Koch asked me in a comment to replicate an experiment done in 1950 by Helen A Crumley et al demonstrating Tobacco seed growth in deuterium oxide (D2O). The experiment is rather simple (and the figure from the paper is shown below) as Crumley placed 100 seeds in differing amounts of D2O (double distilled water, 33%, 66%, and 99.8% D2O) and analyzed the growth.
So here I am planning the experiment. I will change some things from their experiment. First they placed the seeds on wet cloths (paper towels?), and I will submerge the seeds in the water amounts they used. They also used a variety of plant species (tobacco, clover, radish, Kentucky bluegrass), where I will just use tobacco seeds (but I will try two different species). Finally they talk about their results in terms of percent germination, but it isn’t clear from the paper if they mean number of plants that have exhibited germination, or if they are referring to some amount of growth exhibited by each plant. I will look for both possibilities and report the results as I find them.
In a preliminary experiment I will submerge the plants in water in petri dishes and seal it with parafilm. I will be looking into a more airtight solution as time goes on. I also won’t do 100 seeds but probably on the order of 33 seeds per sample. And in the future I will look into figuring out a way to measure the seed growth.
Bill Hooker suggested (and rightly so!) that I document what I consider to be typical specimens. I found, today that the zoom feature on the webcam I’m using is quite sufficient for getting close enough to demonstrate this and so I snapped a picture and tried to document. Powerpoint messed me up a little bit, but this is good enough.
The orange box is highlighting a seed that is showing no signs of germination. From my studies I’ve found that the the seed coat becomes slightly transparent just before radicle (the pre root) penetration and you can see the precursor to puncture. The seed coat usually gets lighter too and in this case is really dark in color.
The pink box (hehe) is what I would count as germination when I’m counting the seeds. I see the tip of the radicle penetrating the seed coat and so germination is officially underway.
In the very near dead center of the image is a seed that has what I referred to in an earlier post as an extended radicle. Basically I meant that the radicle was pushed through and the root is now forming. The root will continue to grow until the seed coat has come off, which over to the right it has (there is a green leaf that is cropped on the edge of the image).
It’s a new day and that means a new Crumley Trial. This is the 3rd try and hopefully one of the last trials (if everything goes smoothly). As always, I’m looking to repeat the results Crumley got back in 1960 growing tobacco seeds in D2O. Only I’m doing it better (no disrespect to Crumley, but there were some flaws in the original setup).
After getting the results of the water evaporation experiment I decided to go with the DOW Corning Vacuum Grease to seal the chambers. The product claims it has low reactivity with water and provides a great seal (according to my results). Since I want as little interaction with water as possible this seems like a viable option. With that said, let’s move on to the setup.
I prepared 8 samples for this trial using a combination of DI water, DDW, and D2O. I’m using Cuban Havana 2000 seeds from the Tobacco Seed Co (because the idea of growing Cuban cigar seeds in the lab sounds awesome!).
I counted 30 seeds per sample and placed them on weigh paper for addition to the samples after the water step (see below). Using weigh paper helped considerably when dealing with the static attraction the seeds tend to have.
1 set of seeds (unknown amount) was placed in 8mL of DI water and left to presoak for 30 min. The water from this sample would be used as a control to monitor fungal and mold growth.
I placed the seeds aside and prepared the water for the samples. The 8 samples include: (1)di water (no seeds), (2) di water, (3) 33% d2o in di water(2mL d2o, 4mL di water), (4) 66% d2o in di water, (5) 99.9% pure d2o, (6) 33% d2o in ddw, (7) 66% d2o in ddw, and (8) 99.9% pure ddw.
6mL of each water type was added to an analyslide (see Experiment Product page at top). The presorted seeds were then added to the water. The analyslides were then closed and sealed with Dow Corning Vacuum Grease.
For the control sample, seeds were removed before closing and sealing the chamber.
Sealing the chambers is quite a task that I have turned into an art. When I put the analyslide cover on I put it on loosely at first. Then I tilt it vertically so the air bubble moves toward the slight opening at the top. I begin to press around the edges with my thumbs until it is closed completely. Finally I spread vacuum grease around the rim (at first with the no cotton end of a long qtip, but then I used some plastic innoculating loops we had because it was thinner) for the final seal.
I will provide a post on the sealing process later today or tomorrow depending on who can show up to take pictures of me doing this.
For those counting along with me, the final counts won’t be out of 30. When I seal the chamber I end up crushing 1-3 seeds in the process and a couple more end up in the outer fringes where it is tough to discern sprouting or not. So I will count up the seeds in the interior circle (the fragmented one) and count the seeds that sprout from that total.
Here is the results after 24 hours. I haven’t looked in detail yet, but it’s interesting to note that there are 2 seeds in the 66% d2o in di water sample that appear to have sprouted already.
Also for those counting along with me, the final counts won’t be out of 30. When I seal the chamber I end up crushing 1-3 seeds in the process and a couple more end up in the outer fringes where it is tough to discern sprouting or not. So I will count up the seeds in the interior circle (the fragmented one) and count the seeds that sprout from that total.