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 Interesting links and a question about UV 
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Joined: Sun Oct 17, 2010 2:27 am
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Post Interesting links and a question about UV
If you're interested in mutagenesis, you could probably get some results with seeds and chemical mutagens like sodium azide or EMS. Here are some interesting links.

http://www.plant-biology.com/EMS-mutagenesis.php you could possibly get some results.

http://www.faculty.ucr.edu/~jkzhu/artic ... /yskim.pdf

http://lecturer.ukdw.ac.id/dhira/BactGe ... agens.html

http://fruitfly4.aecom.yu.edu/labmanual/16a.html

Question--one of the links refers to using ultraviolet light for mutagenesis. Of course, for safety reasons, it would be important to be careful with UV light, but has anyone experimented with it? UV lights can be purchased commercially, and miniature ones are even included in toothbrush sterilizers. Anybody know how much light would be required, and for how long, to get mutagenesis in a reasonably short period of time?


Sun Oct 17, 2010 6:38 pm
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Post Re: Interesting links and a question about UV
Why would you induce random mutations in organisms?


Sun Oct 17, 2010 7:17 pm
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Post Re: Interesting links and a question about UV
While I've never used Sodium Azide, I have use EMS, which is a very effective mutagen. Both of these compounds should not be used unless you have (1) Proper training (2) Proper Facilities (3) Proper means of disposal. Both are highly toxic. EMS is a powerful carcinogen/mutagen and needs to be handled under a fume hood at all times.

UV light, though it has its dangers, is not only more practical, but far safer. If you are going to attempt mutagenesis, stick to UV light unless you have the proper facilities. The exact protocol is going to vary depending on the organism you are mutating, so something to remember when doing this is to always specify what it is you are wanting to mutate.

Quote:
Why would you induce random mutations in organisms?


Random mutagenesis has been the basis of genetic research for decades. The idea is that you randomly mutate an organism. You then observe for mutant phenotypes in subsequent generations and isolate them. You can then map the mutant gene causing the mutant phenotype. This tells you that this gene somehow affects that process.

For example, if you mutate a mouse and you find a mutant that doesn't grow a tail. That tells you that the mutated gene is involved with the development of tails in mice. It give you a function for that gene in the broader sense. Its one of the most powerful methods in genetic research there is and is commonly referred to as Forward Genetics.


Sun Oct 17, 2010 9:42 pm
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Post Re: Interesting links and a question about UV
"To create a random mutant library that contains enough variants to give you a good chance of obtaining the altered enzyme you desire."

See:

http://bitesizebio.com/2007/12/12/8-app ... tagenesis/


Sun Oct 17, 2010 9:45 pm
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Post Re: Interesting links and a question about UV
chadn737 wrote:
While I've never used Sodium Azide, I have use EMS, which is a very effective mutagen. Both of these compounds should not be used unless you have (1) Proper training (2) Proper Facilities (3) Proper means of disposal. Both are highly toxic. EMS is a powerful carcinogen/mutagen and needs to be handled under a fume hood at all times.

UV light, though it has its dangers, is not only more practical, but far safer. If you are going to attempt mutagenesis, stick to UV light unless you have the proper facilities. The exact protocol is going to vary depending on the organism you are mutating, so something to remember when doing this is to always specify what it is you are wanting to mutate.

Quote:
Why would you induce random mutations in organisms?


Random mutagenesis has been the basis of genetic research for decades. The idea is that you randomly mutate an organism. You then observe for mutant phenotypes in subsequent generations and isolate them. You can then map the mutant gene causing the mutant phenotype. This tells you that this gene somehow affects that process.

For example, if you mutate a mouse and you find a mutant that doesn't grow a tail. That tells you that the mutated gene is involved with the development of tails in mice. It give you a function for that gene in the broader sense. Its one of the most powerful methods in genetic research there is and is commonly referred to as Forward Genetics.



As I mentioned in another post, I am curious about the possiblity of mutating cannabis seeds. Any comments on a possible protocol?

Your observation about the importance of handling, or better yet, avoiding, carcinogens is completely true. Is there any chance that pharmaceutical medications that are known to be highly hepatoxic couild be ground up and dissolved into a solution that a pouch of seeds could be soaked in to induce mutagenesis? Or--how about a highly toxic (but easy to handle) solution made by soaking cigarette tobacco in water?


