[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:

Originally Posted by spurr

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
https://www.molecular-plant-biotechno...t-breeding.htm



Anther and Microscope [sic; should be Microspore] Culture
https://www.molecular-plant-biotechno...pe-culture.htm



Pollen Culture
https://www.molecular-plant-biotechno...en-culture.htm



Protocol for Pollen Culture
https://www.molecular-plant-biotechno...en-culture.htm


Oval Culture
https://www.molecular-plant-biotechno...le-culture.htm



Haploids from Unpollinated Ovaries
https://www.molecular-plant-biotechno...ed-ovaries.htm

[spurr]

.

[mofeta]

Originally Posted by spurr

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.

Originally Posted by spurr

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.

Originally Posted by spurr

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]

Originally Posted by Bobby Stainless

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.