After allowing the silicone to cure for 24 to 48 hours at room temperature, remove the silicone sheet from the Petri dish using clean forceps and move it to a clean, flat surface. Remove the wires from the silicone sheet.
To ensure the channels are open on one side, use fine-point forceps to clear the thin layer of silicone blocking the open side of the channels. Cut the silicone sheet perpendicular to the channels into three-millimeter strips using clean scissors. Place each strip in an aluminum foil envelope and seal it with autoclave tape.
Autoclave the strips at 121 degrees Celsius for at least 30 minutes before storing them until required. To sterilize and vernalize the seeds, take one milliliter of 50%bleach solution containing 0.1%Tween 20 in a 1.5-milliliter microcentrifuge tube and suspend up to 100 arabidopsis seeds in it. Incubate the seed suspension at room temperature for five to 10 minutes.
Post-incubation, aspirate or pipette out the bleach solution under sterile conditions. Rinse the seeds with one milliliter of sterilized distilled water. Be sure to invert the tubes adequately while rinsing the seeds.
To remove any bleach solution left at the top of the tube, repeat the rinsing four times. Leave approximately 250 microliters of water with the seeds in the tube, and store the seeds in the dark at four degrees Celsius for three days. Next, before plating the seeds, place the sterile strips on the surface of the prepared MS plate to guide the seed alignment with the channels on the strips.
Then, working under sterile conditions, use a 20-microliter pipette tip to aspirate and transfer the appropriate number of prepared seeds onto the plate. Once the seeds are plated, remove the sterile strips. Cover the plate.
And seal one of the sides of the plate parallel to the rows of seeds with parafilm. Wrap breathable tape on top of the parafilm and around all the other edges of the plate to allow for air exchange during incubation. Carefully orient two plates vertically with their parafilm-sealed sides facing down.
Separate them at the bottom by horizontally placing a 15-milliliter centrifuge tube in between, while securing them together on top with a rubber band. Ensure the plate surfaces form an angle of 100 to 110 with the benchtop surface. Store the plates in this orientation for 72 hours in total darkness at 21 degrees Celsius to allow seedling hypocotyls to grow about five millimeters in length.
After which, remove the setup from the dark and keep it under a 16-hour light and eight-hour dark cycle for two to four days at the same temperature before grafting. Graft the seedlings between five and seven 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 remains at the bottom of the silicone strip to prepare the seedling for cutting. Working in a sterile hood with the aid of a dissection scope, prepare the scions for grafting. Using a fresh scalpel blade, make a straight, clean, perpendicular cut across the hypocotyl.
Avoid pressing down and pushing the seedling into the agar. Next, remove the chute and keep the cut portion of the chute hydrated by ensuring contact with the media surface. Alternatively, the chute can be moved to a Petri dish filled with sterile distilled water.
To prepare the rootstocks, carefully grasp the root within the space between the closed forceps and turn gently to pull it, leaving the cut section of the rootstocks in the middle of the strip. Then, gently pick up the desired chute using the fine-tipped forceps and insert it into the top of the the channel. Visually, confirm the contact between the scion and rootstock to obtain a successful graft.
After all the grafts have been made, wrap the plates with parafilm and breathable tape, and set up the plates the same way as demonstrated before without disturbing the seedlings or silicone strips. Carefully move the plates to a growth chamber set at 26 degrees Celsius with a 16-hour light and eight-hour dark cycle. After seven to 10 days, evaluate the grafted seedlings under sterile conditions by using forceps to carefully peel off the silicone strip from one side and free the seedlings from the channels.
Remove any a adventitious roots growing from the scion by cutting them with a fresh scalpel blade or crushing them with fine-tipped forceps. Once again, visually evaluate whether the rootstock has become firmly attached to the scion forming a successful graft. Move the successful grafts to seedling propagation soil and cover the soil with transparent plastic for a few days while the seedlings get established.
Allow the plants to grow under the previously mentioned light and dark cycles at 21 degrees Celsius. Grafting trials to determine the optimal grafting protocol revealed an overall grafting success rate of 25%when the seeds germinated directly on the silicone strips with enclosed channels. Reorienting the seeds to keep the embryos'cotyledons and radical pointing downward during germination slightly increased the grafting efficiency to 33%Upon removing the silicone closing on the channels to allow contact between the seed and the growth medium, 85%of the seedlings germinated successfully.
However, the grafting success rate remained at 31%indicating that optimal seedling growth could not be achieved with on-strip germination. Interestingly, the demonstrated protocol in which seedlings were cultivated vertically on the grafting plate before being inserted into the open channels in the strips yielded an overall grafting success rate to 48%during the first two trials. Considering the lower success rate of the third and the relatively small trial, which may be attributed to slight variations in grafting sites, the overall grafting success was found to be 45%This is comparable with the method where the seedlings were cultivated on horizontal plates, cut on a solid surface, and inserted into grafting strips.
However, the demonstrated protocol is comparatively less time-consuming. Using this protocol, labs new to seedling grafting can rapidly create large numbers of grafting support devices while being able to customize the device to fit their specific needs easily.
