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?