Chromosome doubling using colchicine or oryzalin

[Helen]

Feb 12, 2014 13:40:05 GMT -5 Graham said:

I suggested chromosome doubling skirret. Polyploidy usually results in larger/thicker plant parts and could increase the overall size of the root. Cant say for sure it would work but I think it's worth a try.

Is that something an amateur could do or more in the realms of crop scientists?

[Graham]

If you can obtain the necessary chemicals it's a fairly simple processes. Colchicine is the most commonly used substance, but it's extremely toxic so must be used with care. Unfortunately, none of the chemicals that can induce polyploidy are available in the UK so I can't put my idea to the test.

[gregmartin]

Graham, is oryzalin not available for legal reasons? Here in the US they sell it as a preemergence herbicide. I picked up a brand called Weed Impede that is 40.40% oryzalin. Skirret is on my list for the reason you mentioned.

[Graham]

Greg, the UK government likes to "protect" us from ourselves by banning or restricting access to a lot of things. That's why people here refer to it as a nanny state. It could be worse, but it's a pain at times.

[papabearjay]

Dec 30, 2014 11:20:41 GMT -5 gregmartin said:

Graham, is oryzalin not available for legal reasons? Here in the US they sell it as a preemergence herbicide. I picked up a brand called Weed Impede that is 40.40% oryzalin. Skirret is on my list for the reason you mentioned.

I think the main issue here though with Weed Impede, would likely result from the solvents used.  It might be 40.40%, but you've no idea what they're using as a solvent which could completely interfere with the process of mitotic inhibition for our purposes.  Even at the diluted concentrations needed to induce doubling.  Though, don't let me stop you!  I'm all for science!  Generally, most experiments aim for 90% kill using chromosome-doubling agents.  That's generally due to the ineffectiveness achieved by using lower concentrations.

[gregmartin]

Good thought papabearjay. The label doesn't give you the identity of the remaining 59.6% so I went to its MSDS. It doesn't tell you how much of each, but the rest of it is propylene glycol and glycerin. While I was looking up MSDS sheets I thought I might as well check Surflan and it has the same two inert ingredients. Surflan has been used with success so we should be good, but I'll try to remember to post some results when I get to this. For me Weed Impede was the way to go because I was able to buy it in an 8 ounce bottle very cheaply.

[papabearjay]

Jan 5, 2015 8:32:41 GMT -5 gregmartin said:

Good thought papabearjay. The label doesn't give you the identity of the remaining 59.6% so I went to its MSDS. It doesn't tell you how much of each, but the rest of it is propylene glycol and glycerin. While I was looking up MSDS sheets I thought I might as well check Surflan and it has the same two inert ingredients. Surflan has been used with success so we should be good, but I'll try to remember to post some results when I get to this. For me Weed Impede was the way to go because I was able to buy it in an 8 ounce bottle very cheaply.

Surflan has been used with success as a chromosome doubling agent, or weed killer?  And of course, expense is always a consideration.

I would recommend either doing something along the lines of zero rate (control), a number of intermediate rates, and the full label rate.  See where the effectiveness lies.... Or do a serial dilution set.

EDIT:
Nevermind, I just figured out Surflan.  I didn't realize it was oryzalin.  Excellent.

[gregmartin]

"Surflan has been used with success as a chromosome doubling agent, or weed killer?"

Both I suspect! I like the serial dilution idea as it appears from the available literature that the ideal concentration differs by the species you're applying the oryzalin to.

Here's an article on tetraploid induction of Rhododendrons using colchicine and oryzalin in the form of diluted Surflan:
www.ces.ncsu.edu/fletcher/staff/tranney/polyploid.pdf

I like the method they used with the agar. Here's a few quick quotes from that paper:

"oryzalin has also been effectively utilized as a doubling agent and is considerably less toxic than colchicine (van Tuyl, 1992). Both agents have a similar mode of action: inhibiting microtubule polymerization and arresting mitosis at metaphase"

"Oryzalin is more effective at lower dosages than colchicine due to a higher specificity for tubulin binding sites in plant material"

"Undesirable side effects of colchicine, including sterility, abnormal growth, and deformed tissue can be avoided when using oryzalin"

"The preemergent herbicide Surflan® A.S. (40.4% oryzalin) was diluted to produce a suspension containing 50μM oryzalin with 5.5g/L agar at 50 °C. Concentrations of oryzalin and agar were based on preliminary studies (data not presented). A single drop (2-4 μL) of warm (~40 °C) oryzalin suspension was then pipetted on top of the cotyledons of each seedling to cover the emerging shoot. Pots were placed in a high humidity (approximately 100% relative humidity) growth chamber at 23ºC under constant light to preserve the integrity of the agar droplet."

