Doubled haploids

[mofeta]

(Edit: this thread sprung from this thread)

Hi Spurr!

Here is a new thread to discuss doubled haploid breeding. My appointments all canceled this morning and I have some free time to spend!

You said:

As I understand things, one can make DiHap strains from a female and a male (using TC and mutagens), then cross once they are grown out as mature DiHap plants. The resulting seed will not be DiHap, it will be 2N, but will be fixed for (some) goal traits. Feminization, and selfing, is/are route(s) some academics have taken, IIRC.

My knowledge of this is minimal, I haven't studied DH directly, only tangentially in relation to something else I was looking into. This is my understanding of the process:

Homozygous diploid plants are created using haploid cells from one plant, instead of two parents. This is done two ways:

1: By outcrossing to something distantly related to the plant you are working with, something similar enough to get viable seed, but distant enough so that the haploid contribution from the other plant is not expressed, resulting in a haploid plant. These haploids are then treated to double the haploid to homozygous diploid. I don't think this is of any use in pot.

2: By culturing gametes. Pollen and ova are, obviously, haploid. If you can coax either one of them into growing into a plant by themselves, you end up with a haploid plant that is sterile. Using chemicals, you can induce them to double this single haploid contribution into a homozygous diploid capable of reproduction. Usually it is the pollen that is used, as culturing individual ova is much more difficult.

Illustration of above.

Most of the plants that are bred using DH are monecious, like cereal grains. Being self fertile simplifies the process.

The use of DH in diecious plants seems to be limited to crops that being all male is a plus. Asparagus is a good example. Hemp too, maybe?

DH in Asparagus from good book on DH breeding.

The way I understand it (I could be totally wrong), is that the reason for this is (in a diecious plant) that when you double a haploid that is female you get XX. When you double a male haploid you get either XX or YY. When your haploid plant comes from a pollen grain that would have produced a female plant in normal reproduction, it will be XX when doubled. If the pollen grain was one that would have produced a male plant in normal reproduction it would be YY when doubled, and although fertile, would be incapable of contributing a X to offspring, making all seeds from the cross male. Of course you could reverse the females and breed those.

Part of good book on DH breeding that tells of how it is particularly useful in self-pollinating (monecious) plants.

Another part of the same book that discusses DH in diecious plants. Click the "Page 28" link to see the whole page.

Good paper on DH in rapeseed (mustard like plant that canola oil comes from). Papers like this discouraged me from thinking about trying this at home.

 

[spurr]

Hello,

Nice you got the morning off, and good post

Sadly, I have to head out very soon. For now, all of this is off the top of my head, so I can't guarantee it's 100% accurate until I have time to check my notes.

As I wrote in the other thread, I don't want to publicly share too much specific details about my plans to try and create DiHap C. spp. strains, but I am open to general discussion. I am still learning ...

I don't plan to use donor plant(s), for males I plan to try anther culture (where pollen is formed in 'microsporangium') and pollen (i.e., microspore) culture. And for females I plan to try ovule culture. I will use a (considerably) more safe and more effective mutagen than colchicine. I think C. spp. is better defined as subdioecious than dioecious.

I also plan to test inducing female DiHap to male (to facilitate crossing of plants), and male DiHap to female. I need to do more research for the latter goal.

Here are good sections of a useful web site; check out the topics (by tab) on the left and right hand side of the web page(s):

Use of Haploids in Plant Breeding
http://www.molecular-plant-biotechn...nd-uses/use-of-haploids-in-plant-breeding.htm



Anther and Microscope [sic; should be Microspore] Culture
http://www.molecular-plant-biotechn...on-and-uses/anther-and-microscope-culture.htm



Pollen Culture
http://www.molecular-plant-biotechnology.info/haploid-production-and-uses/pollen-culture.htm



Protocol for Pollen Culture
http://www.molecular-plant-biotechn...tion-and-uses/protocol-for-pollen-culture.htm


Oval Culture
http://www.molecular-plant-biotechnology.info/haploid-production-and-uses/ovule-culture.htm



Haploids from Unpollinated Ovaries
http://www.molecular-plant-biotechn...d-uses/haploids-from-unpollinated-ovaries.htm
​

 

[spurr]

.

 

[mofeta]

As I wrote in the other thread, I don't want to publicly share too much specific details about my plans to try and create DiHap

OK. If I tread on what you want to keep secret, let me know.

for males I plan to try anther culture (where pollen is formed in 'microsporangium') and pollen (i.e., microspore) culture. And for females I plan to try ovule culture.

I can be pretty dense sometimes, so bear with me please. I still don't see how you can get around the "supermale" thing. Anther culture will provide both male and female plants that would be fertile when doubled. But, the males will all be supermales, YY, no? Culturing ova and doubling them will produce all females also. Like I said before, you could just self the females after reversing, but I don't see how you could get female offspring using a YY supermale that you would get from DH.

and male DiHap to female.

This hits on the point I'm making. I think you could get a female looking flower on the DH male, but it would only have Y to contribute, so any seed would be male.

Thanks!

 

[mofeta]

I thought about it some more. Although the offspring of the DH male and the DH female would all be male, they would be normal XY males. I think? You could use those.

 

[mofeta]

So, you could do this

1) culture pollen to get haploid individuals, ones from a pollen grain with an X would be female haploids that would become diploid XX females when doubled, ones from a pollen grain with a Y would be male haploids that would become diploid YY supermales when doubled.

2) select females from above for the traits you want- they will be homozygous for any trait displayed by virtue of being a doubled haploid

3) cross the selected females with a few of the supermales- the result will be all male, but normal XY

4) use the normal males made in step 3 to cross back to your selcted females from step 2, grow out offspring, the ones that come out the way you want will tell you which supermales and their XY offspring have the traits you want- Hello homozygosity!

 

[Bobby Stainless]

What else can be used, other than colchicine treatments/treated seeds?

 

[Bobby Stainless]

anyone?

 

[mofeta]

What else can be used, other than colchicine treatments/treated seeds?

Hi Bobby-

Colchicine is not used on the seeds in this process, it is used on plants.

First, plants are made with only one set of chromosomes, instead of the normal two. These plants are "haploid". One of the ways these plants are made is by growing pollen. Using tissue culture techniques, pollen can be sprouted in a petri dish, and then grown into a plant with roots and leaves. These plants will be sterile, unable to reproduce.

The haploids are then treated with colchicine or a similar chemical that makes the single set of chromosomes turn into two sets, like a normal plant, hence the name "doubled haploid". Now they can reproduce.

The whole point of doing this is to produce a plant that is homozygous, and a doubled haploid is about as homozygous as you can get.

This technique is really difficult, expensive and time consuming. I've never done it, and probably never will. I was only posting on it because Spurr brought it up in another thread, and I find the subject interesting.

 

[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.

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.

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

edit: btw spurr stopped posting here.

 

[mofeta]

Hi Bobby

I think we are talking about two different things here.

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).

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!

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]

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.

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.

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]

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.