Category Archives: Arabidopsis Adaptation

Arabidopsis growth after 2 weeks

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.

Some notes:

  • 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.

Arabidopsis growth setup

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):

  1. Place seeds in microcentrifuge tube.
  2. Wash with 4:1 ethanol to bleach solution. (I used 1ml of this mixture)
  3. Let sit for 10 min.
  4. Pipette out mixture.
  5. Wash twice with 100% ethanol, and discard ethanol.
  6. 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).

  1. Measure 50ml of D2O and 50ml of DDW
  2. Add 0.22g of MS media to each tube
  3. 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
  4. Add 0.15g agar (to make 1% gel)
  5. Heat to dissolve agar
  6. Pour 5ml amounts into test tubes
  7. 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.

Arabidopsis growth update

Ever since I added a timed incandescent bulb to the system, the plants have been flourishing! That’s a gross over exaggeration, but they’ve been growing faster and better than prior. Check them out! I think next week I’ll add some 20% D2O plants and start charting their growth.20130109-125256.jpg

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Arabidopsis growth update

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:

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Arabidopsis growth try 2 setup

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Since I had leftover MS salt-water mixture leftover from the first experimental setup, I decided to use the remaining bit to make this batch. I did add some water since last time I had over 1% gel. I added about 5ml to the remaining solution, which is some unknown amount. Obviously this isn’t the best experimental setup, but I essentially just want to see if I can grow plants so accuracy isn’t too important.

I have received some replies from some UNM Biologists and hopefully can meet with some students this week to work out some experimental details. More information to come as I know it.

Arabidopsis Growth Week 4 – End

So it seems that my plants of 3 weeks growth somehow stunted their growth and are beginning to die. Because of that I’m starting a second round of plants. Hopefully this time I get some growth. If not, I may need to buy a grow lamp.

I’ve also reached out to some plant biologists here at UNM in hopes they could teach me the basics of plant growth. Let’s see what turns up. Later today I’ll write up my experimental details once I get the experiment started.

Arabidopsis Growth Week 3

It’s the start of the third week of arabidopsis growth. Sadly there hasn’t been much growth difference from last week. Some notes:

  • I noticed that the seedlings in just about every sample aren’t doing too well.
  • Generally, planting on top of the agar wasn’t the ideal way to plant the seeds.
  • The seedlings in the most media and planted in the agar are doing alright.
  • The seedlings in the small jar (with about 4ml of agar) are all dry and the agar is all dried up as well. That sucks.
  • It’s hard to find a balance between keeping the plants covered and exposing them to air. I fear the lack of air flow is hindering their growth, but leaving them exposed also increases the evaporation rate. Having to constantly replace the water makes this experiment expensive for D2O/DDW studies.

Arabidopsis Growth

I haven’t inspected deeply, but a lot of the seeds in all my samples have started to sprout. This is good news indeed 🙂

Arabidopsis growth day 1

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!

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Arabidopsis seeds in test tubes. The four samples on the left have the seeds on top of the growing medium, while the samples on the right have the seeds suspended in the gel.

 

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Arabidopsis seeds in 4ml jar.

 

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poorly setup experiment with seeds in DI water (on left), and seeds in MS salt water (2 on right)