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.

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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.


References:

• www.youtube.com/watch?v=I2k2g00onL0
• www.edisontechcenter.org/Fluorescent.html
• www.obspm.fr/IMG/article_PDF/On-the-outside-of-the-International-Space-Station_a2525.pdf

Mutation Breeding

The natural genetic diversity found in a crop species was formed over long periods of time by the slow accumulation of mutations in different lineages. We can simulate this process, in shorter time-frames, by dramatically increasing the mutation rate. This can be done by exposing the limited germplasm we have to ionizing radiation (such as X-rays) or any of the many chemicals that damage or modify DNA, thus causing mutations.

"Taihei" (center) and mutants (ring).
Adapted from fig2 on p259 at [link]
and image at [link].

Once we have this newly-made genetic diversity, we can then select towards whatever our goals are.  (Well, we can if some of those mutants happen to be of the sort we find interesting.) Mutation breeding was used to generate the diversity of colors in chrysanthemum seen at right. Another such project is responsible for the intensely red grapefruit types we've gotten used to.

Really, mutation breeding has been used to improve crops in all sorts of ways.

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X-rays and Gamma-rays are extremely energetic types of light. They're so energetic that they break DNA when they make a direct hit. When used for mutagenesis, they tear chromosomes apart. Cells can heal this damage, but errors (such as mutations and chromosomal rearrangements) are often introduced in the process. They're relatively easy to generate (even for a relative amateur), but they're difficult to handle. They'll pass through almost anything. It takes heavy lead shielding to keep them contained.

High-energy particle beams (think neutrons or electrons) wreak havoc to DNA like X-rays. With advances in technology, such particle beams may eventually be easier to come by. However, for now, they are limited to research institutions with much larger budgets than mine.

Lots of chemicals can damage DNA and hence cause mutations. Ethyl methanesulfonate (EMS) is commonly used because it is relatively easy to handle (including neutralization for disposal) and effective in generating mutations. The problem with mutagenic chemicals is... well, that they're mutagenic. Things being mutagenic in the lab often translates to them being carcinogenic (cancer causing) in real life. I don't feel like mutating today. Do you?

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If I wanted to do some mutation breeding in my home lab/garden, I would want a way to make mutations that I could turn on and off. It is feasible to build a device to generate X-rays (with some intensive electrical engineering), but ideally I'd want something that would only impact what was in a very small space.

Ultraviolet (UV) light causes mutations. (That is essentially what a sun-burn is, after all.) UV-C in particular is referred to as germicidal-UV because it causes large numbers of DNA breaks, resulting in the destruction of chromosomes and death of cells. This germicidal-UV is used to sterilize surfaces/water/etc. in various situations in the lab, hospital, or restaurant. All it takes to generate UV-C is some slightly fancy fluorescent bulbs. Since these can be turned on and off with a switch and the fancy light they produce can easily be contained in a box, this seems like it might be a way to go.

What isn't clear is if UV-C light can be effectively used to induce mutations in seeds. UV-C is readily absorbed by organic materials, so large doses will be probably be needed to impact the embryo protected within any but the smallest of seeds. There seems to be very little research available on this topic, so I'll have to do some experimenting.


References:

• https://en.wikipedia.org/wiki/Mutation_breeding
• https://en.wikipedia.org/wiki/Germplasm
• EMS etc.
• https://en.wikipedia.org/wiki/Ethyl_methanesulfonate
• http://openwetware.org/wiki/Lidstrom:EMS_Mutagenesis
• https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&uact=8&ved=0ahUKEwjK_vTkwdHLAhXltoMKHSvTC_MQFggyMAI&url=http%3A%2F%2Flabs.bio.unc.edu%2FKieber%2FMethods%2FEMS%2520Mutagenesis.doc&usg=AFQjCNHse3TaH_9G9_BFdZGazd5oV1xmIg&sig2=EHY2MTJ1r6sdBj-36F2vLA
• Germicidal UV bulbs:
• https://www.google.com/search?q=germicidal+uv+bulbs&gws_rd=ssl#q=germicidal+uv+bulbs&tbm=shop
• https://www.sciencedaily.com/releases/2013/06/130620132412.htm
• http://www.fao.org/docrep/012/i0956e/i0956e09.pdf
• http://file.scirp.org/Html/1-2700095_4887.htm

Transgenic Sweet-Potatoes

One of the first tools available to biologists seeking to make transgenic plants was a natural bacteria called Agrobacterium. The bacteria infects plants and induces the production of auxins and cytokinins, plant hormones that together trigger the growth of a disordered plant tissue called a gall. The gall provides nutrients that the bacteria then uses for its own growth. The bacteria pulls off this trick by inserting genetic information into plant cells. It can do this because it carries a plasmid that can function both in its own cells and in the cells of a plant. When the bacteria infects the plant, the plasmid is transferred into the plant's cells. Once there, the plasmid integrates into the genome of the plant and activates. The bacteria are natural genetic engineers.

Genetic remnants can be left behind whenever the bacteria does this trick and nowadays we can do genome sequencing to find such remnants. Kyndt et al found remnants of this process in sweet-potatoes (Ipomea batatas). In particular, they found such a fragment inserted into an intron of an F-box gene in the sweet-potato genome. Interestingly, this interruption is found in all domesticated sweet-potatoes they examined and not in wild related species.

The insertion contains multiple complete genes and results in the interruption of the F-box gene. F-box genes are generally involved in protein degradation and in plants they're involved in the regulation of development during growth. It isn't clear that the insertion they found results in a change in the development of the plant associated with domestication, however. They are still working on this.

Oh yes, did I mention they found this fragment in all samples they examined of domesticated sweet-potatoes? This means that every single sweet-potato is a transgenic organism, a GMO. We didn't make it a GMO, but there is no way around it, every sweet-potato is a GMO.

Every sweet-potato is a GMO.

Are you against GMOs? Then don't eat any more sweet-potatoes. Are you actually against the business practices of Monsanto surrounding their GMO and herbicide products? Then rail against those business practices instead of the red herring that GMOs are in the overall story.



References:

1. Agrobacterium: en.wikipedia.org/wiki/Agrobacterium
2. Kyndt et al, 2015: m.pnas.org/content/early/2015/04/14/1419685112.full.pdf
3. F-box genes: en.wikipedia.org/wiki/F-box_protein
4. Red herring: en.wikipedia.org/wiki/Red_herring