Here are the pictures of my samples after setup. Note that there are three additional samples of seeds in water. These samples are basically the equivalent of my original DDW Effects on life experiment. But now that I think of it, I didn’t use DDW so I don’t know exactly what I’m looking for… d’oh!
It was discussed a long time ago that adapting arabidopsis to D2O could reveal some interesting finds. So in an effort to do that I’m going to grow some example plants to test the setup and try and produce seeds.
The one issue I worry about is evaporation. Since this cannot be stopped, evaporation could become a costly component of the experiment (D2O and DDW cost about ~$100 per 100ml). The first experiment will be using DI water. Here is my setup:
This is based on the combination of these two protocols:
I mixed 0.2192g (4.3g/L) of MS salt with 50ml of DI water.
I then put 30ml in a beaker with 0.5g of agar, heated, and stirred. I was supposed to make 1% gel and measured 0.5g for the 50ml that I made instead of for the 30ml that I added to the beaker. When the agar dissolved, I added 10ml of MS salt water to the solution to reduce the gel percentage (now at 1.25%).
I then put the MS agar solution in test tubes: 2 @ 1ml each, 2 @ 2ml each, 2 @3 ml each, and 2 @ 5ml each. I also put ~4ml into some random (but clean) glass jar thing. I bought these a long time ago when I was looking at clear, glass jars for the tobacco and arabidopsis seed DDW experiments. The reason for the various volumes is to determine how much medium the seeds need to grow efficiently and healthily.
While I waited for the MS salt-agar solution to cool (and thus solidify) I poured a bunch of arabidopsis seeds into a petri dish and added some DI water to it. I did not steralize these seeds, because I’m just timing the growth and learning about the pollination process and judging how well the seeds will grow in the lab. For the actual experiments I will steralize the seeds.
Once the growing media cooled, I used a pipetter to collect a few seeds (~5 seeds per sample) and place them in each sample. There are two sets of test tubes (one volume of each set, ie 1ml, 2ml, 3ml, 5ml).
For the first set, seeds were placed on top of the growing medium.
For the second set, seeds were plunged into the gel.
For the random 4ml jar, seeds were plunged into the gel.
Once seeds were set, rubber stops were placed over top of the test tubes to minimize evaporation.
I’m sure this process has tons of kinks, which will be worked out when the real experiment begins, but this is the purpose of an experiment right?
C. R. Bhatia and H. H. Smith, “Adaptation and Growth Response of Arabidopsis thaliana to Deuterium,” Planta, vol. 80, no. 2, pp. 176–184, 1968.
Looking for papers on yeast adaptation (of which I’ve found precisely one!) I came across this paper where they attempted to adapt arabidopsis to D2O. I was instantly intrigued and downloaded the paper for brain consumption. I will now take notes on the paper with the intent of setting up an experiment that can run with minimal effort on my part.
A paper that is mentioned in the intro that reviews all the genetic and cytological effects of D2O, seems like an interesting place to check out next for work with the yeast project.
Flaumenhaft, E., S. Bose, H. L. Crespi, and J. J. KATZ: Deuterium isotope effects in cytology. Int. Rev. Cytol. 18, 313–361 (1965).
“Concerning the possible biological action of deuterium, it is known that substitution of deuterium in hydrogen bonds of essential macro- molecules (nucleic acids and proteins) changes the properties of these molecules by increasing bond strength and also by a general retardation of reaction rates.“
seeds were surface steralized in 3% hydrogen peroxide and 90% ethanol (a 1:1 mixture) for one minute
seeds were sown into mineral media mixed with 0.78% agar – I’ll have to find out what mineral media is available in 2012. Also I find it strange that in this paper they say what products they use, except for the mineral media. If it’s made in house, why don’t they say what the composition is?! Argh!! It’s always the important detail that is left out…
seeds were grown in test tubes, and closed with glass caps.
