Hi,
Just wondering if anyone out there in the community has tried to use doubled haploid method to produce true breeding cultivars?
Or if anyone has got a good method for anther or ovule culture?
Thanks!
Chronisseur
3 Likes
I havenât tried any of the methods, just spent way too long reading about them, so I can try and recall what I found if itâs of any use.
This is a holy grail of breeding that is used for various other food crops. It is done a few different ways, two of which you have mentioned, ovule and anther culture to cultivate the haploid plant followed by gene doubling with colchicine or something else.
Both ovule and anthers culture require plant specific protocols that use varying amounts of plant hormones. I have yet to find a paper that has done this successfully, though anther culture seems to be the most trialed approach. One approach that I think holds promise is to use a wide cross and then use embryo rescue. The idea here is to use a distantly related plant species, say Japanese hops for example as the pollen donor, although fertilization occurs, the pollen donorâs chromosomes are eliminated during early cell divisions. Typically the plant would then abort the undeveloped seeds; the breeder instead rescued the undeveloped embryo and cultures it to get a haploid plantlet that is then gene doubled. These things are experimental and take some pretty serious laboratory work, and Iâm not aware of this being successfully done in the real world for cannabis.
4 Likes
Ha! Iâm keen. Letâs push back the boundaries
2 Likes
The lack of drive is not the issue itâs more often than not a âfundingâ issue and a thing like this takes serious dedication and an almost âmaniacâ type of personality to see it through to completion
3 Likes
I wouldnât be asking if I wasnât seriously considering how to do it!!
It would be so convenient to make true breeding strains this way, if we had a good method.
3 Likes
I prefer the âtried and trueâ old school method myself it allows for a more complete genome and from my understanding you will get the same result just by selfing a plant PS as this method here pss not hating just saying
2 Likes
Not quite the same, you get a completely homozygous plant in one step using haploid doubling, but you still end up with the issue of not having males/females of the variety, so it is most useful for example for making true F1 varieties. There is a fair amount of research going on for this approach because in theory it massively reduces the amount of time and effort required to produce an IBL, and would lower the risk of losing traits for later generations. You essentially are snapshoting a specific phenotype and making it breed true from that point forward.
I think itâs the technical challenge that is the issue on this technique being more common. The process requires a full clean lab, specific chemicals/hormone ratios and protocols that are not publicly available as far as I can tell.
A good start would be to check out embryo rescue techniques. Also there is some research done on pollen grain culture that Iâll see if I can find. A lot of the equipment and setup is similar to tissue culture, though a good stereo microscope is also required.
4 Likes
Thatâs exactly the point of doing this.
I want to end up with genetically complete identical sets of information on both sex chromosomes, then the cross is completely homozygous.
The biggest issue I can see is that you need to revert a male anther culture into a doubled haploid female, which in cannabis might be a bit tricky. I donât know if ethylene is effective, but it might be possible to eg leave some banana skins in the tent during the flowering cycle or vent gas in a bag.
Thatâs the reason ovule culture is probably the better route but itâs much harder as you have to do tissue culture on the internal part of the seed, which means surgery and then implantation into a medium, which is much more tricky than just culturing pollen dust onto a plate in tissue culture.
Maybe one way to do it would be to use silver nitrate or thiosulfate to make pollen on a female, then culture the female pollen, then doubled haploid using colchicine. These plants can then be crossed back to the original female, but itâs not perfect because youâre still going to have a heterozygous combination with the original. What to do?
3 Likes
Here is an article on pollen culture that is worth a read. Cannabis is regarded as a ârecalcitrantâ species for this kind of culturing, so it is going to take a lot of experimenting.
Unfortunately there is more involved than simply culturing pollen as the pollen grains are only able to be cultured when they are immature, so they would need to be extracted before the flower has opened. This is micro plant surgery any approach you take.
3 Likes
Would you need to do constant genetic testing or could you safely assume that you were getting the outcome that you wanted?
3 Likes
@Ecnylabs1 check it out bro have you ever heard or tried this yet?
2 Likes
Well if you succeeded despite all the massive hurdlesâŚ
It depends how far down the science bud path you want to go, I guess.
For me personally it would just be a really useful way to preserve that âexactâ strain. Iâve heard of people doing tissue culture, this is the next level along from that, where it becomes genetic engineering.
2 Likes
@Chronisseur I tagged @Ecnylabs1 because embryo rescue is what he does he helped @Upstate with the Green Hornet
3 Likes
If you culture the pollen, or ovules, embryos etc. you get half the chromosomes, so a haploid. It would die or be weedy if you grow it like this.
If you use colchicine you are basically copying that single set of chromosomes and doubling to make it a normal plant again. and then creating a completely homozygous true breeding plant in one step.
Edit:
I reckon a wide cross using hop pollen is worth a look, the pollen fertilizes the egg cell and forms the zygote, but the pollen chromosomes fail to integrate with the egg and you get a haploid embryo, if you leave that on the plant it will abort, it might just die or even grow an empty seed etc.
But if you rescue the embryo before it aborts, you should be able to culture it and double it for a homozygous copy of the mother.
2 Likes
I have not tried this technique, but It would take a lot of time to dedicate the R & D toward a completely true breeding line using that method.
On another note: A buddy of mine is doing some cool projects reversing males. He selects the best males he finds in a pack of old
Seeds, and then reverses the male. He then runs those s1 male seeds to see mainly which males heâs going to select - then he runs that s2 seed line (which are regular seeds this generation) and continues to work the line using the best males working them into the s1 generation to get true breeding seeds - Essentially what he is trying to do is create a pure breeding pure, all male line in seed form.
3 Likes
Iâve tried this how did he pull it off? He actually got germination?
1 Like
Why is that an issue?
The Y Chromosome is highly degernated and as far as i read only posseses genes that are important for the male.
Female x Female is the way.
Double Haploid is the Goat
All male is impossible As far as i know.
1 Like
Tell me how to revert a male please!!!
2 Likes
