We are trying to demonstrate a general method for delivery of dye or DNA into plants using carbon nanofiber arrays. Many plants are recalcitrant to traditional transformation methods. Our method can solve this problem using carbon nanofiber arrays on flexible rigid substrates.
Our technique is fast and user friendly. It can be applied to virtually any plant species or plant organ. It can also be used to deliver other reagents, such as dyes and proteins.
Begin by preparing vertically aligned carbon nanofibers, or VACNFs. To transfer the VACNFs to a flexible substrate first, place the individual chips in acetone to separate the flexible substrate from the rigid substrate. Then wash SU-8 films with IPA for five minutes, followed by washing with water for five minutes.
Place the chips in a commercial box with a sticky pad when transporting them. Prepare a silicone rubber PET holder by mixing the elastomer and the crosslinker from a two part kit. Cut out a square of PET and tape it down in a clear plastic dish.
Then, pour a very thin film of silicone rubber on top of the PET and cure it at 80 degrees Celsius for 1 to 2 hours. Transfer the SU-8 film to the PET holder using a pair of tweezers and place the fiberless side of the SU-8 film on the silicone rubber. Press on the edges of the SU-8 square to help it stick to the silicone rubber PET and avoid breaking off fibers.
Then place a 1 microliter droplet of dye onto the fiber side of the SU-8 film and let it dry for 10 minutes. After drying, place the VACNF film onto the plant surface using a pair of sharp tweezers. Then, gently roll a small makeup applicator over the VACNF film, mark the areas where the flexible substrates are placed with a soft tipped marker.
Then, remove the flexible substrates from the plant surface using tape. Repeat VACNF application for controls, such as placing dye on the fiberless side of a film, using the film without dye, and using the fiberless side of a film. The on-chip method was used to deliver fluorescein dye to curved surfaces, such as strawberries in which a strong fluorescein signal was observed immediately.
Whereas in apples, a strong fluorescein signal was seen after two hours of delivery. Similarly, delivery of a plasmid containing yellow fluorescent protein was achieved in apples. Also, a successful transient transformation of onions was done by delivering the plasmid containing yellow fluorescent protein using VACNF films.
To begin, remove the photoresist from the VACNFs by washing with 100%acetone for five minutes, followed by a five minute wash with 100%IPA, and then double distilled water for five minutes. Then, place the plant tissue to be impaled on a hard surface for support. Place a 1 microliter droplet of dye onto the surface of the plant tissue.
Place a VACNF chip with a rigid substrate on the top of the droplet with the fibers oriented such that they will come into contact with the droplet. Using the flat side of a pair of tweezers, tap the chip, mark the area of the plant that the chip came into contact with using a soft tipped marker. Remove the VACNF chips after delivery.
Repeat these steps for controls, such as placing dye on the fiberless side of a film, using the film without dye, and using the fiberless side of a film. Store intact plants, or excised plant organs in humid chambers in long day conditions. For excised organs, use a plastic Petri dish with wet paper towels.
Using this method, fluorescein dye was delivered to Arabidopsis leaves. Images taken at different time points, showed that the fluorescein dye was observed immediately after delivery. Similarly, delivery of a plasmid containing yellow fluorescent protein was achieved in Arabidopsis leaves.
