Tuesday, April 19, 2016

Mutation Breeding 2

1. Spare microwave.
In a recent post, I talked about how ultraviolet light could be a useful mutatgen. It damages DNA effectively, is easy to generate, is easy to keep contained, and there's no cleanup required. This post is going to be something of a departure from what I typically post, as this is just a short little story of how I built a device to try and mutate seeds with germicidal-UV light, starting with a spare microwave oven.

2. Dangerous bits.
It took a little work to get into the guts of the microwave. The outer shell was held in place with security screws, intended to keep the unwary away from some very dangerous components. The large transformer and capacitor on the bottom together increase the voltage supplied by the wall current from 120 V to something like 4 kV. This higher voltage is then fed into the magnetron (at the top), which generates microwaves. These parts are able and quite willing to kill you if power is running through them. If you don't have years of experience with electronic components, don't mess with the guts of a microwave.

3. Neutered and running.
Once the microwave was neutered, I had to make sure the other aspects of the device were still working. when the "microwave" is running, theory says the two lines running to the dangerous parts should essentially be wall current. This is dangerous, but much more in the realm of the familiar (to anyone who has rewired a lamp, installed a fluorescent ballast, or many other minor tasks). When it isn't running, there shouldn't be anything running through these lines. My multimeter showed otherwise. When running, it is straight wall current at 120 VAC. When it isn't running, but plugged in, there is roughly 7 VAC running through it.

4. Rewired for UV.
After unplugging the device and making sure there was no unexpected voltage remaining in the system, I installed the ballasts and receptacles need for a pair of fluorescent light bulbs. Germicidal-UV bulbs are similar to regular fluorescent bulbs, they all use a small amount of ionized mercury to produce copious amounts of UV. Regular bulbs are lined with phosphors to convert the UV into visible light and are made with UV-absorbing glass to minimize the amount of UV that escapes. Germicidal-UV bulbs don't have any phosphors and are made from UV-transparent glass to maximize the UV that "escapes".

5. Door disassembled.
The front door of a microwave includes a metal layer gridded with holes. The holes are small enough that quanta of microwave energy just can't fit through them. Ultraviolet, on the other hand, can get through the holes just fine. To remedy this, I decided to replace the clear front plastic panel with a piece of opaque orange acrylic.

6. The ultraviolet oven.
Now that everything is put back together, I have an ultraviolet oven. I don't have a direct photo of the bulbs in action. I didn't feel like focusing intense ultraviolet light onto my retina. The final photo at left shows some intense UV bouncing through the exhaust port at left. The opaque acrylic front lets you see when the bulbs light up, but should be blocking sufficient UV to make the machine not be hazardous. I need to find something that strongly fluoresces in UV-C for testing purposes. Inside the oven, or adjacent to the exhaust, the object should fluoresce. In front of the door, the object should remain dark. If the door is passing much UV-C, I'll have to reassess the safety protocols I'll have to keep in mind while using the device.

The next step would then be to start experimenting with mutagenizing batches of seeds. To maximize the number of mutant plants which get produced, the basic idea is to dose the seeds at a high enough level to reduce germination by roughly 50%. It will probably take several mutations to actually kill a seed, so dosing them at this level will ensure that most of the seeds that germinate and survive will also be carrying mutations.

What sort of exposure can my new ultraviolet oven produce? Some really rough calculations indicate that seeds held within an inch of the bulbs for about five minutes would be approximately the same exposure that seeds on the space-station received over a year in direct sunlight. Seeds protected from UV, but otherwise exposed to space, showed higher survival. A few minutes in the ultraviolet oven should have a significant impact of germination, at least with smaller seeds.

Another thing to be tested is the effect on seeds that are dry vs. ones that have been soaked in water overnight. Plant embryos that have woken up and are metabolically active will be more able to repair DNA damaged by UV (a necessary part of converting damaged DNA into new mutations), compared to those that are completely asleep in dry seeds.

After I dose a few different batches of seeds, it will take another couple weeks to determine what impact on their germination was had. I'll probably do some really high doses, just to make sure I am likely to cover the range from no-impact to strong-impact. Once I get a basic idea of the range of doses that are interesting, I'll do more test exposures at intermediate levels to better resolve the dosage-response curve. All of this will take me several weeks further, so the next posting on this topic will probably be a while.