Biomimetic Replication of Root Surface Microstructure using Alteration of Soft Lithography

Biomimetics has been previously used as a tool to study leaf-microorganism interactions. However, no such tool exists for roots. Here, we develop a protocol to form synthetic surfaces mimicking root surface microstructure for the study of root-environment interactions.

This protocol makes it possible to develop a synthetic system mimicking the root surface microstructure. The system can be used for studying root-environment interactions in a control manner with emphasis on surface structure. This is a simple technique that can be performed in any lab studying root-environment interactions.

Additionally, this technique enables the formation of the surface from various materials. The most difficult step is the removal of the natural root from the negative mold. This step requires gentle handling of the root to avoid tearing the root hairs.

Visual demonstration will enhance the viewer's ability to replicate this procedure. Begin by removing the plant from soil after three weeks of growth. Cut the root system from the plant at the point of interaction with the stem and put the rootless plant in a beaker with water.

After a few days, cut the adventitious roots that emerge from the stem and use them for replication. To prepare seed germinated roots, wet a filter paper inside a Petri dish with water and put several M82 seeds on the paper. Incubate the dish at 25 degrees Celsius and hydrate the paper every day.

Approximately five days later, the germinating roots will be long enough to use for replication. Prepare negative replica solution by adding 9.49 grams of diurethane dimetharylate to a 50 milliliter cup. Add 1.45 milliliters of ethyl methacrylate to the cup and store at room temperature until the solution becomes homogenous and looks clear.

Add three milliliters of the plasticizer and stir the solution for another hour. Then add 300 microliters of the photo initiator and stir overnight or until all bubbles are removed. To generate the negative replica of the root, take a clean glass slide and pour one milliliter of the negative replica solution on it.

Place two to three roots over the solution, making sure that the roots are not fully covered. Keep the slide under an eight watt ultraviolet lamp for eight to 10 minutes, then switch off the UV lamp. Remove the replica from the glass slide and put it in a Petri dish filled with ethanol to remove unreacted monomer.

Use forceps to slowly and gently remove the root from the replica. To prepare the PDMS solution for the positive replica, place 10 grams of dimethyl siloxane in a cup. Add one gram of curing agent and mix thoroughly.

Heat the mixture in a desiccator under vacuum for two hours to remove air bubbles. Place the polyurethane negative replica in a Petri dish and pour the PDMS mixture on top of the negative replica. Apply vacuum for two hours to assure coverage of the microstructure and keep the Petri dish at room temperature overnight.

On the next day, separate the cured positive replica from the negative replica using forceps. To prepare the positive replica from ethyl cellulose, add 20 milliliters of ethanol to a 100 milliliter cup. Then add 1.32 milliliters of plasticizer and 3.3 grams of ethyl cellulose.

And stir the solution at room temperature for two hours. On the next day, pour the ethyl cellulose solution on top of the negative replica in a Petri dish and leave the dish under the hood overnight. Then slowly remove the positive replica from the negative replica with forceps.

A good negative replica can be generated if the protocol is followed correctly. This replica will show the cell structure of the root surface as well as holes representing the location of the root hairs. If the root is submerged in the polyurethane solution, it will become entrapped in the hard polymer and remain within the negative replica after it is cured.

When curing with the UV light, exceeding the recommended curing time, will result in an extremely hard polyurethane mold, making it impossible to remove the root without breaking it. Sometimes the root pieces are so small that microscopy is required to see them. Positive replicas were generated with PDMS and ethyl cellulose.

The root hairs are in the elongation zone where they begin to emerge. Their lengths varies along the root surface as they become longer, similar to the natural root. Some of the hairs on the ethyl cellulose replica are even visible under the light microscope.

When placing the root over the negative replica solution, it is important to choose the right side and avoid the submergence of the root inside the solution. This technique was developed to supply a new avenue to research root surface environment interactions. Specifically, the physical forces caused by microstructure and material properties of the surface.