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teonanacatl

For sale- Oryzalin (Used to induce polyploidy)

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Oryzalin is a much less toxic alternative to colchicine for inducing polyploidy in plants (including cacti).

Using appropriate safety this chemical can be easily used by the home plant breeder to induce polyploidy.

Its also used as a pre-emergent herbicide, but I bought it to induce polyploidy.

Ive got 10mL bottles of 40.4% Oryzalin in propylene glycol, $5+ postage.

With the concentrations required 10mL of this solution will last for ever.

For more information see:

http://members.tripod.com/~h_syriacus/oryzalin.html

* I disagree with his calculations.

The MSDS:

www.southernag.com/docs/labels_msds/Ms1240.pdf

And the attached paper.

PM me.

Chromosome doubling in cacti.pdf

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teo, I am out of my depth here, and don't understand some things entirely.

I got a microscope on loan, but doubt I could notice this way if I have doubled my chromosones.

could, visual inspection as well, give you an idea if one was successful?

I know you don't post much latly, but I invite you to start a thread, about this using more layman terms.

as well I would struggle to, mix up those dilutions, but it could be easy, if given some thumb rules, like one single drop out of an eye dropper, diluted with x amounts of millilitres of sterile water.

in other words, I can't mix one drop of oryzalin with 10.000 drops of water, as it's too labour intensive. what I would do is, find out how many eye dropper drops make 1ml, and than take it from there...., is this a good method??

Edited by planthelper
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Oops sorry guys I didnt see this here!

Thanks Naja :)

Ill admit I have not tried this yet. Lets just say a small taipan gave my year a setback! :)

How are you using it on seed or on plants?

Basically if its on plants or seed and they survive then there is a chance it has worked.

The paper above says the following:

2.7. Chromosomecounting

Chromosome number was confirmed using pollen mother cells (PMCs) of flower buds which developed from the putative poly- ploid branches. Flower buds 5–6 cm long were collected, dissected, and fixed in 3:1 ethanol:acetic acid. After 24 h flower buds were transferred to 70% (v/v) ethanol. Squash preparations of PMCs were stained with 2% (w/v) aceto-carmine. Slides were observed under an AxiosKop 2 light microscope (Zeiss) and photographed with AxioCam camera (Zeiss).

So there is that option (which isnt that hard) or you just wait and see what happens to the plants. There will be visible signs Im just not sure what they may be.

The chance of success is low, that paper recorded a max of 5% success so this is only worth it on bulk seed / plant material.

Ill also post this link though Im not sure how helpful it will be.

http://www.shroomery.org/forums/showflat.php/Number/4233548/fpart/all/vc/1

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Does anyone have any interesting results to report? Any pics of crazy-funky-mutant-plants?

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5 hours ago, Smiling said:

Anyone got any freaky results from these experiments?

 

Pending.  I did mine in-vitro and am having trouble deflasking them

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On 1/17/2018 at 5:36 AM, Darklight said:

 

Pending.  I did mine in-vitro and am having trouble deflasking them

 

 

Fuggit. That wasn't the Surflan solution I got from Teon that I used for that experiment, it was some old pure Oryzalin stock for micropropagation. Threw it in some callus regen mix I had for a species I have a full callus regen protocol for, but because the species is variable in morphology while in culture I need to wait until plants are deflasked and in the ground to monitor for gross changes of horticultural interest. While they're in the jars they could be anything. The kill curve was nice over the Oryzalin gradient tho, so I'm hoping for some happy results.

 

Since I don't have a flow cytometer handy I thought I'd save time and check the stomates under a microscope against the controls, but that wasn't possible with my very limited microscopy skills. Couldn't see a damn thing. So we're back at wait-and-see. For a bunch of reasons results should be in by Xmas

 

 

I only just finished running the wetwork for the Surflan experiment a minute ago, and fuck it's the most virulent orange solution I've ever seen. Fair burned my eyes out with colour. I ran it using in-vitro apical tips, because this other species doesn't have a working callus culture protocol yet. And I used tiny tips because I don't want to give any cells the chance to encourage other cells to revert back to diploidy.

