[mofeta]

Hi Bobby

I think we are talking about two different things here.

Originally Posted by Bobby Stainless

I understand the process. Actually seed treatment is the safest and most effective way to apply colchicine.

I am wondering what other substance can be used, that also inhibits chromosome segregation to the daughter cells. Which in turn creates larger daughter cells w/ multiple chromosome sets.

In the above quote you are talking about making polyploid plants (more than two sets of chromosomes). Colchicine is indeed applied to seeds to make polyploids.

The doubled haploid thing I was talking about uses colchicine to increase the ploidy of a haploid plant (not seed) from n to 2n (diploid).

Originally Posted by Bobby Stainless

I think the whole process is a bit silly, as far as the pursuit of homozygous plant is concerned. Most breeders just put in the work.

I agree. I think that for our purposes this technique would actually be more work and more difficult than good old selective breeding. Big companies with professional breeders and deep pockets might find it attractive though, if it is ever legal. Or maybe Spurr will do it in his basement!

Originally Posted by Bobby Stainless

Regardless, I was just wondering if there was some other substance besides the highly poisonous cochicine that would create the same effect.

I'm not sure how many substances are used to alter ploidy, I know that there is another one besides colchicine that is supposed to be less toxic to animals called oryzalin (trade name Surflan). It is an herbicide.

[mofeta]

....

[Bobby Stainless]

I got trolls...

Sounds like something you need an ointment for.

Thanks for clearing that up for me.

[Nunsacred]

Ok I've been taught a bit about DH breeding
and I agree that it generally doesn't sound any easier than inbreeding for say 5 generations of backcrossing.

However, I was also very surprised to hear that doubling up often occurs spontaneously so there's quite likely NO NEED FOR COLCHICINE, and the question remains : is there a close relative of cannabis (not necessarily a CENH3 mutant) which does it all naturally?

I believe it's worth a concerted effort to find out.
The problem then becomes identifying the DH individuals.......and the rate of efficiency.

I'm giving the matter some serious thought and wondering how to find out about centromeric relatedness of plant species....

Edit: by the way that BUMP gif above ^^ is fkn priceless

[mofeta]

Originally Posted by Nunsacred

However, I was also very surprised to hear that doubling up often occurs spontaneously so there's quite likely NO NEED FOR COLCHICINE,

What's wrong with colchicine? It works great, is easy to use, and contrary to popular belief is not very toxic, especially in this context. Colchicine is routinely consumed by gout sufferers at a level several orders of magnitude higher that any exposure that would occur doubling a haploid plant.

Anyway, it is the production of the haploids that is hard, the doubling is relatively trivial.

Originally Posted by Nunsacred

and the question remains : is there a close relative of cannabis (not necessarily a CENH3 mutant) which does it all naturally?

This outcrossing technique works best in a crowded area of the phylogenetic tree, like Poaceae/Gramineae, Solanaceae etc. Even then, it is quite difficult. The search for the inducer lines is long and tedious, and even then the production of viable seed is shaky, and the rate of haploid induction low.

In a plant like pot, out on that lonely little monotypic twig of a small taxon like Cannabaceae sesu stricto, I don't know if you would have much luck. Of course a big (expensive) program of systematically testing weed relatives, including even plants from Cannabaceae sensu lato, might turn something up, but my feeling is that the reproductive barriers are just too high. I think that any successful haploid production would require in vitro rescue of the embryo, you might as well just culture gametes. That is why I dismissed this technique in the first post of this thread.

A related technique I failed to mention in my first post was the use of irradiated pollen from the same species. Quite a few dioecious outcrossers have been worked this way, like melons, cucumbers, squash etc., I bet if would work with pot. But, it also usually requires in vitro embryo rescue, and of course the irradiation process is not something you would think of as something done by a hobbyist in his home.

Originally Posted by Nunsacred

I believe it's worth a concerted effort to find out.

Yeah, it would be real cool to have a haploid inducer. I just think that the only method practical for an amateur/hobbyist is gamete culture.


Germinating Onion Flower Bud




Haploid Onion Plant From Flower Bud Culture





Haploid Cabbage Embryo From Microspore Culture


I hope I haven't discouraged you, this technique will eventually be used (if it isn't already, by the big professional outfits like GW) on Cannabis. Once the inducer line is developed, by whatever technique, (only has to be done once) we will be golden.

Maybe you will do it! What if you found a hackberry that was a haploid inducer for pot? People would be composing poems and songs about you, and erecting statues of you in their town squares!

