Measuring Endoreduplication by Flow Cytometry of Isolated Tuber Protoplasts

The overall goal of this experiment is to evaluate levels of endoreduplication within potato tubers. This method can help in answer key questions about potato tuber morphology, such as the developmental stage in which tuber tissues undergo endoreduplication. The main advantage of this technique is that it provides much clearer results compared to typical flow cytometry preparations.

Demonstrating the procedure will be Parker Laimbeer. A grad student from my laboratory. After preparing solutions and buffers according to the text protocol, surface sterilize potato tubers by immersing them in 71%ethanol for at least five minutes.

Remove the plant materials from the ethanol and allow them to air dry. Prepare and label sterile 50 milliliter conical tubes for each sample and aliquot 15 milliliters of filter sterilized plasmolysis incubation solution or PIS, into each. Using either a sterilized scalpel or a cork bore sample approximately one cubic centimeter of the desire to sterilize tissue.

Use the sterilize scalpel to coarsely chop the tissue into approximately three cubic millimeter pieces. And transfer the tissue to a conical tube containing PIS. Then incubate the tissue at four degree Celsius overnight.

To generate potato protoplasts begin by preparing and filtering an appropriate amount of enzyme solution or ES with 0.71 molar mannitol, three millimolar calcium chloride, one millimolar monopotassium phosphate. One millimolar magnesium chloride, 4%Onozuka R-10 cellulase, 0.8%Macerozyme R-10, 1%hemi cellulase and 10 millimolar MES buffer, pH 5.8. Using a serological pipette, aspirate off the PIS from the samples in the tubes.

Add 10 milliliters of the ES to each sample and invert the tubes two to three times. Then incubate the samples at 29%Celsius and 180 RPM overnight. On day three, to harvest and wash the protoplasts, remove the samples from the shaker and allow them to settle for approximately 10 minutes.

With a serological pipette, aspirate off as much of the ES solution as possible, taking care to avoid the digested tissue. Then using a micro pipette and while holding the tube at an angle, remove any remaining ES solution again avoiding the digested tissue. It is critical to remove as much of the protoplast wash solution as possible to avoid damage to the nuclei.

Holding the tube at an angle may help with the removal of the solution while leaving the protoplasts. Add 15 milliliters of PWS to each sample. And invert the tube gently two to three times.

Allow the protoplasts to settle for 10 minutes. Then use a serological pipette to remove the PWS. And a micro pipette to remove any remaining liquid.

While keeping the samples on ice, add 1.5 milliliters of ice cold FCB to each tube. Briefly shake or vortex each sample to break up aggregated tissue. It is important to break up the clumps of tuber tissue so that the FCB may fully permeate the cells and release the nuclei.

If a control is to be added beginning at this step use a razor blade to finely chop a small leaf in 1.5 milliliters of ice cold FCB. Control samples should be the same ploity as the experimental samples preferably the same genotype. Insert a 1.5 milliliter microcentrifuge tube with the tip cut off and 106 micrometer metal mesh melted to the bottom into a two milliliter micro centrifuge tube.

Then pass one milliliter of the FCB tissue suspension through the filter. Add 250 microliters of the RNA solution to each sample. Then invert the tubes and incubate them for 30 minutes at room temperature.

Add 125 microliters of previously prepared propidium iodide or PI solution to each sample. Invert and incubate the tubes on ice for 30 minutes. Use samples as soon as possible and within two hours two avoid degradation.

Create two dot plots using logarithmic scale of forward scatter versus side scatter and PI versus side scatter. Also create a histogram with PI on the x axis. Logarithmic scale is required to ensure all events are within scale as nuclei may differ vastly in fluorescence.

Load a tube of a known control sample such as an in vitro leaf tissue from a sample genotype. And adjust the voltage so that all events are on scale. Make note of the channel of the 2C peak in the control sample.

The 2C peaks of the experimental samples should fall in the same location. Load an experimental tuber sample and again ensure that all events are on scale. If adjustments are required repeat the voltage adjustment of the control sample to identify the channel of 2C peaks.

Manually gate the protoplast nuclei using the side scatter versus PI plot. Then set the PI histogram to only show the gated protoplast nuclei region. Collect the desired number of events from each sample.

Frequently researchers use 10, 000 gated events for flow cytometry. However, 2000 events are used here for tuber protoplast samples to accommodate more samples and samples with low concentrations. The generation of protoplasts is necessary to achieve repeatable flow cytometry results from potato tubers.

The general morphology of which is displayed here. The separation between pith and perimedullary parenchyma is illustrated with black lines. The vascular ring, which separates parenchyma from the cortex, is denoted with a black arrow.

Isolated protoplasts should be spherical and symmetrical with the plasma membrane intact. Note the size difference between leaf and tuber protoplasts as well as the differences in size within a tissue which may indicate different levels of endoreduplication. This figure displays the variation in results that maybe encountered in both the flow cytometry scatter plots and histograms.

Intact nuclei show clean separation between peaks to quantify relative abundance of cells, whereas peaks integrated samples show shifted wide and overlapping bases. In the corresponding scatter plots the black boxes indicate nuclear event gating for histograms and the clustering of events is reflected in the width of the histogram peaks. This experiment analyzing endoreduplication levels or EI confirms previous results that pith tissue exhibits higher EI than cortex tissue.

Surprisingly the parenchyma tissue's profile was similar to cortex and was also significantly different from pith tissue. While attempting this procedure, it is important to remember to maintain sample sterility for the first two incubations as well as keep the samples on ice for as much as possible after the protoplasts have been lysed. Following this procedure, other methods like genetic manipulation can be performed in order to answer additional questions, like what genes influence tuber endoreduplication levels.

After watching this video, you should have a good understanding of how to extract protoplasts from potato tubers and prepare them for flow cytometry to investigate their endoreduplication levels. Don't forget that working with propidium iodide can be extremely hazardous and precautions such as proper PPE should always be taken while performing this procedure.