Having fun with colchicine ?

[nicollas]

Hi,

I may have access to a person with skills and equipment to work with colchicine. This may be a great help in  some interspecific crosses that generate sterile hybrids whose fertility can be restored by creating polyploids.

But i was wondering if there are other fun projects that can be made with colchicine ? Graham suggested doubling chromosome of skirret and it seems a fun project. Can i do this for every specy or are there some guidelines to know what are the more relevant tries ? I dont want to annoy too much this person as it is a friend of a friend, so i'll try to give precise instructions for dilution and time, but is there a rule of thumb for such numbers, based on the size of the seeds maybe ?

Thanks for any reply, i really want exploit this opportunity the more powerful and relevant way !

[nicollas]

And i suppose i need to keep seeds of untraited seeds from the same original batch to use it as the standard to try to spot polyploids seedling based on vigor and leaf size as Joseph Lofthouse is doing for his tetraploid watermelons ?

[billw]

It takes a lot of experimenting, unless you can find previous research. There is a wide variance in response to both concentration and exposure time, so you might try 10 different concentrations at 3, 6, 12, 24 hours in order to find the combination that produces doubling without killing the culture. Colchicine is used at concentrations from <1uM to almost 40,000 uM, so there is no one-size-fits-all approach. Some plants also respond better to different chemicals - sometimes oryzalin or trifluralin are more effective than colchicine.

The only fast and reliable way to know if it has worked is to do chromosome counts under the microscope. Otherwise you have to wait until the plant grows large enough to evaluate. You may need to treat hundreds of cultures to get a few successes.

I think skirret would be a very interesting project for doubling. Tetraploid and hexaploid carrots are said to have larger roots, which seems like cause for a bit of optimism.

[nicollas]

Thanks for the answer. If there is no rule of thumb i'll should search extensively in the literature because i dont know if the person will do a lot of treatments for me. Is chromosome counting hard to do with the adequate microscope or is it just counting some black lines ? If so i could do it myself if it is just a matter of time.

[billw]

It takes a little practice but kids do it in first year biology, so it ought to be possible for any enthusiastic hobbyist. There are a number of different dyeing processes that work better for some plants than others, but if you are willing to spend some time experimenting, you should be able to get good results with most plants. Generally, the larger the roots, the easier it is. Plants with very small roots can be hard to prepare. Happily, root crops do not have this problem.

[Joseph Lofthouse]

With watermelon I settled on using oryzalin at 2 PPM and 24 hours of treatment of seeds with a 6 hour presoak in water. Those conditions were determined experimentally as giving the best (apparent) conversion with the least seedling death or deformity.

2PPM is a concentrated solution of oryzalin. There is no point using anything more concentrated than that. I didn't test lower concentrations than that. Oryzalin is readily available over the counter at any fine nursery and at disreputable poison peddler's everywhere. (Currently 37 offerings on eBay). Colchicine is so hard to obtain that if it isn't a controlled substance it is treated as one.

I estimate about a 1/3 conversion rate for watermelon seeds under those conditions.

[raymondo]

Jun 28, 2014 10:06:03 GMT -5 billw said:

It takes a little practice but kids do it in first year biology, so it ought to be possible for any enthusiastic hobbyist. There are a number of different dyeing processes that work better for some plants than others, but if you are willing to spend some time experimenting, you should be able to get good results with most plants. Generally, the larger the roots, the easier it is. Plants with very small roots can be hard to prepare. Happily, root crops do not have this problem.

In first year biology, they tend to use onion roots because onions have large, easy to see chromosomes. Various Cucumis species, for example, are renowned for having very small chromosomes, difficult to count. Indeed, I tried, and failed, as part of a research project I did at university. You'd need access to a good lab with good optical gear and various dyes and fixers. It would be interesting to try though.

[billw]

That's a good point. You have two problems to contend with: chromosome size and uptake of dye. Many plants dye well and have chromosomes that are distinguishable at 1000x. Many others don't. In most cases, you can find prior research that will save you the trouble of experimenting, but in the cases of less common crops like skirret, you may just have to try it and experiment with several different dyes.

I'm working with ulluco right now, which has chromosomes probably about half the length of onion, but still big enough to count.

[templeton]

Colchicine can be extracted from autumn crocus. I wouldn't go near it myself.
T

[cesarz]

Hi nicollas,

I have a tissue culture laboratory and have been experimenting with colchicine. Instead of using seeds which give very high mortality rate, why not just use a mature plant and put a drop of 10ppm solution of colchicine on the growing point of the plant. I have very high success with this and the results are very noticeable because the plant habit changes dramatically when the colchicine worked.

I used this method mainly on my orchids but have also used it on my diploid brassicas and diploid potatoes to make them tetraploids.

Hope this helps.

Cheers,
Cesar Z

[nicollas]

Thanks for the anwsers,

i would prefer seeds because it seems more convenient for me and my friend's friend, but i'll give a try with plant for more precious material. skirret seeds are cheap/abundant so i was thinking of trying a lot of it to add chances of mutations.