 

Everything survived the 1:200 soak for 24 hrs ( I used the lower level for all explants- in-vitro plants have their stomates more open as they are adapted to a high humidity environment ). Actually I was surprised how much they were in good nick, given I didn't throw any nutes or sugar in there and if I'd spent 24 hrs in a place that orange I would have died of bleeding eyeballs :)

 

I wasted a fuckton of time looking for an optimal protocol for this then realised I should go for the simplest one as I haven't run the experiment at all before, so I'm not expecting superlative results. First time is indeed lucky if something good happens :) There are a couple of minor things I'd do differently but overall it was just a cut and soak on the orbital shaker for 24 hrs, 3x rinse in water, recut ends and place in media

 

Normally I eschew anything with this much liquid handling if I can avoid it, throwing round multiple liquids over multiple explant batches is a prime way to introduce contam. Fingers crossed we didn't get any.

 

Writing up the notes now. There were 46 explants treated. Interested to see if there's a kill rate. Wish me luck :)

 

Thanks Teonacatl for the chance to give this a whirl <3

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Just now, Darklight said:

 

 

Fuggit. That wasn't the Surflan solution I got from Teon that I used for that experiment, it was some old pure Oryzalin stock for

 

The main issue here being sterility. I've read that Oryzalin doesn't survive autoclaving and so the pure Oryzalin I used for the callus experiment was dissolved then filter sterilised and added to media post-autoclave.

 

Given that I'm out of 0.2uM syringe filters, and I'm crossing my fingers that the Surflan is as close to sterile as practicable, being dissolved in something hostile to life like DMSO. So I just pipetted it raw into some sterile distilled water flasks using sterile tips.

 

This'll be the first place things can fuck up if the Surflan has fugly spores of anything in it.

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Update: all explants from the 1:200 Surflan soak treatment remained sterile despite the lack of filter sterilisation for the Surflan solution

 

This is not atypical for chem stocksolutions, many of which don't support microscopic life if kept reasonably stored and unopened, but it's good to know. Also I don't need to run the experiment again- how good is that :)

 

The thing that hasn't happened, and that I was hoping for, is a kill rate. It shows uptake of the Surflan and gives a good indication of the effectiveness of the application rate. I didn't run a gradient ( multiple concentrations simultaneously, or same concentration for different time points ) because I couldn't be arsed. A kill rate of about 30-50% at some concentration would be expected to provide some level of ploidy- tho it might only leave you with survivors and you'd still need to monitor for gross morphological changes and signs of chimerism.

 

1:200 would have been a pretty high rate from previous experience with Oryzalin solution ( Oryzalin is the active ingredient in Surflan ) in the agar media and using callus as a starting material rather than apical tips- I've seen that in two other species. But good quality callus is difficult to optimise media for in this species and I was going for a fast and dirty protocol, so I tried the web-standard protocol. Looks like it's not as effective.

 

All explants are fine- no Surflan damage at +9 days and under lights. There's still time for damage to show, but it's becoming less likely as time progresses. Not expecting new growth to show for another month

 

Tl:dr: If I want to try apical node soak again I'll need to run the soak at higher concentrations- ideally with a kill curve plan

 

The actual lab phase for this stuff is facile and fast. It's the monitoring and logging which kills it for mutation work.

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On 3/7/2018 at 10:13 AM, Darklight said:

The actual lab phase for this stuff is facile and fast. It's the monitoring and logging which kills it for mutation work.

 

Rightio, the above sums it right up.

 

Interesting conclusions have been drawn, experiments from both the Surflan and pure Oryzalin batches  above are still underway

 

The regen plants from the pure Oryzalin batch are of a different species to the Surflan batch. So yeah, some difference in response to be expected. Also, the first species had undifferentiated ( not embryonic ) callus as it's parent material. The latter species had apical nodes as it's donor material. This has def affected the selection process, I might not explain it well enough to do it justice, pls see below.