Sun Oct 17, 2010 9:58 pm
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Post Re: Interesting links and a question about UV
UV radiation isn't the typical route used with plants. Usually EMS or insertional mutagenesis is used. The fact that you want to do Cannabis, which has legal issues, makes it harder to find a protocol as basic genetics is not often done on it.

Your best bet would be to mutagenize the pollen. Collect a bunch of pollen and expose it to UV light and then use the pollen to fertilize your flowers.

Since I really don't have a basis for Cannabis to recommend a specific protocol, I would suggest collecting several different "pools" of pollen. Expose each pool for different lengths of time. Say 1 min, 2 min, 3 min. Then fertilize your plants.

Now, here's a bit of very basic genetics to keep in mind. The first offspring (called the M1) are going to be heterozygous, since only the pollen was mutated. Chances are they will appear normal. You will have to self-fertilize each of the M1 plants and obtaining a second generation (called the M2) before you start seeing mutants, as most mutations will be recessive.

Here is another problem. There are thousands of genes. Whats more, plants tend to have multiple copies of the same gene. They are redundant. You will have to go through a lot of plants in order to start finding mutants, because most will look normal.


Mon Oct 18, 2010 12:35 am
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Post Re: Interesting links and a question about UV
Wow...that is really cool.


Tue Oct 19, 2010 1:27 am
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Post Re: Interesting links and a question about UV
chadn737 wrote:
UV radiation isn't the typical route used with plants. Usually EMS or insertional mutagenesis is used. The fact that you want to do Cannabis, which has legal issues, makes it harder to find a protocol as basic genetics is not often done on it.

Your best bet would be to mutagenize the pollen. Collect a bunch of pollen and expose it to UV light and then use the pollen to fertilize your flowers.

Since I really don't have a basis for Cannabis to recommend a specific protocol, I would suggest collecting several different "pools" of pollen. Expose each pool for different lengths of time. Say 1 min, 2 min, 3 min. Then fertilize your plants.

Now, here's a bit of very basic genetics to keep in mind. The first offspring (called the M1) are going to be heterozygous, since only the pollen was mutated. Chances are they will appear normal. You will have to self-fertilize each of the M1 plants and obtaining a second generation (called the M2) before you start seeing mutants, as most mutations will be recessive.

Here is another problem. There are thousands of genes. Whats more, plants tend to have multiple copies of the same gene. They are redundant. You will have to go through a lot of plants in order to start finding mutants, because most will look normal.


Hmmm. I may understand, or I may not.

Let me ask this. I'll go through step by step to make sure I'm following you. First, I introduce the mutated pollen to a female plant. That plant produces seeds. I plant and grow the seeds into plant M1. Then plant M1 is pollinated, and it produces seeds, which are then planted, producing plant M2.

If that is not correct, please let me know. If it IS correct, my question is this--what is the source of the pollen that is introduced to female plant M1 to produce seeds? Obviously that pollen is going to contribute to the genetic inheritance of M2...so where should it come from?

Second question--let's say a mutant is produced at the M2 step that we want to MAINTAIN as a strain, i.e., produce a bunch of seeds that will grow that type of mutated plant. How could that be accomplished? It seems to me that there is going to have to be a cloning (before the plant establishes a sex) at some point in the process--and that a female version of the clone would then be pollinated by its male version to produce those seeds. Am I on the right track?


Tue Oct 19, 2010 2:18 am
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Post Re: Interesting links and a question about UV
snubdubs wrote:
If that is not correct, please let me know. If it IS correct, my question is this--what is the source of the pollen that is introduced to female plant M1 to produce seeds? Obviously that pollen is going to contribute to the genetic inheritance of M2...so where should it come from?
The whole first generation is called M1. You get M2 by breeding M1 with itself.

snubdubs wrote:
Second question--let's say a mutant is produced at the M2 step that we want to MAINTAIN as a strain, i.e., produce a bunch of seeds that will grow that type of mutated plant. How could that be accomplished? It seems to me that there is going to have to be a cloning (before the plant establishes a sex) at some point in the process--and that a female version of the clone would then be pollinated by its male version to produce those seeds. Am I on the right track?
You breed plants that have the trait you want with themselves until they produce offspring that all have the trait every time. Then you know that the strain has the same allele in both corresponding loci. A strain like this is called true-breeding.

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Sun Oct 24, 2010 11:14 pm
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