[Nunsacred]

Thanks mofeta,

from some initial googling it does look as though there are 2 main candidates to try as an initial shot-to-nothing :

Celtis australis ('Hackberry tree' as you said, lol, how well-named for this purpose)
Humulus lupulus (Common hop - not easy to germinate but root cuttings can be obtained)

Both of these should grow well enough in my garden, both have been reported to have the same number of chromosomes as cannabis (2n=20) so I'll see how easy it is to find or get them growing for a bit of fresh pollen.

I agree that colchicine isn't all that poisonous in the big scheme of things. I'm not against it per se, it's just that it's not necessarily available to all of us, and I was surprised to hear from breeders that they often don't need it.

I assume that people have grown hops and cannabis together before without noticing any significant cross pollination success there...

If someone said they were seeing their guerrilla grow plants seeded but with no obvious males around I'd assume they just haven't found the male cannabis flowers on their own plants and that the seeds are probably not worth growing from a hermie parent.
Now though, I really hope that's not always the case.....

[mofeta]

I like your enthusiasm!

I think considerable efforts have already been made to make a Cannabis/Humulus inter-specific hybrid, and no seeds are produced (I think). That doesn't mean that a haploid embryo wasn't produced, though. If you tried this and no seeds were produced, you could dissect the right parts out and try to culture them.

Female hop rhizomes are easy to get, male not so. You could dust female hops with pot pollen, and see if you got any offspring. Then maybe one of those could be used as a pollen donor haploid inducer. Or you could obtain hops seeds to get male plants. The seeds are sold sometimes, and you can also find them in low quality hops. I used to smoke hops when I was at University, I would buy real cheap ones and there were lots of viable seeds. Also, depending where you live, there may be wild hops. I collected wild hops from the mountains of SE Arizona several times.

On the colchicine availabilty, I think in the old days people used macerated Colchicum corms to good effect.

[GMT]

I think the efforts into haploid plants and DiHaploid plants later on, are overlooking a very important stage that comes later, namely meiosis.

[mofeta]

Originally Posted by GMT

I think the efforts into haploid plants and DiHaploid plants later on, are overlooking a very important stage that comes later, namely meiosis.

Hi GMT

Well, the whole point of DH is to avoid the "allele shuffling" of meiosis.

DH makes for perfectly homozygous plants, something that can't be achieved with standard breeding techniques (you can get close though, with massive work and time).

This technique, although powerful, has limited use, and is only appropriate in the right context.

A good illustration of where you could use this is in the much talked about "GS Cookies" cut. If you cultured gametes from this cut, either ova or pollen from reversal, and then doubled them, some of them would show the "Cookies" trait. The ones that did would be perfectly homozygous for the trait. That would be useful in developing a true breeding cookie line.

I hope this is a useful reply.

Thanks,

mofeta

[GMT]

ah mate, I say this only to clarify my position, and not to be a smart arse I promise, but I meant meiosis, not mitosis. Mitosis being the standard parent to daughter cells as the plant grows, where the shuffling takes place (and the occasional miscopy) and meiosis being the creation of the haploid cells. In meiosis a lot more than reshuffling takes place. It's when the rewrites take place. The doubling of segments of dna, the deletion of segments, and the random swaps and passing down the line of whole genes. And all randomly, and different in each haploid cell produced. Now I accept that in alot of cases, the changes make no diff to the outcome, but still the haploid cells produced in any plant, will not only be diff from the original, but also from each other. Now I do accept that these diffs will be far smaller than in say crossing a haze to an afghani, (just to take things to their extreme). But you still aren't getting seeds that contain the exact same instruction sets or the same order that existed in the mother DH plant. Lets look at the process steps.
1. pollen grown into haploid plant
2. poison added to double the haploid cells into diploid cells (assuming you don't kill the plant)
3. plant flowered and found to be female (super male makes things impossible)
4. clones taken and one reversed for pollen
5. now mutated pollen applied to dh mother
6. diploid seeds created which inherit (and I do accept the concept but still) diploid not DiHaploid DNA strands, one from original DH mom and one from pollen producing mom. And the process is the same in producing the ovums you pollinate, so each strand of dna is now different from the original DH mom's. And these will most certainly never be identical changes in both the pollen and the ovums that meet to produce the seeds.
7. you have female only seeds which each contain two strands of dna that is different in each seed and different from the DH mom.
You still don't have a line of identical plants to the ideal DH, and if you want to make them into a male female line, you essentially throw away all that work.
And all of that ignores the fact that the DH mom will be different from both the father and the grandmother, (if using male pollen, though I would assume you'd reverse a desirable female in order to have any chance of getting anything like what you wanted) making it a crap shoot as to whether any of the resulting seeds will contain any of the desired traits you were aiming for in the first place.