 

Kill curves which escalated over doseage for the first species. A good sign. Progressive failing at 30 days was observed in the second species, which was only treated at a single rate. Also a good sign. Something's whacked em, especially obvious since untreated controls from both groups showed no variance from normal growth in-vitro

 

Both treatment batches above maintained sterility after exposure- no further worrying about dirty Surflan needed.

 

However none of the Oryzalin/ callus batch are showing signs of morphological changes at deflask. And the Surflan/ apical nodes are definitely showing morphological stress that only a few are starting to recover from now.

 

Since Surflan is just Oryzalin powder + PEG, there's not the scope on this work to discuss the possible contribution of the PEG to the difference in results. I'm not convinced this is even an issue. For the purposes of the screening process I'm looking at two very different sets of results ( species and donor material aside ) and it seems to come down to exactly how the survivors of the treatment are selected for monitoring.

 

This could be because of the length of time both treated parent stock could survive exposure. The callus regens were grabbed pretty much as they showed up, over a 2-4 week period, and the remaining callus died off ( possibly exposure rate for the callus was too high )- and thus while the kill curve looks great- what's persisted is Surflan survivors rather than transformed polyploids.

 

Callus is a bit more sensitive to external influence than organised tissue is.

 

I can't remember the application rates or whether they were comparable for the two species/ treated tissue types. Will look it up later

 

Deflasked plants for the Oryzalin batch ( total of 18 survivors. 2 controls, 14 low range Oryzalin and 2 high-range Oryzalin  ) for this species/ treatment will go in the ground for long term monitoring of gross morphological changes to indicate polyploidy. It's the best I can hope for in this species, I've fucked around enough with wretched bloody microscopy teks to check the leaves, but my microscopy is shit, it's fiddly, and after all it won't make any difference to plant sales unless ploidy confers distinct traits which are visible to the buyer ( the entire point of the experiment in this plant- and yes, such distinctions do matter at the start when you're setting these things up )

 

In contrast, ALL of the 1:200 24hr Surflan/ apical node treatment species showed significant distress not long after my last post. Discolouration from the Surflan hit them to varying degrees, and this became even more pronounced after a temperature rise on the grow shelves ( hint- keep your Surflan-treated tissue at a constant temperature. Growth almost completely ceased even after 2 subcultures to try to get them away from any excreted Surflan metabolites

 

Growth in some of the remaining Surflan treated apices is now slowly resuming. Control, untreated apices from that experiment are fine and kept growing normally during this time. All leaves from apices in the treated group dropped or severely discoloured, new growth is scarce and severely stunted. About 30% failed at +30 days and another 20% failed really quickly after a temp rise. However root development is occurring in a few surviving apices and I'm hoping leaf regrowth will follow

 

This Surflan batch is a way off deflask yet, hopefully the tells of ploidy will be more visible in this species.

 

It could well be that cells/ tissue which has been rendered polyploid by these treatments takes a while to recover- that by grabbing the earlier survivors of the first ( Oryzalin ) treatment I was unlikely to get any polyploid individuals. The four month recovery rate for the second apical ( different species and parent material, Surflan treated ) batch would bear this out. At this point this idea is just that, but I have read ( mostly informal )  accounts of bothvigorous early, and of late recovery from Oryzalin/ Surflan treatment being indicative of ploidy. As a result of my ( minimal, early stage ) project here I'm more inclined to suspect it's the latter. But we'll see

 

Am confident the application rate for 1:200 Surflan/ apical node batch is sufficient- maybe a tad high if anything. I woulda/ shoulda/ coulda run a lowed dose batch alongside the controls but CBF. Maybe next time

 

So yeah, there it is... it's not just the actual wetwork, its the monitoring and logging which is the killer here. I won't know about the Oryzalin batch for 2 years. The second species isn't even close to deflask yet, so maybe 2 years from today- if there are any survivors

 

Apologies for the lack of organisation or succinctness in the post, I only got my head around some of it last week

Edited by Darklight
clarity, a substance produced from claret.
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