The overall goal of the lateral root inducible system is to synchronously induce the initiation of lateral roots in plants using hormone treatments. Plant roots are the hidden and are known to be extremely difficult to study. We have developed an in vitro system that allows to study the process of lateral root initiation on a cytological, histological, and molecule level.
Generally, scientists new to this method might struggle because the plants have not grown into proper contact with the medium, preventing a homogenous induction of the lateral roots. Visual demonstration of this method helps showing the critical steps that should be taken into account to ensure a homogenous induction. Although this system was originally developed in a little plant Arabidopsis, it can also be applied to other systems such as maize, rice, or barley.
To begin, apply a 20-micron nylon mesh to NPA-supplemented growth medium using a pair of sterile tweezers. Gently push the mesh onto the growth medium using a drigalski to make sure it is in good contact with the medium. Next, pinch a sterile toothpick into the agar to create a sticky surface on the toothpick.
Use the toothpick to evenly distribute a batch of gas-sterilized seeds onto the prepared plates. Seal the plates with breathable adhesive tape and store them at four degrees Celsius in the dark for at least two days and a maximum of one week. This is needed for stratification and ensures synchronized and efficient germination.
On the third day, move the plates to the growth room for two days of germination followed by three days of growth at an angle of 80 to 90 degrees. On day eight, identify the roots that have grown entirely in contact with the mesh and on the contrary, roots that are not in contact with the mesh. Remove all the seedlings which roots are not in contact with the mesh.
Transfer the seedling-containing meshes to plates supplemented with 10 micromolar NAA. Once on the plate, skim the mesh over the medium surface if necessary to make sure that it is in good contact with the growth medium. To ensure a homogenous induction in all seedlings, it is important to discard the seedlings that have not grown into contact with the mesh on NPA-supplemented medium and to properly skim the mesh if necessary on NAA-supplemented medium.
Seal the new plates with breathable tape and place them in the growth room in a vertical position for the desired amount of time after induction. When ready to collect the samples, use a scalpel to cut the root segments all at once directly on the mesh and sample them by gently scraping the surface of the mesh. We will now show some steps of the lateral root inducible system in maize.
Begin by sterilizing the maize kernels by first placing the necessary number of kernels into a sterile glass beaker. Then, add 100 milliliters of 6%sodium hypochlorite in water to the beaker and add a magnetic stir bar. Place the beaker on a magnetic stirrer and stir at a low speed for five mintues at room temperature.
Gently remove the bleach solution. Replace it with 100 milliliters of fresh sterile water and rinse the kernels for five minutes. Repeat this step five times.
Put on a pair of gloves and tear off two stretches of hand paper towels, placing them on top of each other on a clean surface. Then, fold the sheets double over the length. Use tweezers to distribute 10 kernels approximately two centimeters from the top, over the entire length of the paper while keeping an interspace distance of eight centimeters between each kernel and eight centimeters free at both ends.
Make sure the radical of the kernel is facing down and toward the paper. Gently roll the paper over the length while keeping the seeds in place. Spraying the paper with sterile water can facilitate this step if necessary.
Put the rolls into sterilized glass tubes and combine additional tubes into the rack. Pour 125 milliliters of a 50 micromolar NPA solution over the paper roll in the tube. The paper roll will absorb the solution and become completely soaked.
Place the paper roll system in a growth cabinet for three days. Make sure that the paper rolls remain soaked during incubation by adding extra NPA solution as needed. To ensure homogenous induction of lateral root initiation, it is important to check that the paper rolls remain moisturized in NPA solution, but that the roots are not submerged in the liquid itself.
After three days on NPA, the maize seedling has a small shoot and a root of approximately two to three centimeters. On day four, gently squeeze out most of the remaining NPA solution from the paper rolls and place the rolls in sterile water until they become saturated. After about five minutes, gently squeeze out most of the water from the paper rolls.
Repeat the wash step three times. Following the final rinse, squeeze out most of the water and place it back into the tube. Pour a 50 micromolar solution of NAA over the paper rolls in the tubes until they become completely soaked.
Then, place the paper roll system in the growth cabinet for the desired duration after induction. In comparison to oxen response marker line pDR5-GUS seedlings that haven't grown on NAA or have grown for one hour on NAA, the seedlings grown for two hours on NAA show an oxen response which is one of the first events triggering lateral root initiation. These divisions start moving up from the tip starting at around two hours, and increase with prolonged exposure to NAA.
Using the cell cycle marker line pCYCB1;1-Gus, the first cell divisions that lead to lateral root formation can be seen starting at around six hours after the start of NAA treatment. Microarrays and quantitative rt-PCR can be used to evaluate expression of various genes throughout the different regions of the Arabidopsis and maize roots during lateral root induction. In both Arabidposis and maize, when the plants grow for a longer time, the induced divisions will give rise to the development and outgrowth of lateral roots.
As proof of concept for the system on maize, here are seedlings grown under different conditions for comparison. Seedlings grown for 10 days on water have lateral roots that didn't initiate synchronously and are widely spaced along the axis. Seedlings grown for 10 days on NPA contain no lateral roots at all.
Seedlings grown for three days on NPA, followed by an NAA treatment, show a massive and synchronized induction of lateral roots. In this video we have shown you how to set up a lateral root inducible system that has turned out to be very essential in the discovery of fundamental new insights into the process of root branching. We have shown you how it works for Arabidopsis and maize, but it can easily be adapted to other plant species of choice.
Good luck.
