This will probably be the last update. I’m going to (1) need to take a break until after I defend, and (2) need to start a new experiment because the plants are running low on media. I did just buy these awesome 1in diameter test tubes which should give the plants all the media and water they could need for a longer period of time. Anyways let’s go to the pictures:
Here is the update for my arabidopsis growth. It still seems to me that the plants in 10% D2O are doing better than the plants in DDW. I’ll have to make a huge purchase of D2O and DDW (still don’t understand why DDW is more expensive than D2O since one is a by product of the other) and try lower concentrations of deuterium (like 0-10% amounts).
Notice that the seeds in 99% D2O germinated (mostly likely due to D-exchange), but haven’t grown in 3 weeks.
I wanted to take images of the root growth, but my camera couldn’t see clearly enough in the agar to take decent pictures.
These pictures are a bit late, but better late than never. They are an update of the growth progress of arabidopsis in varying amounts of D2O. The group shot is inconsistent with the others because the sample captured for 0% D2O (DDW, deuterium depleted water) is different than the sample used in the individual 0% D2O image.
With that said, there are a couple morphological things I would like to point out. In my opinion the plants are growing better (bigger, faster, etc) in 10% D2O than they are in DDW. They also appear to be slightly more green. And finally there are little hairs on the leaves that seem to be more prominent than in the other samples.
Meanwhile, in 60% D2O the plants are very yellow in appearance. It is interesting that more plants sprouted in that sample, but they are significantly behind in development.
I’ll update tomorrow for the weekly update. And this time I’ll upload the images in real-time. Promise!
These pictures are a few days delayed. I tried to take the images on Monday, but realized my iPhone camera was glitching (purple spots on CCD). I’ve got a brand new iPhone now and retook the images on Tuesday and finally got them into WordPress for you all to see. Let’s talk about what we are seeing here.
If you recall, I setup this experiment a couple of weeks ago. The first few pictures are from the original arabidopsis growth experiment about 2 months ago. The rest are from the most recent protocol. This experiment was inspired by a paper I read a few months ago where they grew arabidopsis in heavy water and cultivated seeds from each generation growing it in higher amounts of heavy water. The purpose was to test for adaptation.
- The paper reported visible morphology changes in the plant growth in high amounts of D2O. I don’t remember the details, but the obvious changes include leaf discoloration and flowering at earlier stages. The expected (and observed) change was delay in growth of plant in presence of high levels of D2O.
- For the most part I’m not observing these color changes. The plants are still too young to determine flowering capabilities. The delay of growth in D2O has been noticed, although growth in 10% D2O has been pretty similar to growth in DDW. Perhaps that level of concentration results in very little effect.
- The most astounding thing is that the seeds germinated in 99% D2O media. In my Repeating Crumley experiment this never occurred. There could be several factors related to this, but the most obvious factor is deuterium exchange. Since the test tubes are sealed with breathable tape, there is air transfer between the samples and the environment. From what I’ve seen D-exchange is mostly a surface interaction type problem and since the seeds are near the surface there would be considerably more exchange during early germination than at any other point in the life cycle. I expect that as the roots grow it will become incredibly difficult for the plant to sustain life. The interesting thing to mention is that Bhatia and Smith (of the aforementioned paper) note they never were able to grow seeds in anything over 70% D2O. I don’t understand how.
- In the 99% D2O samples, one plant has grown its first leaves (cotyledon), while the other samples have begun to sprout a root (radicle). I’ll keep observing to see if these plants develop further.
- I’m actually very surprised to see the 60% D2O plants do fairly well.
- I’m wondering if the presence of the MS media has given the plants an advantage that Gilbert Lewis didn’t think of.
- Evaporation of my gel is a huge problem. The two month old sample is running out of media and there is no way that I know of to replenish the supply. The other samples will have this happen in time. I will test a small batch of samples to see if I can use a rubber stopper to get the plants to grow in an effort to reduce evaporation. Other setups have the advantage of keeping the plants in a moisture controlled environment. If I had $1,000,000, I could create a grow house that cycles heavy water mixtures into a chamber to keep the plants in an environment to reduce evaporation. Maybe this would be a worthwhile grant?
- Equipment to create my own D2O and DDW from tap water would be crucial so I wouldn’t need to spend thousands buying 100g bottles from Sigma. Think of the experiments I could run!
I think that’s all I have for now. I’m potentially about to come upon some money so I hope to spend a bunch of it on water before I run out and then I’ll run a series of experiments that should take me through the semester.
I don’t believe I have time this semester to adapt Arabidopsis to D2O, but I might as well get started and hope I can carry out my experiments this summer and beyond. With that said, I’ll also be looking for morphological affects of D2O on the plants. So I’m starting the growth now and let’s see what observations I can make by March.
Cleaning the seeds (protocol provided by Pedro Nunes):
- Place seeds in microcentrifuge tube.
- Wash with 4:1 ethanol to bleach solution. (I used 1ml of this mixture)
- Let sit for 10 min.
- Pipette out mixture.
- Wash twice with 100% ethanol, and discard ethanol.
- Let the ethanol evaporate.
The seeds will sink to the bottom so it is fairly easy to pipette any liquid in the tube. After step 6 I’ll add some water so I can pipette the seeds into their growth media.
Preparing the growth media:
I originally intended to grow seeds in 5 different mixtures of D2O/DDW, but I spilled one so now I’m doing 4: 0% D2O, 10% D2O, 60% D2O, and 99.9% D2O (I spilled the 5% D2O mixture).
- Measure 50ml of D2O and 50ml of DDW
- Add 0.22g of MS media to each tube
- Mix water in 15ml amounts in small beakers/flasks
- 0% D2O – 0ml D2O, 15ml DDW
- 10% D2O – 1.5ml D2O, 13.5ml DDW
- 60% D2O – 9ml D2O, 6ml DDW
- 99.9% D2O – 15ml D2O, 0ml DDW
- Add 0.15g agar (to make 1% gel)
- Heat to dissolve agar
- Pour 5ml amounts into test tubes
- Allow to cool for gel to solidify
There are three samples per water mixture. Make sure you label your tubes.
Planting the seeds:
After cleaning the seeds use a pipetter to add 3 seeds to each sample. The seeds are small enough to fit in the tiny tips. I’ve read that you can place the seeds on the surface of the growth media, but in my (limited) experience that doesn’t work. As an alternative, I plunge the tip a few millimeters below the surface and drop the seeds there.
This batch of arabidopsis seeds seems to be faring much better. I added an incandescent bulb to my lighting on Monday and the plants appear to be taking much better than with the fluorescent alone. I need to get another timer to put that light on so the plants can have a full 16 hours of both lights.
I also added some micropore tape (used for wounds and stuff) to seal my samples. This allows the plants to breathe and hopefully minimize evaporation. The corks that I was using before seem to do a good job, but I was always fearful that I was hindering air transfer. This method gives me a little better peace of mind.
Here are some seedling images for your viewing pleasure: