Identifying Polyploids

Oryzalin treated Passiflora edulis

The image above shows a tentatively doubled Passiflora edulis ‘Sweetheart’ seedling, attempted using oryzalin. In my previous post I discussed how this was accomplished. There is an untreated seedling growing at the base of the larger plant. This will be ideal to have as comparison to the treated plant.

The first step in identifying doubled candidates is noticing the visually apparent differences. If you look at the plant above, especially if you can compare it to an untreated specimen, you will notice some peculiarities. The leaves are curled, atypical for P. edulis. Growth of the plant has been stunted, and the leaves are noticeably thicker. To test this, you pinch the leaves and feel the thickness and then feel the leaves of an untreated plant; the treated plant’s leaves should feel thicker. This doesn’t guarantee that it has worked, but it’s a step in the right direction.

In order to see if they are truly doubled I will likely have to do a chromosome count using the root tips, by staining and squashing and looking at them under the microscope. The method I used only treats the growing tip and does not affect the ploidy of the roots, so that won’t work. I would have to cut off the treated portion and try to get it to generate its own roots before I can do that, so it’s not impossible.

Another method is to look at the stomates, the breathing holes on the underside of the leaf. They are microscopic, so you’ll need a microscope, and then there are two methods you can use. First method: apply some clear nail polish to the bottom surface of the leaf (you’ll also need a control leaf to compare against), and when dry, carefully peel it off. Examine under a microscope and measure the length (not width) of the guard cells (there are two guard cells on each stomate). If it’s doubled the guard cells will be twice as big, more or less. The second method is to count the chloroplasts of the guard cells. Guard cells typically have fewer chloroplasts than normal leaf cells, and doubled cells will have twice as many.

Here’s a grainy image from Por Qué Biotecnología:

I have never attempted those methods, but the method I have used to identify tetraploids is called flow cytometry. Flow cytometry is a method of looking at the size of the nucleus. This is accomplished by chopping up the leaf, staining the nuclei, and then running the sample into a machine called a flow cytometer. These are big, expensive machines, and not easily accessible. Certain labs have them, and use them for… I don’t know, cancer research? I tried to see if I can buy a used one of off eBay, and they are thousands of dollars, even old ones. They can be finicky, and have to be calibrated routinely, and not generally commonly found. I imagine there are labs that you could send you samples to to have them tested, but I work in a lab that has one.

Flow cytometry is advantageous because it is fast and you can lump samples. When counting the chromosomes in a root tip, it is laborious and time intensive. It takes a while to prepare each sample and then to try to find and count the chromosomes. With flow cytometry, you chop up a piece of leaf and in about a minute you have a nice readout. Or you can group samples, chop up ten leaves and then if your sample gives multiple peaks break it down to five and narrow it down. It’s much easier and time effective, if a little less accurate, which I will discuss with following result.

Here is a result from some leaves of Passiflora caerulea, one is treated and one is not, from the same plant (an untreated sprout came from the roots):

Passiflora caerulea diploid Passiflora caerulea tetraploid?

The important information is where the peak is. The diploid peak (left) is at 236 (see ‘Mean’ in upper right hand corner). The putative ‘tetraploid’ peak (right) is at 376. The size of the peaks is not important, as this is relative to the number of cells in the sample. Let’s look at the diploid graph for a moment: if you look left of the peak, you’ll see a bunch little peaks, which is cellular garbage (chloroplasts, etc). On the right of the peak, just under 500, there are some little peaks. These are the cells undergoing mitosis, so these nuclei have twice the amount of DNA. This is the area to look for putative tetraploid peaks. Now, look at the putative tetraploid: its peak is at 376, shy of the 472 the diploid graph infers what a tetraploid should be. So is it a tetraploid? Maybe.

Sometimes when treating with chemicals you can get a chimaera, where some of the cells are doubled, and some are not. I don’t think this is the case, as I would see multiple peaks, and it clearly has a single peak.

Another thing that might have happened is incomplete doubling. A diploid P. caerulea is 2n=2x=18, so a tetraploid should 2x=36. This treated specimen could be 2x=30 or something, somewhere between a diploid and a tetraploid. I’ll let it grow and we’ll see.

P. caerulea oryzalin treated

This is the Passiflora caerulea plant I treated with oryzalin. The shoot on the left is the putative tetraploid, while the leaves arising from the center are diploid shoots from the root. The leafless stem on the right also yielded some putatively tetraploid leaves that I tested earlier, but has been defoliated due to Gulf Fritillaries.

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