Category Archives: Arabidopsis Adaptation

Arabidopsis Growth Try 4: Week 5

The latest updates are in. Between last week and this week, plants grown in 0% D2O, 30% D2O, 70% D2O, and 80% D2O have died. While this is expected for the larger concentrations, I can’t explain why the 0% D2O sample or 30% D2O sample has died. But I did just think of an interesting effect:

Because the plants are more-or-less sealed from the environment, I have minimized the effects of evaporation and transpiration. I’m not sure if transpired water is harmful to the plants because it basically is their excrement. Regardless, the water in the solid media (the agar) should have a slight rise in D2O concentration due to transpiration processes in the leaves and evaporation of water from the solid media. I’ll have to find the reference for a paper I have that verifies this.

Arabidopsis Growth Try 4: Week 5

Arabidopsis Growth Try 4: Week 3

Arabidopsis Growth Try 4: Week 2

The plants are one week old! They seem to be doing really well, for the most part. As expected, as the concentration of D2O increases the plants develop slower. Also as expected the plants also exhibit leaf decolorization. Notice how in 60, and 70% D2O the leaves are a pale green. In 80% the plants are too small to notice growth. It also seems that the plant in 5% D2O is growing the fastest, which seems to be in line with my observations from the last trial run, and also with hypotheses from my dissertation (to be posted as soon as it is all finished). I will have to record some observations of the plants in DDW and compare them to 5% D2O and 10% D2O.

Arabidopsis Growth Try 4: Setup

I’ve got larger test tubes (1in diameter and about a ft in height), I’ve got plenty of water, and I’ve got a PhD. Looks like I’m ready to grow some plants! Here is the protocol ( adapted from Jan 22):

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 growing seeds in 10 different mixtures of D2O/DDW: 0% D2O, 5% D2O, 10% D2O, 20% D2O, 30% D2O, 40% D2O, 50% D2O, 60% D2O, 70% D2O, and 80% D2O. I’m not doing a 99.9% D2O sample this time. Each sample will have 20ml of water total.

  1. I used 1 bottle of D2O (100g), 1 fresh bottle of DDW (100g), and one old bottle of DDW (~50g, from 1/22/13, stored in desiccator).
  2. Add 0.22g per 50ml of water of MS media
  3. Mix water in 20ml amounts in test tubes
    • 0% D2O – 0ml D2O, 20ml DDW
    • 5% D2O – 1ml D2O, 19ml DDW
    • 10% D2O – 2ml D2O, 18ml DDW
    • 20% D2O – 4ml D2O, 16ml DDW
    • 30% D2O – 6ml D2O, 14ml DDW
    • 40% D2O – 8ml D2O, 12ml DDW
    • 50% D2O – 10ml D2O, 10ml DDW
    • 60% D2O – 12ml D2O, 8ml DDW
    • 70% D2O – 14ml D2O, 6ml DDW
    • 80% D2O – 16ml D2O, 4ml DDW
  4. Add 0.2g agar (to make 1% gel)
  5. Heat to dissolve agar
  6. Allow to cool for gel to solidify

Planting the seeds:

Normally, I use a pipetter that one would use for small volumes to deposit the seeds, but because the tubes are so much bigger than the ones in the past I need to alter my method. Today I used a glass Pasteur pipet with a very nice long tip. I used the wrong kind of bulb, so it was more challenging than expected, but worked well enough.

Also because my test tubes are much bigger than before, I made the mistake of not purchasing a test tube holder. So I had to fashion one from spare lab components. Check out my system:

Test tube holder fr 25mm diameter tubes.
Test tube holder for 25mm diameter tubes.

It’s made from the breadboard that has become my plant station, screws, and nuts. Pretty simple, and works amazingly well.

Last Arabidopsis Update

The media is dwindling and the plants will die shortly. I’ll be restarting this experiment in larger environments with more media. Hopefully the plants can do better.

Yesterday Koch noticed that the roots in 10% D2O were much longer than those of the plants in DDW. Something interesting.

Arabidopsis Update (From March 6, 2013)

I took these images several days ago and again forgot to post them. Stupid dissertation…

Anywho, here they are. I’ll try and remember to update tomorrow when I take the images. Tomorrow will be the last day I update this experiment. I’ll be going into full dissertation mode and will be starting a new experiment when I return. I bought some larger test tubes (1in wide), which should give the plants all the room they need and should be large enough to provide more media to keep the plants alive for longer.