Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

The overall goal of this procedure is to produce the reversal of the geomagnetic field or GMF with helm Holts coils controlled by a power supply. Ultimately, it is shown that GMF reversal affects the morphology and gene expression of plants. This method can help to answer key questions in a plant evolution field, such as could the reversal of the earth magnetic field have played a role in plant evolution and adaptation.

The main Advantage of this technique is that it can be used to test for potential biological effects of weak magnetic fields in a simple model organism that can be grown with many replicates, all under precisely controlled conditions. Demonstrating this procedure will be Alberto Bocco and under guided student from my laboratory, and also Kiara aa. She's a PhD student in my laboratory and CIA Bertea, who is an associate professor in my group.

To begin turn on the three DC power supplies that are connected to the three couples of Helm Holt's Coil pairs, and turn on the three axis magnetometer whose probe is inserted in the triaxial coils. Turn on the computer and launch the magnetometer software that allows data to be collected from the three axis magnetometer. The power supplies are also connected to a computer via A-G-P-I-B connection for control.

Set the voltages of the power supplies to generate the desired magnetic field with a reversed magnetic fields vector as described in the text protocol. Place the seeds of Arabidopsis Aliana ecotype Columbia, zero into a 1.5 milliliter tube surface, sterilized by treatment with a calcium hypochlorite solution for 10 to 12 minutes at 25 to 28 degrees Celsius with continuous shaking. Then rinse the seeds twice with 80%ethanol, followed by a wash with a hundred percent ethanol.

And finally, a rinse with sterile distilled water. Prepare one liter of Murrah Shiga and SCOG modified medium as described in the text protocol before solidification. Pour 80 milliliters of medium into each square Petri plate, so 30 sterile seeds on a plate.

Then seal the plates with a waxy film. Vern lize the plates horizontally in darkness at four degrees Celsius for two days to potentiate and synchronize germination. Prepare sodium vapor lamps by covering the spotlight with a blue gelatin film to reduce the red component of the lamps following vernalization.

Prepare to expose Petri plates to either normal or reversed.GMF. Expose the seeds in a climate controlled environment at 22 degrees Celsius in a vertical position. In parallel experiments both outside the triaxial coils and inside the triaxial coils.

Under a photo period schedule of eight hours, darkness, and 16 hours light. After exposure, take pictures of the Petri dishes to calculate the root length and leaf areas. First, measure the side of the Petri plate.

Then open the image of the Petri plate in image J software and use the straight line option to draw a line that exactly crosses the plate side. In the analyze menu, select set scale and insert the actual distance in the known distance box. Then insert the unit of length.

And finally click the global option to make settings available for all measurements for root length. Carefully follow the shape of the root by using the freehand tool. Measure the length by using the measure option in the analyze menu.

Continue to measure all roots in the picture and save the file for further statistical analysis. For leaf area from the image menu, use the adjust option, followed by color threshold, select the individual leaf, and in the analyze menu, select analyze particles, save the individual measurements for statistical analysis. Collect 30 shoots and 30 roots separately, and immediately freeze them in liquid nitrogen.

Then grind in liquid nitrogen with a mortar and pestle before isolating total RNA as described in the text protocol, perform all experiments on a real-time system using cyber green one With ROX as an internal loading standard, perform the reaction with a 25 microliter mixture. Using the primers listed in the text protocol include non reverse transcription controls to monitor for genomic DNA contamination, as well as non template controls. Read fluorescence following each a kneeling and extension phase.

The two magnifying glasses represent the data collected, including a kneeling and dissociation curves. Navigate to the analysis and setup screen and ensure that the dataset collected during the dissociation segment of the experiment is selected. For analysis for all runs, perform a melting curve analysis from 55 to 95 degrees Celsius by including the dissociation segment in the thermal profile.

Use the dissociation curve screen access through the results tab. To view the dissociation profile. Analyze all amplification plots with the real-time PCR instrument software.

To obtain CT values, calibrate and normalize relative RNA levels with the level of the best housekeeping genes. Access the amplification plots screen through the results tab. Select the ramp or plateau for which data should be analyzed using the analysis, selection, and setup screen.

And then select the baseline corrected normalized fluorescence from the fluorescence menu on the command panel. Access the plate sample value screen through the results tab. To display CT values for the sampled wells.

Use four different reference genes to normalize the results of the real-time PCR. Select the top ranked gene using the analysis software and use the most stable gene for normalization. Briefly organize the input data on an Excel sheet with the first column containing the gene names and the first row containing the sample names.

Then select the analysis software from the menu bar. Use the dialogue box to select the input data. Next, check the field sample names, gene names, and simple output only.

Click on the go button To perform the analysis, select the top ranked gene as the candidate gene most stably expressed. After 10 days of exposure control plants show a significantly greater root length and more expanded leaflets as compared to plants that were exposed to reversed GMF conditions after 10 days of exposure. The effect of reversal of the GMF was a drastic change in the gene expression of all tested genes, including crucifer in three copper transport protein one and redox responsive transcription factor one bars indicate standard error asterisk indicate significant differences between plants exposed to reversed and normal GMF conditions.

The effect of reversal of the GMF induced no significant changes in shoot gene expression. However, a drastic downregulation was observed in root gene expression of plants grown. Under reverse GMF conditions, these genes include thal, aate peroxidase, aate peroxidase, one iron superoxide dismutase, one N-A-D-P-H, respiratory burst oxidase, protein, and catalyst three.

Once master, this technique can be done from minutes to today's, according to the experiment required if it is performed properly. After watching this video, you should have a good idea of how to reverse or alter the geomagnetic field and to perform morphological and genetic studies on exposed plants. While attempting this procedure, it is important to remove any magnetic field sources from the region surrounded with helmet coils.

It is important that the helmet coils and their generators do not generate heat or vibrations. The investigator should be blinded as the magnetic field exposure condition until the final data have been recorded. This technique has enabled researchers in magneto reception to explore the effect of magnetic fields on plant evolution, animal behavior, and navigation, and on human cell lines.

In future, we hope to perform similar studies in microgravity in order to evaluate how the alteration of the magnetic field will impact in space condition on plant growth. Working with systems that generate weak magnetic fields is not thought to be hazardous. However, you should take the normal precautions and you should seek the advice of a competent electrician before working with equipment such as the power supplies for the helm holds coil pairs.