samples were cold treated for 5 days at 4C and then moved to a climate controlled box maintained at 24C and under constant illumination (they use some fluorescent lighting and I’m assuming they are 1960’s grow lamps)
they did 2 experiments: one was a germination experiment much like the experiments I did in the repeating crumley series, the other was the full adaptation experiments, in all experiments seeds were grown at 0, 10, 30, 50, 70, and 90% D2O
in both experiments they looked for morphological differences from normal plants
in the first generation of plants, they found germination rates to be increasingly delayed with increasing concentrations of D2O. This is pretty consistent with with the results I linked to above, although they counted germination from the emergence of the first leaves, whereas I looked for any emergence from the seed coat.
they also discovered that the green-ness of the plant decreased with increasing D2O conc. and the survival rate of the plants also decreased. No plant produced seeds above 50% D2O in this first generation.
flowering rates were also lower in samples with higher d2o amounts. the authors subtracted out the germination delay to determine an average flowering time and saw that that increased with increasing d2o concentration.
as for the adaptation experiment, they say they screened over 850 plants, but only have growth information for plants grown in 50% D2O. They find that after 6 generations of growth on 50% D2O seeds can survive to maturity at 70% D2O and seeds obtained from these plants grow normally in the first generation on H2O media. They conclude that the D2O adaptation is not genetic because of this. I would like to add that I’m highly skeptical of the adaptation efforts included in this paper. There are no counts of the number of plants per generation, the number of seeds planted, etc.
they hint that they would are taking measures to develop mutants that can grow at concentrations of D2O that are above 70%, I’m guessing they failed because the papers that cite this paper are not by the authors.
This paper has been cited twice according to the internet:
Brown, B. T. (1972) A new screening procedure for detecting plant growth regulating compounds. Pesticide Science 3(2)
Foston, Marcus B. (2012) Deuterium incorporation in biomass cell wall components by NMR analysis. The Analyst 137(5)
This paper must have been one of the last studies on organism development in D2O, especially because the papers that cite this paper are in unrelated fields and this is one of the most recent papers I’ve seen in this field. With that said, I’m a little skeptical of the effort to obtain D2O adapted arabidopsis.
I am impressed that they wanted to answer the question of whether H2O affects D2O adaptations in similar ways that D2O affects H2O adaptations. I don’t think their study was thorough enough and growing plants in 50% D2O still leaves the room for plants to get the H2O requirements. I think growth at levels above 70% would be key to obtaining true D2O adapted plants.
Their methods seem pretty simple and I could work on my own version of this experiment pretty easily and have already looked up product information regarding plant growth in the lab:
I asked Koch to take pictures of the plants for me while I am out of town. Here are his notes regarding the experience:
Wasn’t sure of your camera settings. I had trouble with depth of focus, so I chose aperture priority (“A”)
and F=22, which seemed to work OK. I didn’t want to change your zoom, so I didn’t. I took pictures of bottoms
until half way through when I realized to take tops too (so two pictures each for arabidopsis). This was a
mistake, but since I don’t know if the photos are useful, I’m stopping now.
Photos are labeled TYPE CUVETTE_NUMBERS UP/DOWN and I left in the actual photo number, as follows
I can’t tell in the havanna seeds sample, but the virginia gold tobacco seeds in di and tap have tiny hairs. This is not alarming because I noticed this in prior trials. It is obvious that the hair growth in the ddw samples far exceeds that of the growth in these two samples.
Steve and I have decided to try and publish the results of this study. I’ve made a mindmap that will allow us to coordinate the planning of the paper and upcoming experiments to include in the paper. This page will be updated frequently so check here often.
Here are the cropped photos. I used a program called JPEGcrops to mass crop the images from day 7. It was sweet and quick. I also included (uncropped) photos of the DI and tap water samples from Trial 3 of the DDW experiment. I don’t know if the curviness is real or not so I’ll let you comment below if you think it’s real. My money is on yes!