[gregmartin]

Opps, missed your edit...
Oh well, hopefully folks will find the paper interesting that I posted the link to.

[Joseph Lofthouse]

I did diploid to tetraploid conversion experiments by soaking watermelon seeds in aqueous solutions of oryzalin. Prior to treatment, the seeds were soaked for 6 hours in water. The conditions that worked best were soaking in a 2 PPM solution for 26 hours. That is somewhat more than the solubility of oryzalin in water, so I didn't gain anything by using more concentrated solutions. Those conditions gave about 40% conversion. If I do this again, I will use a 1 PPM solution. I work with PPM because it makes the math trivial and I don't have to look up molecular weights. I had previously tried soaking germinated seedlings in the solution, but that killed everything. At such low concentrations I am not worried about the side-effects of the 'inert' ingredients.

Presumably converted (tetraploid) watermelon on the left. The diploid version of the same variety on the right. I crossed some of these presumed tetraploids with diploid melons last summer. If some of the offspring are seedless that will be another confirmation that the conversion was successful.




Diploid on the left. Presumed tetraploid on the right. Larger leaves is on of the archetypical effects of tetraploidy.

[Joseph Lofthouse]

Counting chromosomes by root-tip squash is within the reach of amateur scientists. The tools and reagents are for sale on eBay.

[papabearjay]

Jan 6, 2015 12:59:20 GMT -5 Joseph Lofthouse said:

Counting chromosomes by root-tip squash is within the reach of amateur scientists. The tools and reagents are for sale on eBay.

Very true, the issue is working with a species that doesn't have a multitude of chromosomes initially.  I'm aware of the range of chromosomes typically ranging from as few as 5 to as many as 200, but apparently Wikipedia says there are higher counts.  I wouldn't want to count 308 chromosomes, or attempt any chromosome doubling trials.

[darrenabbey]

I would want to take a photo of the spread chromosomes, then mark them off as I count them.

Really, I'd rather avoid the whole process unless I needed it. Unfortunately, one of the projects I'm thinking about (the-biologist-is-in.blogspot.com/2015/01/hybrid-sterility-and-speciation.html) will probably require chromosome doubling and the associated detailed examinations.

[gregmartin]

Darren, have you considered using garden strawberries as well as wild? That would likely complicate things, but you have to love the size. Keep us in the loop. Ever since I read Burbank's notes I've been thinking about this one too....very curious what we'll get....would be great to get strawberries up off the ground while injecting some raspberry flavors into them. I wonder if blackberry can cross with strawberry too. Thanks.

[darrenabbey]

I've had a great deal of difficulty finding specific ploidy information for available strawberry (or raspberry) varieties. At the moment I only have the wild forms I mentioned and I was able to look up the ploidy for those, so that knowledge drove my thinking.

It occurs to me that it would be nice to start with tetraploid (or multiple of 4n) parents, so the resulting hybrid would automatically have paired chromosomes and be fertile. If the ploidy of either parent isn't a multiple of 4n, then there will be infertility issues due to unpaired chromosomes. Using diploid parents would help reveal what was the cause of the infertility that Burbank observed, so is probably worth doing. However, as any sort of chromosome count issue would be resolved by chromosome doubling, the ploidy of the parents really doesn't matter if you're willing to do a chromosome doubling experiment.

Bloom time overlap might be an issue (as it was for Burbank), so some continuously flowering garden types would be ideal for multiple reasons. My imagination has been providing me with images of black strawberries, so who knows what could come out of it. If the cross works with raspberries, then I expect it would probably work with blackberries.